No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon
Abstract
Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP-IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.
Supplementary resources (5)
... Therefore, trypsin activity remained lower in the pyloric caecal tissue of the TRP-2*92 salmon (Figure 2) because the total trypsin activity per fry ( Figure 2) as well as in sum of pyloric caecal tissues and lumen (Rungruangsak Torrissen and Male 2000) were similar between the fish with different trypsin genotypes. The results were also in line with the observations by Male et al. [1995] that trypsin genes seemed to be stable, and although the pattern of expressed genes varied extensively, the expression of trypsinogen mRNA was quantitatively similar between individual salmon. ...
... Cloning and characterization of trypsin isozymes in Atlantic salmon have been performed, and five clones containing near full-length transcripts (four encoded anionic forms and one encoded cationic variant) have been revealed (Male et al. 1995). So far, trypsin clones and trypsin isozymes have never been matched, probably due to too few differences in sequences and the knowledge of gene expression is still limited. ...
... Trypsin is a major digestive enzyme in the large family of serine proteases, and its well known structural information in mammals has made it an excellent protein model for studying the relationship between sequence, structure, and function of different isoforms of trypsin in Atlantic salmon (Male et al. 1995). Trypsin genes in mammals (Craik et al. 1984;Fletcher et al. 1987) have similar structure to chymotrypsin whereas their differences in substrate specificities are due to differences in the substrate-binding pockets, two supporting loops (Hedstrom et al. 1992) and certain contributing amino acid residues (Hedstrom et al. 1994a(Hedstrom et al. , 1994b. ...
It is an Open Access Chapter and can be downloaded for reading at
https://novapublishers.com/wp-content/uploads/2019/05/978-1-63117-570-1_ch6.pdf
Atlantic salmon, Salmo salar L., is an anadromous species living in fresh water during the first year(s) of life until it smolts, and later in the sea from post-smolts until maturation before returning to fresh water to spawn. The early life period in fresh water and the first sea winter are critical periods when genetics, food qualities, and environmental conditions can affect growth rates. As a carnivore, dietary protein is a very important key nutrient for growth, and trypsin is the key protease that activates other pancreatic zymogens including chymotrypsinogen. This has made trypsin important for genetic studies on protein metabolism and the protease activity ratio of trypsin to chymotrypsin (T/C ratio) the important factor for digestive efficiency and growth.
Trypsin has different isoforms. Variations in genetic expression of trypsin isozyme patterns (trypsin phenotypes) of individual Atlantic salmon result in different abilities of the fish to digest the same protein and optimize food utilization and growth. Changes in trypsin phenotypes can be induced by temperature (at egg incubation and first feeding periods) and by dietary quality at the very early life stage. Changes in environmental conditions affect trypsin gene expressions at molecular and protein levels, regardless of genetic expression of parents. The effects can be maintenance ration, consumption, digestion, absorption and transport of free amino acids, insulin secretion, protein growth efficiency (ratio of protein to lipid – P/L ratio), health, maturation, and behaviour during the whole life cycle. Fish possessing different trypsin genotypes have different temperature preferences for optimizing food digestion for utilization and growth, which result in different growth rates and temperature distributions of the fish in natural ecosystems. Genetically manipulated fish, such as triploid Atlantic salmon, have less food utilization efficiency than their diploid counterparts. Molecular cloning and characterization of trypsin isozymes show very little genetic variation in trypsin genes. So far, trypsin clones and trypsin isozymes have never been matched, probably due to too few differences in sequences and the knowledge of gene expression is still limited. Studies on trypsin genotypes have provided new insights that can be exploited and integrated into other research fields to elucidate genetics of growth performance quality through food utilization under different environmental conditions. * Corresponding Author address: Matre Research Station, N-5984 Matredal, Norway; Email: Krisnart@imr.no. No part of this digital document may be reproduced, stored in a retrieval system or transmitted commercially in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.
... The primary structure of trypsin is available for enormous number of species including fish. Pancreatic cDNA libraries from Atlantic salmon have been constructed and screened, and five clones containing near full-length transcripts have been characterized showing three of them possessing very similar sequences and might be encoded by the same gene locus while the other two were probably encoded by separate gene loci [14]. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms, and the fifth clone represented a cationic trypsin, with around 70% identical primary sequence between anionic and cationic forms [14]. ...
... Pancreatic cDNA libraries from Atlantic salmon have been constructed and screened, and five clones containing near full-length transcripts have been characterized showing three of them possessing very similar sequences and might be encoded by the same gene locus while the other two were probably encoded by separate gene loci [14]. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms, and the fifth clone represented a cationic trypsin, with around 70% identical primary sequence between anionic and cationic forms [14]. Multiple alignments of amino acid sequences of trypsin from different species, and the relationships between trypsin sequences evolutionally presented as a phylogram are illustrated in Rungruangsak Torrissen and Male [1] and Male et al. [14]. ...
... The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms, and the fifth clone represented a cationic trypsin, with around 70% identical primary sequence between anionic and cationic forms [14]. Multiple alignments of amino acid sequences of trypsin from different species, and the relationships between trypsin sequences evolutionally presented as a phylogram are illustrated in Rungruangsak Torrissen and Male [1] and Male et al. [14]. Trypsin has been purified and characterized with the N-terminal amino acid sequences identified in different species (Table 1). ...
It is an Open Access Chapter, and can be downloaded for reading here.
http://www.novapublishers.org/catalog/product_info.php?products_id=38114
... Animal trypsins usually have cationic and anionic forms, such as bovine (Walsh, 1970), dog (Pinsky, Laforge, & Scheele, 1985) and rat (Fletcher, Alhadeff, Craik, & Largman, 1987). Molecular cloning study has identified the existence of these two forms of trypsin in Atlantic salmon (Male, Lorens, Smalas, & Torrissen, 1995). However, to our knowledge, cationic trypsin from freshwater fish has not been reported. ...
... The N-terminal amino acid sequence of trypsin B was analysed and a sequence of IVGGYECEAH was determined ( Fig. 6). This sequence, although short, exhibits high similarities with trypsins from common carp (Cao et al., 2000), Japanese anchovy (Kishimura, Hayashi, Miyashita, & Noami, 2005), Atlantic salmon (Male et al., 1995), tongol tuna (Klomklao et al., 2006), dogfish (Titani, Ericsson, Neurath, & Walsh, 1973), Atlantic cod (Asgeirsson et al., 1989), cow (Walsh, 1970), pig (Hermodson, Ericsson, Neurath, & Walsh, 1973), dog (Pinsky et al., 1985) and rat (Craik et al., 1984). Though the N-terminal sequence of trypsin A was not successfully determined, our present data clearly demonstrated that the two enzymes from the pyloric caeca are most likely members of the trypsin family. ...
... Alignment of the N-terminal amino acid sequence of mandarin fish trypsin B with trypsins from other species. Common carp trypsin A and B (Cao et al., 2000), Japanese anchovy (Kishimura et al., 2005), Atlantic salmon (Male et al., 1995), Atlantic cod (Asgeirsson et al., 1989), tongol tuna (Klomklao et al., 2006), dogfish (Titani et al., 1973), bovine (Walsh, 1970), porcine (Hermodson et al., 1973), dog (Pinsky et al., 1985) and rat (Craik et al., 1984). Same amino acid residues are boxed. ...
Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21kDa and 21.5kDa, respectively, as estimated by SDS-PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35°C and 40°C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca(2+) and Mg(2+) and inactivated by Fe(2+), Zn(2+), Mn(2+), Cu(2+), Al(3+), Ba(2+) and Co(2+) to different degrees. Apparent Km values of trypsin A and B were 2.18μM and 1.88μM, and Kcat values were 81.6S(-1) and 111.3S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.
Copyright © 2008 Elsevier Ltd. All rights reserved.
... The enzyme preparation was highly active between pH 7.0 and 9.0 with an optimum at pH 8.0 when incubated for 15 min at 60°C. The relative activities This characteristic likely contributes to its physiological role in intestinal tissue, where pH is high (Male et al. 1995), and is a relevant aspect that enables its use in liquid detergent formulations, as the pH of laundry detergents is commonly alkaline (Maurer 2004). The optimum pH for zebra blenny proteases was similar to that reported by Bougatef et al. (2008) for proteases extracted from the viscera of Sardinelle (Sardinella aurita). ...
... The enzyme preparation proved sensitive to ions, mainly Zn 2+ , Cu 2+ , Mn 2+ and Hg 2 + . It is known that Hg 2+ is among ions that act on sulfhydryl residues in proteins (Male et al. 1995). The inhibition caused by this ion suggests the relevance of sulfhydryl residues for the catalytic action of most proteases (Bezerra et al. 2005). ...
The present study describes the characterization of crude protease extract from zebra blenny (Salaria basilisca) and its evaluation in liquid detergent and shrimp waste deproteinization. At least five caseinolytic proteases clear bands were observed in zymogram. The crude alkaline protease showed optimum activity at pH 8.0 and 60 °C, and it was highly stable over a wide range of pH from 6.0 to 11.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (5 % Tween 80 and 5 % Triton X-100) and oxidizing agents (1 % sodium perborate), and relative stability towards anionic surfactant (1 % Sodium dodecyl sulfate (SDS)). They also showed high stability and compatibility with various laundry liquid detergents from Tunisian market. Furthermore, the crude enzyme was stable towards several organic solvents and retained more than 50 % of its original activity after 30 days of incubation at 30 °C in the presence of 50 % (v/v) dimethylsulfoxide (DMSO). Further, proteases from zebra blenny viscera were found to be effective in the deproteinization of shrimp wastes. The protein removal after 3 h at 40 °C with an enzyme/substrate ratio (E/S) of 5 U/mg protein was about 77 %.
... The presence of proteases and lipases in carnivorous species is justiWed by the fact that they consume fat and protein-rich live prey, being carbohydrates the less macronutrient utilized (Chakrabarti et al. 1995;Dhont and Van Stappen 2003;Lubzens and Zmora 2003). Alkaline proteases secreted by the exocrine pancreas, such as trypsin, chymotrypsin and elastase (Gudmundsdottir et al. 1993;Male et al. 1995;Douglas and Gallant 1998;Suzuki et al. 2002), take charge of protein digestion during the early stages of larval development, when gastric glands are still not developed and, therefore, there is no pepsin activity for acid digestion yet (Darias et al. 2005a). Trypsinogen is the inactive form of trypsin and several studies revealed the existence of diVerent isoforms showing temporal and spatial variations in their expression patterns that indicate functional diVerences along larval development Pérez-Casanova et al. 2006). ...
... In the present work, only one type of trypsinogen could be isolated, which presented similarities with two trypsinogens, depending on the species compared. Thus, red porgy trypsinogen showed higher identity to trypsinogen II from winter Xounder (Douglas and Gallant 1998) and Atlantic salmon (Male et al. 1995) and to trypsinogen I from Japanese Xounder (Paralichthys olivaceus) and Atlantic cod (Gudmundsdottir et al. 1993). Concerning to the partial sequence studied at amino acid Red porgy trypsinogen sequence (a) and bile salt-activated lipase sequence (b) corresponding to PCR ampliWcations using primers (fdp2/fdp3 and BAL5ЈN/BAL3Ј, respectively) from winter Xounder. ...
The fish larval stage is a critical step since only those specimens that survive will reach the adult stage in future. Knowledge
related to fish larval nutritional requirements and digestive enzymes capacity is still scarce, although necessary to obtain
satisfactory survival and growth rates. Trypsinogen is the precursor of trypsin, the main proteolytic enzyme acting during
the early larval stage. Bile salt-activated lipase (BAL) is a multi-substrate digestive enzyme that hydrolyzes carboxyl ester
bonds of acylglycerols, cholesterol esters and fat-soluble vitamin esters. The goal of this study was to determine the pattern
of trypsinogen and BAL expression during larval development in red porgy (Pagrus pagrus, Pisces, Sparidae), reared under standard conditions to provide the basis for future experiments testing the possible transcriptional
regulation for this enzyme under different nutritional conditions. Thus, partial cDNAs for trypsinogen and BAL from red porgy
were isolated. The putative aminoacid sequences obtained for both precursors showed around 80% identity to other fish sequences
from GenBank database. Trypsinogen and BAL were expressed from hatching and specifically located in the exocrine pancreas,
revealed by in situ hybridization. The present study shows that this species is being prepared for protein and lipid digestion
before exogenous feeding starts, exhibiting an ontogenetically programmed pattern for trypsinogen and BAL expression during
the yolk-sac stage.
... The TR-S and TR-P were stabilized by calcium ion from thermal denaturation. These findings suggest that the TR-S and TR-P possess the primary calcium binding site like mammalian pancreatic trypsin and other fish trypsins (Male et al., 1995; Genicot et al., 1996). In conclusion, the TR-S showed almost same characteristics with that of porcine pancreatic trypsin except for unstable below pH 5.0. ...
... azonus) with those of other vertebrates. Antarctic fish (Genicot et al., 1996); Cod (Gudmundsdottir et al., 1993); Salmon (Male et al., 1995); Founder fish (GenBank accession number AB029750); Porcine (Hermodson et al. 1973); Bovine (Walsch, 1970); Human (Emi et al., 1986).Fig. 4. Effects of temperature on the activity of trypsins from true sardine (S. melanostictus) and arabesque greenling (P. ...
Trypsins, TR-S and TR-P, from the viscera of true sardine (Sardinops melanostictus) and from the pyloric ceca of arabesque greenling (Pleuroprammus azonus), respectively, were purified by gel filtration and anion-exchange chromatography. Final enzyme preparations were nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weights of both enzymes were estimated to be 24,000 Da by SDS–PAGE. The N-terminal amino acid sequences of the TR-S, IVGGYECKAYSQPWQVSLNS, and TR-P, IVGGYECTPHTQAHQVSLNS, were found. The TR-S and TR-P had maximal activities at around pH 8.0 for hydrolysis of Nα-p-tosyl-l-arginine methyl ester. Optimum temperature of the TR-S and TR-P were 60 and 50 °C, respectively. The TR-S and TR-P were unstable at above 50 and 30 °C, respectively, and below pH 5.0. Both TR-S and TR-P were stabilized by calcium ion.
... Instability at acidic pH was also observed for trypsins from other fish species (Asgeirsson et al., 1989; Kishimura et al., 2005; al., The enzymes were kept at 30 °C and pH 8.0 for 0–8 h in the presence of 10 mM CaCl 2 (closed symbol) or 10 mM EDTA (open symbol), and then the remaining activities at 30 °C and pH 8.0 were determined. (a) Pacific cod trypsin; (b) saffron cod trypsin.Gudmundsdottir et al., 1993); walleye pollock (Kishimura et al., 2008); elkhorn sculpin (Kishimura et al., 2007); arabesque greenling (Kishimura et al., 2006a); brown hakeling (Kishimura et al., 2006c); Atlantic salmon (Male et al., 1995); Antarctic fish (Genicot et al., 1996); spotted mackerel (Kishimura et al., 2006b); yellow tail (Kishimura et al., 2006c); jacopever (Kishimura et al., 2007); Japanese anchovy (Kishimura et al., 2005); true sardine (Kishimura, Hayashi, et al., 2006); Japanese dace (DDBJ accession number AB445492); lamprey (Roach, Wang, Gan, & Hood, 1997); skipjack tuna (Klomklao et al., 2007); tongol tuna (); yellowfin tuna (); Atlantic bonito (Klomklao et al., 2007c); zebrafish (DDBJ accession number AF541952); bluefish (Klomklao et al., 2007b); tilapia (DDBJ accession number AY510093); rat (Roach et al., 1997); dog (Roach et al., 1997); porcine (Hermodson, Ericsson, Neurath, & Walsh, 1973); bovine (Walsh, 1970); and human (Emi et al., 1986). Amino acid residues different from the Pacific cod trypsin are shaded. ...
... The GM-T and EG-T were stabilised by calcium ion from thermal denaturation. These findings suggest that the GM-T and EG-T possess the primary calcium-binding site like mammalian pancreatic trypsin and other fish trypsins (Genicot, Rentier-Delrue, Edwards, Vanbeeumen, & Gerday, 1996; Male, Lorens, Smalas, & Torrissen, 1995).Fig. 5 shows the N-terminal amino acid sequences of the GM-T and EG-T aligned with those of other animal trypsins. ...
Trypsins from the pyloric ceca of Pacific cod (Gadus macrocephalus) (GM-T) and saffron cod (Eleginus gracilis) (EG-T) were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparations were nearly homogeneous on SDS–PAGE and the molecular weights of both enzymes were estimated to be approximately 24 kDa by SDS–PAGE. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of GM-T and EG-T. The optimum pH and optimum temperature of both trypsins were around pH 8.0 and 50 °C, respectively, using Nα-p-tosyl-l-arginine methyl ester as substrate. The GM-T and EG-T were unstable above 30 °C and below pH 5.0, and they were stabilised by calcium ion. The N-terminal amino acid sequences of GM-T (IVGGYECTRHSQAHQVSLNS) and EG-T (IVGGYECPRHSQAHQVSLNS) were found. The percentage of hydrophobic amino acid in the N-terminal 20 amino acids sequences of these cold-zone fish trypsins was lower (28%) than those of temperate-zone fish trypsins (34%), tropical-zone fish trypsins (37%) and mammalian trypsins (34%). Whereas the content of charged amino acids in the GM-T and EG-T was relatively higher than those of trypsins from temperate-zone fish, tropical-zone fish and mammals. Moreover, the GM-T catalyzed synthesis of Nα-(tert-butoxycarbonyl)-l-alanyl-l-alanine-p-nitroanilide (Nα-Boc-l-Ala-l-Ala-pNA) has been studied by using Nα-(tert-butoxycarbonyl)-l-alanine-p-guanidinophenyl ester [Nα-Boc-l-Ala-OpGu (inverse substrate)] as acyl donor and l-alanine-p-nitroanilide (l-Ala-pNA) as acyl acceptor, respectively.
... Other studies focused on the recovery of trypsin and chymotrypsin from Monterey sardine (50)(51)(52)(53)(54). When it comes to salmonids, trypsin with cold-adapted properties was extracted from Coho salmon (55) and Atlantic salmon (56)(57)(58). ...
The fisheries and aquaculture industries are some of the major economic sectors in the world. However, these industries generate significant amounts of wastes that need to be properly managed to avoid serious health and environmental issues. Recent advances in marine waste valorization indicate that fish waste biomass represents an abundant source of high-value biomolecules including enzymes, functional proteins, bioactive peptides, and omega-3 rich oils. Enzyme-assisted processes, for the recovery of these value-added biomolecules, have gained interest over chemical-based processes due to their cost-effectiveness as well as their green and eco-friendly aspects. Currently, the majority of commercially available proteases that are used to recover value-added compounds from fisheries and aquaculture wastes are mesophilic and/or thermophilic that require significant energy input and can lead to unfavorable reactions (i.e., oxidation). Cold-adapted proteases extracted from cold-water fish species, on the other hand, are active at low temperatures but unstable at higher temperatures which makes them interesting from both environmental and economic points of view by upcycling fish waste as well as by offering substantial energy savings. This review provides a general overview of cold-adapted proteolytic enzymes from cold-water fish species and highlights the opportunities they offer in the valorization of fisheries and aquaculture wastes.
... Some enzyme may have several forms within one species, forms that differ in specificity and activity [e.g. trypsin/trypsinogen in Atlantic salmon [108], Atlantic cod [109], albacore tuna Thunnus alalunga [110], anchovy Engraulis japonicus [111] ] .Di g e s t i v ee n z y m ep r o f i l e sa n da c t i vities vary between species, depending on food preferences. Carnivorous species have higher levels of proteolytic enzyme activity whereas carbohydrases are predominant in herbivorous and omnivorous fish [112,113]. ...
As most fish are ectotherms, their physiology is strongly affected by temperature. Temperature affects their metabolic rate and thus their energy balance and behavior, including locomotor and feeding behavior. Temperature influences the ability/desire of the fish to obtain food, and how they process food through digestion, absorb nutrients within the gastrointestinal tract and store excess energy. As fish display a large variability in habitats, feeding habits, and anatomical and physiological features, the effects of temperature are complex and species-specific. The effects of temperature depend on the timing, intensity and duration of exposure as well as the speed at which temperature changes occur. Whereas acute short-term variations of temperature might have drastic, often detrimental, effects on fish physiology, long-term gradual variations might lead to acclimation, e.g. variations in metabolic and digestive enzyme profiles. The goal of this review is to summarize our current knowledge on the effects of temperature on energy homeostasis, with specific focus on metabolism, feeding, digestion and how fish are often able to “adapt” to changing environments through phenotypic and physiological changes.
... Multiple trypsinogen forms have been attributed to gene duplication in pancreatic serine proteases along the evolutionary history and alimentary selective pressure could justify their maintenance in the genome (Roach et al. 1997;Suzuki et al. 2002). Trypsinogen isoforms may perform different functions due to variations in their molecular structure or kinetic properties of the codifying enzymes (Male et al. 1995;Ásgeirsson et al. 1989;Ahsan et al. 2001). Besides these intraspecific differences, trypsin has shown important differences among evolutionary classes. ...
Persian sturgeon (Acipenser persicus) is a critically endangered species, mainly due to overexploitation for its caviar. The permanence of populations of this species in the Caspian Sea is fully dependent on restocking programs. Accordingly, it is considered as an interesting target for aquaculture for both restocking and commercial purposes. In addition, as a Chondrostei, it exhibits one of the slowest rates of molecular evolution among vertebrates and is propounded as an excellent candidate for phylogenetic analysis and evolutionary biology. In this study, the early ontogeny of some key digestive enzymes precursors was determined at molecular level, aiming to obtain basic knowledge on the acquisition of digestive capacity of this species, and at the same time, to advance in its phylogenetic status from the point of view of digestion. For this purpose, A. persicus cDNAs for β-actin (actb; used as an internal reference gene), bile-salt activated lipase (cel), trypsinogen 1 (try1), pepsinogen (pga), and gastric proton pump (atp4a) were amplified and cloned, and their subsequent expressions were measured by quantitative real-time PCR during the first 34 days post hatch (dph). Two isoforms for pga and at least six for try1 were obtained in this study, probably due to the additional genome duplication which sturgeons suffered along evolution. Phylogenetic analysis of the deduced amino acids sequences from the studied genes demonstrated that this species has a close evolutionary distance to Holostei, coelacanths, and tetrapods, including amphibians, reptiles, birds and mammals. According to our results, expression of all the genes increased gradually over time and reached maximum levels around 18 dph. This pattern, which was comparable to length and weight data, could indicate that around 3 weeks after hatching, the digestive capacity of the Persian sturgeon changes from larval to juvenile mode.
... The N-terminal amino acid sequence (first 25 amino acids) of purified trypsin from cobia was determined to be IVGGYECTPHSQAHQVSLNSGYHFC. Among all marine fish used for these alignments of the N-terminal sequences, Salmo salar, Gadus morhua, Paranothotenia magellanica and Oncorhynchus keta were species with high similarity to cobia trypsin [27][28][29][30]. An exception was Dicentrarchus labrax which had an enzyme with only 10 homologous amino acids residues [31]. ...
A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50 °C and 8.5, respectively. The enzyme was thermostable until 55 °C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38 mM, kcat value was 3.14 s⁻¹, and kcat/km was 8.26 s⁻¹ mM⁻¹. The catalytic proficiency of the purified enzyme was 2.75 × 10¹² M⁻¹ showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93 kcal mol⁻¹ while the resulting rate enhancement of this reaction was found to be approximately in a range from 10⁹ to 10¹⁰-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.
... The enzyme preparation proved completely sensitive to ion Hg 2+ . It is known that Hg 2+ is among ions that act on sulfhydryl residues in proteins [23]. The inhibition caused by this ion suggests the relevance of sulfhydryl residues for the catalytic action of most proteases [24]. ...
The present study describes the characterization of crude protease extract from Arthrobacter ari-laitensis Re117 and its evaluation in solid and liquid detergent. One caseinolytic protease clear band was observed in zymogram. The crude alkaline protease showed optimum activity at pH 9.0 and 50˚C, and it was highly stable over a wide range of pH from 8.0 to 9.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (Tween 80, Tween 20 and Triton X-100) and stimulate activity towards oxidizing agents such as sodium perborate. They also showed high stability and compatibility with various laundry solid detergents from Tunisian market. The pro-tease of A. arilaitensis Re117, was also tested for shrimp waste deproteinization to produce chitin. The protein removal with a ratio E/S of 20 was about 83%. The novelties of the Re117 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition , the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.
... The activity of the protease was potentially abolished by the presence of Zn 2+ , while Cu 2+ and Hg 2+ inhibited completely the activity of the enzyme. It is known that Hg 2+ is among ions that act on sulfhydryl residues in proteins [40]. The inhibition of the enzyme by Hg 2+ suggested the existence of sulfhydryl residues for the catalytic action of most proteases [41]. ...
A plant protease named microcarpain was purified from the latex of Ficus microcarpa by acetonic (20-40 % saturation) precipitation, Sephadex G-75 filtration, and Mono Q-Sefinose FF chromatography. The protease was purified with a yield of 9.25 % and a purification factor of 8. The molecular weight of the microcarpain was estimated to be 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed maximum activity at pH 8.0 and at a temperature of 70 °C. Proteolytic activity was strongly inhibited by dithio-bis-nitrobenzoic acid (DTNB), Hg(2+), and Cu(2+). The N-terminal amino acid sequence of the purified microcarpain "VPETVDWRSKGAV" showed high homology with a protease from Arabidopsis thaliana. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine peptidases family.
... Os diferentes comportamentos da tripsina de cecos pilóricos e do intestino posterior podem ser devido à presença de diferentes formas desta enzima nestes dois segmentos (Figura 5). Em salmão do Atlântico, inúmeros genes da tripsina têm sido reportados e caracterizados (Male et al., 1995). A composição aminoacídica diferenciada entre essas isozimas é fato conhecido, mas nem sempre esta característica traz diferenças marcantes em suas propriedades cinéticas. ...
... Como para cualquier otro rasgo anatómico o funcional, se han descrito variaciones poblacionales en las enzimas digestivas de algunas especies de peces. Concretamente en el salmón se han identificado hasta 4 isoformas de tripsina, tres de ellas aniónicas y una catiónica que muestran evidentes diferencias funcionales (Male et al., 1995, Outzen et al., 1996. La presencia de tales isoformas parece tener repercusiones significativas en la forma en que es digerido el alimento y por lo tanto en el crecimiento final de los peces (Torrissen, 1991), por lo que se abre una interesante línea de trabajo en la que cabría indagar por la presencia de tales polimorfismos en otras especies de interés económico con objeto de utilizar dicho rasgo como carácter con peso en la selección de reproductores. ...
Resumen El estudio de las enzimas digestivas, sus características, funcionalidad y adaptaciones al régimen alimenticio, conforman uno de los campos de investigación más amplios e interesantes en la nutrición de especies acuicultivadas. Un gran número de investigaciones han abordado aspectos que van desde la descripción de los parámetros funcionales de las principales enzimas hasta la forma en que éstas pueden ser utilizadas para modelizar la digestión en una especie concreta o su papel como indicadores de la condición nutricional durante la etapa larvaria. El presente trabajo pretende llevar a cabo un repaso somero a todos estos aspectos, planteando además las perspectivas de futuras investigaciones en bioquímica de la digestión.
... Localizar essas associações corresponde à pesquisa de QTLs (Quantitative Trait LociLiu & Cordes, 2004). Vários QTLs já foram identificados em peixes, alguns relacionados com o peso (O'Malley et al., 2003;Reid et al., 2005) ou comprimento (Borrell et al., 2004).TaoDarias et al., 2005), as enzimas pancreáticas tripsina (Male et al., 2004) e amilase (Douglas et al., 2000;Darias et al., 2006) ou as enzimas intestinais leucina-alanina peptidase, aminopeptidase ou fosfatase alcalina (Cahu et al., 1999;Zouiten et al., 2008), tornaram possível compreender melhor os mecanismos e alterações moleculares que ocorrem a nível digestivo durante o desenvolvimento larvar. A técnica de PCR quantitativo em tempo real é actualmente o método mais exacto de quantificação dos níveis de expressão genética (Fernandes et al., 2008), permitindo obter resultados mais fiáveis e rápidos que as técnicas convencionais e elimina o uso de sondas radioactivas normalmente utilizadas em hibridação do tipo " Northern ". ...
Major gaps in knowledge on fish larval nutritional requirements still remain, what leads to high mortalities and quality problems in marine larviculture. This paper reviews a range of new tools, such as tracer studies, population genomics, nutritional programming, functional genomics and proteomics, as well as some examples of their present use, and potential future applications in the study of fish larvae nutrition.
... However, the addition of Zn 2+ , Cu 2+ and Hg 2+ decreased the activity by 63%, 59% and 74%, respectively. It is known that Hg 2+ is among ions that act on sulfhydryl residues in proteins (Male, Lorens, Smalas, & Torrissen, 1995). The inhibition caused by this ion suggests the relevance of sulfhydryl residues for the catalytic action of most proteases (Bezerra et al., 2005). ...
The present study reports on the characterization and evaluation of a crude acidic protease from the viscera of zebra blenny (Salaria basilisca) for use in gelatin extraction. Using haemoglobin, zymogram analysis revealed the presence of at least one clear band. The crude acid protease was noted to be optimally active at pH 3.0 and 50 °C and highly stable over a pH range of 2.0 to 7.0. The enzymatic extract lost about 87% of its activity after incubation with pepstatin A for 30 min at 4 °C. The acidic protease from the viscera of zebra blenny was noted to be effective in the extraction of gelatin from the skin of zebra blenny, with an extraction yield of 14.65% based on the wet weight of zebra blenny skin. The extracted zebra blenny skin gelatin (ZBSG) was characterized based on its chemical composition, polypeptides pattern, gel strength, textural parameters, and functional properties. ZBSG had high protein (90.6%) and low ash (3.1%) and fat (0.6%) contents. It contained α1 and α2-chains as the major constituents and determined as belonging to type I. The bloom strength of solidified gelatin was 151.3 g. The findings from Fourier Transformed Infrared Spectroscopy (FT-IR) suggested the presence of helical arrangements of ZBSG. The latter showed excellent concentration-dependent functional properties. While emulsion activity index (EAI) and emulsion stability index (ESI) decreased, foam expansion (FE) and foam stability (FS) increased as the concentration of gelatin increased. Overall, zebra blenny endogenous acid protease could open new promising opportunities for the extraction of gelatin.
... Como para cualquier otro rasgo anatómico o funcional, se han descrito variaciones poblacionales en las enzimas digestivas de algunas especies de peces. Concretamente en el salmón se han identificado hasta 4 isoformas de tripsina, tres de ellas aniónicas y una catiónica que muestran evidentes diferencias funcionales (Male et al., 1995, Outzen et al., 1996. La presencia de tales isoformas parece tener repercusiones significativas en la forma en que es digerido el alimento y por lo tanto en el crecimiento final de los peces (Torrissen, 1991), por lo que se abre una interesante línea de trabajo en la que cabría indagar por la presencia de tales polimorfismos en otras especies de interés económico con objeto de utilizar dicho rasgo como carácter con peso en la selección de reproductores. ...
... WP-T and AC-T could have six disulfide-bridges (Cys15-Cys145, Cys33-Cys49, Cys117- Cys218, Cys124-Cys191, Cys156-Cys170, and Cys181- Cys205), because they possessed twelve Cys residues at the appropriate positions in bovine trypsin [35]. WP-T and AC-T completely conserved the catalytic triad (His57, Asp102 and Ser195), the consensus sequence [30]; Arabesque greenling, arabesque greenling (Pleurogrammus azonus) trypsin [28]; Antarctic fish, Antarctic fish (Paranotothenia magellanica) trypsin [31]; Atlantic salmon I, Atlantic salmon (Salmo salar) trypsin I [42]; Anchovy I, anchovy (Engraulis japonicus) trypsin I [29] ; Flounder I, flounder (Paralichthys plivaceus) trypsin I (Accession No.: AB029750 in GenBank); Zebrafish, zebrafish (Danio rerio) trypsin (Accession No.: AF541952 in DDBJ); Tilapia, tilapia (Oreochromis niloticus) trypsin (Accession No.: AY510093 in DDBJ); Bovine cat, bovine cationic trypsin (Accession No.: BC134797 in DDBJ)Fig. 4 Comparison of the activation peptides of WP-T and AC-T with those of other fish and bovine trypsins. ...
Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64 and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20–50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca2+ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca2+-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermostability.
... It has been postulated that higher catalytic efficiencies of cold-adapted enzymes occur at the expense of a more flexible or less stable protein tertiary structure, which reduces the activation energy barrier during catalysis (Hochachka & Somero, 1984). The study of digestive enzymes from sardine (Sardinops melanostica and Sardinops sagax caerulea) detected biochemical characteristics similar to other proteases from cold-adapted fishes (Castillo-Yañez, Pacheco-Aguilar, Garcia-Carreño, & Navarretedel-Toro, 2005; Castillo-Yañez, Pacheco-Aguilar, Lugo-Sanchez, Garcia-Sanchez, & Quintero-Reyes, 2009;Murakami & Noda, 1981), such as Pacific and saffron cod (Gadus macrocephalus and Eleginus gracilis) (Fuchise et al., 2009), Atlantic cod (Gadus morhua) (Palsdottir & Gudmundsdottir, 2008;Stefansson, Helgadottir, Olafsdottir, Gudmundsdottir, & Bjarnason, 2010), Atlantic Salmon (Salmo salar) (Leiros, McSweeney, & Smalas, 2001;Male, Lorens, Smalas, & Torrissen, 1995) and chum salmon (Oncorhynchus keta) (Toyota et al., 2002), including increased catalytic efficiency at low temperatures, lower thermo stability and cold stability. ...
In this work, we report the molecular characterisation of trypsin I (Try I) from Monterey sardine (Sardinops sagax caerulea). Aspects such as thermodynamic activation parameters, molecular model and cDNA-deduced amino acid sequence allow a more in depth understanding of its activity at low temperatures. The analysis of the thermodynamic activation parameters suggests that this molecule is a cold-adapted protease. From the molecular cloning, we deduced the amino acid sequence and predicted a theoretical structural model of sardine Try I with a classical trypsin fold. Cold-adaptation of this enzyme probably comes from amino acid replacement of key residues to improve flexibility at low temperature, thus increasing kcat. The cold-adaptation of sardine Try I opens a wide range of biotechnological applications for this protease and also it is interesting from the structure function relationship point of view of serine protease proteins.
... Um microarray de 16000 sequências de ADN complementar de salmão do Atlântico desenvolvido pelo consórcio GRASP (von Schalburg et al., 2005) e um novo microarray recentemente desenvolvido para a dourada que contém 20000 oligonucleótideos, (Ferraresso et al., 2008) permitirão aos aquacultores pesquisar as alterações na expressão de genes, que ocorrem durante o desenvolvimento e alimentação larvar ou em indivíduos submetidos a experiências nutricionais. A clonagem molecular e a quantificação da actividade de enzimas digestivas de peixes, como a pepsina gástrica (Darias et al., 2005), as enzimas pancreáticas tripsina (Male et al., 2004) e amilase (Douglas et al., 2000;Darias et al., 2006) ou as enzimas intestinais leucina-alanina peptidase, aminopeptidase ou fosfatase alcalina (Cahu et al., 1999;Zouiten et al., 2008), tornaram possível compreender melhor os mecanismos e alterações moleculares que ocorrem a nível digestivo durante o desenvolvimento larvar. A técnica de PCR quantitativo em tempo real é actualmente o método mais exacto de quantificação dos níveis de expressão genética (Fernandes et al., 2008), permitindo obter resultados mais fiáveis e rápidos que as técnicas convencionais e elimina o uso de sondas radioactivas normalmente utilizadas em hibridação do tipo "Northern". ...
Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades
e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais
como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e
fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.
... Kim et al. (1994). (Klomklao et al., 2009a), yellowfin tuna (Klomklao et al., 2006b), tongol tuna (Klomklao et al., 2006a), true sardine, arabesque greenling (Kishimura et al., 2006), Japanese anchovy (Kishimura et al., 2005), cod (Gudmundsdottir et al., 1993), salmon (Male, Lorens, Smalas, & Torrissen, 1995), dogfish (Titani, Ericsson, Neurath, & Walsh, 1975), dog (Pinsky, Laforge, & Scheele, 1985), porcine (Hermodson, Ericsson, Neurath, & Walsh, 1973), and bovine (Walsh, 1970). ...
Trypsin was purified to homogeneity from the viscera of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) through ammonium sulphate fractionation and a series of chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. It was purified to 47.6-fold with a yield of 12.7%. Based on native-PAGE, the purified trypsin showed a single band. The molecular weight of purified trypsin was estimated as 24 kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature for Nα-p-tosyl-l-arginine methyl ester hydrochloride (TAME) hydrolysis were 8.0 and 60 °C, respectively. Trypsin was stable to heat treatment up to 50 °C, and over a pH range of 6.0–11.0. Trypsin was stabilized by calcium ion. The trypsin activity was strongly inhibited by soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone and partially inhibited by ethylenediaminetetraacetic acid. Activity decreased continuously as NaCl concentration (0–30%) increased. Apparent Km value of trypsin was 0.3 mM and Kcat value was 92.1 S−1 for TAME. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGYECQAHSQPPTVSLNA, which is highly homologous with trypsins from other species of fish.Highlights► Novel trypsin from hybrid catfish viscera was purified and characterised. ► The purified enzyme was stable to heat treatment up to 50 °C and over a wide pH range of 6.0–11.0. ► It was also high salt stable which can be used in high salt product.
... The GenBank Acc nos: XaTryp, Xiphister atropurpureus (AAX85688) (Gawlicka & Horn, 2006): CvTryp, Cebidichthys violaceus (AAX83265) (Gawlicka & Horn, 2006): SsTryp, Salmo salar (NP_001117183.1) (Male, Lorens, Smalås, & Torrissen, 1995): DlTryp, Dicentrarchus labrax (CAA07315) (Péres, Zambonino Infante, & Cahu, 1998): PmTryp, Paranotothenia magellanica (CAA57701) (Genicot, Rentier-Delrue, Edwards, Van Beeuman & Gerday, 1996): XmTryp, Xiphister mucosus (AAX85687) (Gawlicka & Horn, 2006): ApTryp, Anoplarchus purpurescens (AAX85686) (Gawlicka & Horn, 2006). provided by Yosapong Temsiripong, Sriracha Moda Co., Ltd., Thailand). ...
Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0– 11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s–1, respectively. The catalytic efficiency (Kcat / Km) was 238 s–1 mM–1.
... Several trypsin genes from Atlantic salmon have been characterised and the expression of trypsinogen mRNA seemed to be quantitatively similar between individual salmon by Northern hybridisations using RNA from individual fish (Male et al., 1995). This in line with the observation demonstrated by Rungruangsak Torrissen and Male (2000) that total trypsin specific activity in the pyloric caeca (in both tissues and lumen) was similar between salmon with different trypsin phenotypes (Table 3). ...
Cultured salmon as escapees should never threaten salmon stocks in the wild as long as they are healthy and are not genetically manipulated. By studying a key digestive protease, trypsin, which is sensitive to environmental changes and influences on food utilisation and growth during the whole life cycle of Atlantic salmon, it is indicated that changes in the phenotypic expression of trypsin can be induced by temperature during egg incubation and the start-feeding period of the alevins. In addition, Atlantic salmon with the same trypsin phenotype showed different feed conversion efficiency and growth rate at different temperatures. Trypsin genes seem to be stable, and although the pattern of expressed genes varies extensively, the expression of trypsinogen mRNA is quantitatively similar between individual salmon in line with the observation that the total trypsin specific activity was similar between the fish with different trypsin phenotypes. However the luminal secretion of the active enzyme, and probably the relative amounts of trypsin isozymes, could be modified by water temperature and food quality. These results indicate that changes in the environmental condition can influence gene expressions of the fish at DNA, RNA and protein levels, regardless of genetic expression of parents. This means that whether the escapees or wild fish is the spawning population, an incidence of the offsprings to have their gene expressions adapted to that environment will be similar. It is the environmental condition that has to be conserved in order to control the genetic structure of animals in the wild. It is naive to think that genetically manipulated escapees such as triploid salmon will not have any impact on wild population, as they are not fertile. Under a more favourable condition for growth, triploid escapees could compete with wild fish on food availability as they have higher consumption rates for higher growth rates, unlike ordinary diploid salmon that could have better food utilisation at a similar consumption rate. This may cause a higher survival rate in triploid escapees and if they spawn, hatching success and survival rate of the offsprings will be low due to low gamete quality for reproduction in triploid fish. This could result in a smaller population of the new generation in that environment in the wild. ________________________________________________________________________
... In aquatic species, trypsin is often present in several isoforms, as in Atlantic salmon, Salmo salar L. (Torrissen 1987). Several trypsin genes have been identified in fish, for instance in Atlantic salmon (Male et al. 1995) and Japanese anchovy, Engraulis japonicus (Ahsan et al. 2001). In Atlantic salmon a wide range of factors has been found to affect trypsin specific activity and isoform expression (reviewed in Rungruangsak- Torrissen & Male 2000). ...
The responses of the digestive proteases trypsin and chymotrypsin and protein metabolism to differences in feed protein quality were investigated in Atlantic salmon (Salmo salar L.). Two sets of experimental feeds were produced. Each set of high and low quality feeds was provided to either 150 g or 2 kg salmon. Protein in the high quality feeds had significantly higher percentages of free (reactive) sulphydryl (SH) groups than the corresponding feeds based on low quality meals. After 90 days feeding, groups given high and low quality feeds did not differ in their specific growth rates (SGR) in either experiment. However, feed conversion efficiency (FCE) was significantly different between the high and low quality feed groups in 2 kg salmon, where the difference between the high and low feed protein qualities was larger, 10% versus 4% SH/[SH + (S–S)] in 150 g salmon. Higher FCE was preceded by significantly higher trypsin and chymotrypsin specific activities on day 60. SGR, in general, changed after the first month and was stable during the last 2 months in both experiments. Concurrently, both trypsin (T) and chymotrypsin (C) decreased with an increased activity ratio of trypsin to chymotrypsin (T/C ratio), and resulting in significantly lower T/C ratio on day 90 in salmon feeding on high quality feeds in both sizes of fish. Differences in FCE were associated with significant differences in levels of total free amino acids (TFAA) in the plasma and the white muscle, as well as in the ratio of essential to non-essential free amino acids (EAA/NEAA ratio), free hydroxyproline, and RNA in the white muscle. Interestingly, after 3 days starvation (day 93), 5–7 h postprandial EAA/NEAA ratio in the plasma was significantly lower in the high quality diet groups in both experiments. Trypsin specific activity inversely correlated with muscle TFAA levels in 2 kg salmon, concurrent with higher muscle levels of RNA, lower free hydroxyproline and higher FCE in fish fed higher quality diets.
... Nevertheless, it is of note that Atlantic salmon of pattern 2' expressing the homozygote of TRP-2 isozyme generally performed less well when betweengroup distinctions were noted. Both teleosts and mammals have been shown to express distinctive forms of trypsin, possessing differing substrate-binding affinities, kinetic properties and catalytic efficiencies (Male et al., 1995). It was of interest, therefore, to examine whether enzyme extracts from the phenotypically distinct salmon differed in their digestive capacity and catalytic abilities. ...
Growth and in vitro digestion was examined in three iso-trypsin phenotypes (patterns 1, 2 and 2') of Atlantic salmon, Salmo salar. Fish grew equally well during the 128 day experiment irrespective of phenotype. At day 64 and 91, weight-specific growth rate of 2' phenotype animals were significantly lower (p < 0.05). At the end of the experiment, significant differences (p < 0.05) were noted between groups with respect to size. An in vitro method was used to examine protein digestion by the different trypsin phenotypes. Three fish meals (FM I > FM II > FM III) of high, medium and low quality, as defined by chemical analyses, were used. Phenotype 1 was able to differentiate between three fish meals ranking FM I > FM II > FM III. In contrast, phenotypes 2 and 2' were not able to separate between meals. In vitro digestion efficiency was dependent upon the three salmon types, ranking phenotype 2 > 1 = 2'.
... The secretion rate of trypsin and chymotrypsin is related to feed intake and filling of the stomach (Einarsson et al. 1996) and the activity ratio of these two enzymes has been suggested as an indicator of the nutritional status of the fish by Rungruangsak Torrissen and Male (2000). Several trypsin genes have been reported and characterized in Atlantic salmon (Male et al. 1995). Individual variation in trypsin isozyme patterns has been characterized using isoelectric focusing techniques, and expression patterns have been found to have a significant effect on growth (Torrissen 1987Torrissen , 1991 ) and feed utilization (Torrissen and Shearer 1992; Torrissen et al. 1998 ). ...
The aim of the present experiment was to screen several biochemical indices in fish and their interrelations in order to select variables for future studies of growth rate and feed conversion. Several parameters [trypsin activity, chymotrypsin activity, free amino acids (FAA) in plasma and white muscle, and RNA and RNA/protein ratio in the white muscle] were measured together with specific growth rate (SGR), feed intake and feed conversion efficiency (FCE) in four groups of diploid or triploid Atlantic salmon (Salmo salar L.) reared under different light regimes. SGR was measured on individually tagged fish, whereas feed intake and feed conversion was estimated on tank basis. A principal component analysis (PCA) explained 80.6% of the variance in the data, using all measured parameters, regardless of ploidy and light regime. Muscle free hydroxyproline showed the highest correlation, alone explaining 55% of SGR variability. The SGR also significantly correlated with trypsin activity (r=0.34), the activity ratio of trypsin to chymotrypsin (T/C) (r=0.39), plasma essential FAA (EAA) (r=0.39), plasma total FAA (TFAA) (r=0.37), the ratio of essential to non-essential FAA (EAA/NEAA) in the white muscle (r=–0.45), muscle RNA (r=–0.45) and RNA/protein ratio (r=–0.41). Tank FCE correlated positively (r=0.97) with SGR, T/C ratio and muscle free hydroxyproline, and negatively (r=–0.90) with muscle EAA/NEAA. The groups reared under continuous light (LL) regime showed significantly higher SGR than simulated natural photoperiod (SNP) groups, and with an apparently higher FCE. A higher growth rate was associated with either a higher consumption rate and/or a higher feed utilization. A negative correlation between muscle RNA concentration and SGR may indicate that increased growth rate under LL regime was not caused by an increased protein deposition rate.
... T, P, T±P inhibition by TLCK, PMSF and by both, respectively; * no inhibition detected; other abbreviations as in legend to I II III I II III I II III IV V VI VII 39.1 T±P T±P 37.7 P 32 P 28.9 P 21 .7 T±P T±P T±P T±P T±P T±P T±P T±P 20.6 T±P T±P T±P T±P T±P T±P T±P T±P 19.5 T±P T±P T±P T±P T±P 17.5 T±P T±P T±P T±P T±P T±P T±P T±P * * * * * T±P 16.2 T±P T±P T±P T±P T±P T±P T±P T±P * * * * * T±P 14.6 T±P (2) or Litopenaeus vannamei (2), while the more intense band (37.7 kdaltons) is heavier than reported for other crustaceans (Table 4). Several forms of serine proteinases have been found in ®sh such as salmon (Male et al. 1995) and cod (Gudmundsdottir et al. 1996). Trypsins from salmon may present either cationic or anionic forms. ...
Proteinase (endopeptidase), trypsin-like and chymotrypsin-like activities were examined throughout the ontogenetic development
of cultured Farfantepenaeus paulensis. Whole individuals from different larval and postlarval stages, and the hepatopancreas of adults were homogenized and assayed
to quantify the enzyme activities of specific substrates. Proteinase activity was identified by substrate-SDS-polyacrylamide
gel electrophoresis. Specific inhibitors for trypsin (TLCK), chymotrypsin (TPCK) and serine proteinases (PMSF) were used to
identify activity zones of these enzymes in gels. Protein-specific activity of total proteinases, trypsin and chymotrypsin
was negligible at the egg stage and at Nauplius III, increasing in the first protozoeal substage (PZ I), and reaching a peak
at PZ III; it decreased again in the subsequent postlarval substages. Different patterns of proteinase activity were observed
in SDS-PAGE zymograms during ontogenetic development. Active bands of 14.6, 16.4, 17.5, 19.5, 22.5, 23.9, 25.8, 28.9, 32.0,
34.4, 37.7, and 42.2 kdaltons were detected in the adult hepatopancreas. Proteolytic activity was detected on gels in PZ I,
and intense activity zones of 16.4, 17.5 and 19.5 kdaltons were found up to Mysis I (M I). Intense bands of 39.1 and 53.5
kdaltons were observed only at PZ III and M I. Band-activity intensity decreased after metamorphosis to the postlarval stage
(PL). The chymotrypsin inhibitor TPCK had no effect on the proteinase bands. Active zones in gel inhibited with both TLCK
and PMSF were considered to represent trypsin. The inhibitory effect of PMSF alone on proteinase extracts indicated chymotrypsin
activity. TLCK and PMSF inhibition also varied during ontogenetic development. The inhibition of bands recorded between 14.6
and 21.7 kdaltons suggested the presence of low molecular weight trypsin in F. paulensis. The 39.1 kdaltons band observed at PZ III and M I were trypsin-like. On the other hand, bands of 28.9, 32 and 37.7 kdaltons
from the adult hepatopancreas seem to represent a chymotrypsin. We conclude that the recorded variation in enzyme activity
may be associated with morphological and behavioral changes during penaeid ontogenetic development. The higher enzyme activity
at PZ II, PZ III and M I may reflect the increased energy turnover associated with intense swimming behavior and food ingestion.
... Putative polyadenylation signal is written in bold-italic. Atlantic cod, Gadus morhua, trypsin I [42]; Antarctic fish, Antarctic fish, Paranotothenia magellanica, trypsin [40]; Atlantic salmon I, Atlantic salmon, Salmo salar, trypsin I [43]; Anchovy I, anchovy, Engraulis japonicus, trypsin I [32]; Flounder I, flounder, Paralichthys plivaceus, trypsin I [Accession No.: AB029750 in GenBank]; Zebrafish, zebrafish, Dnio rerio, trypsin [ The names of sequences are the same inFig. 2. Dashes indicate deletions introduced for maximizing the sequence similarity.Fig. ...
A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25–753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and a mature trypsin of 222 amino acid residues. The AG-T also completely conserved the major structural features common to trypsin such as the catalytic triad (His57, Asp102, and Ser195), the obligatory Asp189 and twelve Cys residues. On the other hand, the AG-T possessed the deletion of Tyr151 and substitution of Pro152 for Gly in the autolysis loop when aligned with the sequence of tropical-zone fish and bovine trypsins. In addition, Val75 concerned in a combination with calcium ion was exchanged for Ala in the AG-T, and the content of positively charged amino acid residues at the calcium-binding site of the AG-T was three times higher than those of tropical-zone fish trypsins. Moreover, the ratio between charged and hydrophobic amino acid residues in the N-terminal region of the AG-T was also higher than those of temperate-zone fish and tropical-zone fish trypsins. Such structural properties of the AG-T would contribute to its low thermostability.
The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures have actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is due to redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.
Two trypsins (A and B) from the liver of albacore tuna (Thunnus alalunga) were purified to homogeneity using a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and Diethylaminoethyl-cellulose. Purity was increased to 80.35- and 101.23-fold with approximately 3.1 and 19.2% yield for trypsins A and B, respectively. The molecular weights of trypsins A and B were estimated to be 21 and 24kDa, respectively, by SDS-PAGE and size exclusion chromatography. Both trypsins showed only one band on native-PAGE. Trypsins A and B exhibited the maximal activity at 60°C and 55°C, respectively, and had the same optimal pH at 8.5 using N(α)-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) as a substrate. Stabilities of both trypsins were well maintained at a temperature up to 50°C and in the pH range of 7.0 to 11.0 and were highly dependent on the presence of calcium ion. The inhibition test demonstrated strong inhibition by soybean trypsin inhibitor and TLCK. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). The N-terminal amino acid sequence of 20 residues of the two trypsin isoforms had homology when compared to those of other fish trypsins.
A clade of trypsins, known as group III, is identified by phylogenetic analysis. These trypsins occur in fish that spend all or part of their lives at temperatures near 0°C and may represent extreme psychrophilic enzymes. A principal component analysis of amino acid compositions distinguishes group III from mesophilic trypsins, as do molecular trees and multidimensional scaling of molecular sequence distances. The primary sequences of group III trypsins, in conjunction with the known structures of mesophilic trypsins, permit insight into function and mechanisms of cold adaption. The techniques employed are broadly applicable to phylogenies characterized by a markedly different, or “fast-evolving,” clade. An updated lactate dehydrogenase molecular tree illustrates an additional fast-evolving clade. Proteins 2002;47:31–44.
Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24 kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist.
One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.
IntroductionProteasesTransglutaminasePolyphenoloxidaseTrimethylamine-N-Oxide DemethylaseLipaseReferences
Anionic trypsin from Pacific saury (Cololabis saira) pyloric ceca was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography. It was purified to 53.7-fold with a yield of 6.1%. The apparent molecular weight of the enzyme was about 24 kDa, as determined by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). On native-PAGE, trypsin showed a single band. The purified anionic trypsin displayed optimal activity at pH 8.5 and 55°C. The enzyme was stable at neutral and alkaline pH and in the temperature range of 20–50°C. The stability was affected by the calcium ion. The activity of purified anionic trypsin was completely inhibited by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and partially inhibited by ethylenediaminetetraacetic acid (EDTA). NaCl (0–30%) decreased the activity in a concentration-dependent manner. The kinetic trypsin constants Km and Kcat were 0.19 mM and 210 s−1, respectively, while the catalytic efficiency (Kcat/Km) was 1105.26 s−1 mM−1. The N-terminal amino acid sequences of anionic trypsin, IVGGYECQAH, were found and were homologous to those of trypsin from other fish species.
Trypsin was purified from the pyloric ceca of hoki fish by (NH 4)2SO4 fractionation, acetone precipitation and affinity chromatography. The purified trypsin migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the isoelectric point was determined as 6.5. The molecular weight was determined as 26,000 Da by SDS-PAGE and as 23,791 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. The enzyme was stable within the pH range of 6.0-11.0, and exhibited optimum activity for the hydrolysis of N-α-benzoyl- dl -arginine-p-nitroanilide (dl -BAPNA) at pH 9.0. The enzyme was stable up to 40C, and its optimum temperature for dl -BAPNA hydrolysis was 60C, and was inhibited by soybean trypsin inhibitor, aprotinin, phenyl methyl sulfonyl fluoride and benzamidine. The apparent Michaelis-Menten constant (Km′) and substrate turnover number (kcat) for the dl -BAPNA hydrolase reaction was 0.06 mM and 0.33 s-1, respectively, while the corresponding values for tosyl arginine methyl ester hydrolysis were 2.08 mM and 19.0 s-1, respectively. The N-terminal 20 amino acid residues of hoki trypsin, IVGGQECVPNSQPFMASLNY, displayed considerable homology with other fish trypsins. © 2007, The Author(s); Journal compilation
Three trypsin isoforms, trypsins A, B and C, from the spleen of skipjack tuna (Katsuwonus pelamis) were purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl-cellulose to obtain a single band on native-PAGE and SDS–PAGE. The molecular mass of all the trypsin isoforms was estimated to be 24kDa by size exclusion chromatography and SDS–PAGE. The optimum pH and temperature of the three isoforms for the hydrolysis of Nα-p-tosyl-l-arginine methyl ester hydrochloride were 8.5 and 60°C, respectively. Trypsins were stable to heat treatment up to 50°C, and over a pH range of 6.0–11.0. All isoforms were stabilised by calcium ions. The trypsin activities were effectively inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid, while E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibitory effect. Activities decreased continuously as NaCl concentration (0–30%) increased. Trypsins A, B and C showed Km of 0.11–0.29mM and Kcat of 57.1–114s−1. The N-terminal amino acid sequence of 20 residues of three trypsin isoforms was IVGGYECQAHSQPHQVSLNS and had high homology to those of other fish trypsins.
Most recent research on teleost digestive enzymes has focused on the immunohistochemistry of flounder and catfish, fewer studies have been done of gene expression. The present study is made an attempt to clarify the expression of digestive enzymes in tilapia (Oreochromis mossambicus) during larval development. This work was done by cloning the digestive enzymes, semi-quantifying the expression of genes by RT-PCR-Southern blot, detecting the expression of proteins (western blot) and investigating enzymatic activity. Under microscopic observation, the mouths of tilapia larvae were seen to open on the third day after hatching and on the fifth day after hatching, the actinotrichia were reabsorbed and the larvae began feeding. The partial nucleotide sequences of pepsinogen, trypsinogen and chymotrypsinogen were obtained. After RT-PCR and southern blotting analysis, the expressing pattern of pepsinogen mRNA appeared on day 2 after hatching, while mRNAs of trypsinogen and chymotrypsinogen were both detected 1 day after hatching. The proteins of larval pepsin, trypsin and chymotrypsin appeared 1 day after hatching, and specific activities of these enzymes were detectable on day 3 (for pepsin) and day 5 (for trypsin and chymotrypsin) after hatching. The transcription of the genes for these three digestive enzymes gradually increased after hatching and that all protein detected on day 1, suggesting that all proteins may result from maternal sources.
Chemical reaction rates often show a strong temperature dependency and a decrease of 10°C from room temperature typically divides the rate by a factor oscillating between 1.5 and 4. The decrease of the rate constant k indeed obeys an equation proposed by Svante Arrhenius as early as in 1889: $$K = A{e^{ - Ea/RT}}$$ (1) in which E
a
is the activation energy, R the gas constant (8.31 kJ mol−1) and T the temperature in Kelvin.
Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.Major gaps in knowledge on fish larval nutritional requirements still remain, what leads to high mortalities and quality problems in marine larviculture. This paper reviews a range of new tools, such as tracer studies, population genomics, nutritional programming, functional genomics and proteomics, as well as some examples of their present use, and potential future applications in the study of fish larvae nutrition.
Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.
The presence of structural calcium in trypsin and subtilisin does not play a role in catalysis, but instead, stabilizes the structures. Trypsin and subtilisin are two large families of serine proteases with different folds that take advantage of the stabilizing effects of binding calcium ions. Representative structures of the different calcium binding sites found in each protein family are described along with the implications for the presence of the ions. This article reviews the details, the energetics, and the roles that calcium plays in the structures of both these families of enzymes.
Cationic trypsin was purified from Pacific saury (Cololabis saira) pyloric ceca by the successive steps of ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. The purification and yield were 90-fold and 8.9%, respectively. The molecular weight was determined to be 24 kDa using Sephacryl S-200 and SDS-PAGE. Cationic trypsin was stable at pH 7–11 for 30 min at 30C, and its maximal activity against L-arginine methyl ester hydrochloride (TAME) was at pH 8.5. Thermostability for cationic trypsin was up to 50C for 15 min and its temperature optimum was 60C. Cationic trypsin was stabilized by calcium ion. Activity decreased as NaCl concentration (0–30%) increased. Inhibitor susceptibility analysis revealed that the enzyme was inhibited effectively by soybean trypsin inhibitor and N-p-tosyl-L-lysine chloromethyl ketone. The Km and Kcat of the enzyme were 0.17 mM and 200 s−1, respectively. The N-terminal amino acid sequence of cationic trypsin was partially determined as IVGGYECQPH- and was very homologous to other trypsins.
Trypsin is a major member of the serine proteases, which constitute a large family of biologically important enzymes. Trypsins from various sources catalyze the hydrolysis of peptide bonds on the carboxyl sides of arginine and lysine. Hence, it is expected that like other trypsins, Pacific saury trypsin would also be used for processing aids in industry applications. Most trypsins are used for a variety of products in the food industry including baked foods, beer, wine, cereal, meat and fish products, and for production of protein hydrolysates and flavor extract. Trypsins are also used in mammalian cell culture to disaggregate adherent cells for research and the production of recombinant proteins, in diabetes diagnosis and therapy, in detergents and bating of leather.
Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S-200 and Sephadex G-50. The purification and yield were 20-fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against Nα-p-tosyl-L-arginine methyl ester was pH 8.0. Trypsin was heat-stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N-p-tosyl-L-lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin-like serine protease. N-Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.
Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent Km and Kcat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S−1, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.
The objective of the present study was to describe the histological and physiological development of the gastrointestinal system in Atlantic halibut from the time of first-feeding until metamorphosis. At first-feeding, the gastrointestinal (GI) tract is divided into anterior, mid and hindgut regions. The liver is present, as is the pancreas. During development the pancreas changes from a compact organ to a diffuse tissue interspersed through much of the mesentery surrounding the GI tract. Functional gastric glands are not present until approximately 66 days post-hatch (dph). Using primers based upon winter flounder digestive enzyme gene sequences, we were able to amplify partial sequences for bile salt-activated lipase (BAL), trypsinogen (TRP), and pepsinogen (PEP) from RNA extracted from whole larvae and juveniles using Reverse Transcription-PCR (RT-PCR). PCR products were sequenced and the sequences used to design halibut gene-specific primers for BAL and PEP. RT-PCR analysis revealed that BAL and TRP gene expression was evident at least from the time of first-feeding but PEP gene expression was not detectable until 80 dph. In situ hybridization using molecular probes from winter flounder sequences localized expression of BAL and TRP to the exocrine pancreas. PEP expression was only localized to the glandular regions of the stomach. These data provide a first step toward understanding the molecular events governing the ontogeny of digestive capacity in Atlantic halibut.
Trypsin was purified from the pyloric ceca of walleye pollock (Theragra chalcogramma) by gel filtration on Sephacryl S-200 and Sephadex G-50. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular mass of the enzyme was estimated to be 24 kDa by SDS–PAGE. Trypsin activity was effectively inhibited by serine protease inhibitors, such as soybean trypsin inhibitor and TLCK. Trypsin had maximal activities at around pH 8.0 and 50 °C for the hydrolysis of Nα-p-tosyl-l-arginine methyl ester hydrochloride. Trypsin was unstable above 30 °C and below pH 5.0, and was stabilized by calcium ions. Walleye pollock trypsin was more thermally unstable than trypsin from the Temperate Zone fish and Tropical Zone fish. The N-terminal amino acid sequence of the trypsin, IVGGYECTKHSQAHQVSLNS, was found, and the sequential identity between the walleye pollock trypsin and Frigid Zone fish trypsin was higher (85–100%) than with Temperate Zone fish trypsin (75–90%), Tropical Zone fish trypsin (75–85%), or mammalian trypsin (60–65%).
Trypsins from the pyloric ceca of jacopever (Sebastes schlegelii), TR-J, and elkhorn sculpin (Alcichthys alcicornis), TR-E, were purified by gel filtration on Sephacryl S-200 and Sephadex G-50. The molecular weights of TR-J and TR-E were estimated to be 24,000 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. TR-J and TR-E revealed optimum temperatures of 60 and 50 °C, respectively, and showed the same optimum pH (pH 8.0) for hydrolysis of Nα-p-tosyl-l-arginine methyl ester. TR-J and TR-E were unstable at above 50 and 40 °C, respectively, and were more stable at alkaline pH than at acidic pH. Thermal stabilities of TR-J and TR-E were highly calcium dependent. These purified trypsin enzymes were inhibited by serine protease inhibitors, such as TLCK and soybean trypsin inhibitor. The N-terminal amino acid sequences of TR-J and TR-E were also investigated. The N-terminal amino acid sequences of TR-J, IVGGYECKPYSQPHQVSLNS, and TR-E, IVGGYECTPHSQAHQVSLNS, were found, and these sequences showed highly homology to other fish trypsins.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Isoelectric focusing of Atlantic salmon, Salmo salar L., trypsin resolved three major isozymes designated TRP-1, TRP-2 and TRP-3, with three variants, TRP-l(9l), TRP-l(75) and TRP-2(92). The TRP-1 and TRP-2 may represent duplicated loci which exhibit either tetrasomic or disomic inheritance from pairing of homeologous chromosomes. A significant difference in growth during 3 months after first-feeding was observed in the families exhibiting a high frequency of the isozyme TRP-2(92) (f≥0·5). Studies of three salmon populations suggested a correlation between the presence of this isozyme and better growth rate. No difference in egg quality was observed between the families possessing and lacking the trypsin isozyme TRP-2(92).
We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.
We have isolated a clustered gene family in D. melanogaster that codes for trypsin-like enzymes. The gene family has been localized to 47D-F by in situ hybridization to polytene chromosomes. The four genes in the family are transcribed in alternating orientations, and code
for 1000 nt mRNAs. Transcripts are present at all stages of the life cycle. In situ hybridization to mRNA in tissue sections of third instar larvae showed that transcripts were restricted to the mid-gut. One
gene was sequenced. The translated amino acid sequence of the proposed active enzyme is 42% homologous to bovine trypsin.
Regions of functional importance are more strongly conserved. These include the active site residues asp102, his57, serl95, and the residue aspl89 which is reputed to bind the basic residue at the substrate cleavage site. The activation peptide is not homologous to that
of most vertebrate trypsins, suggesting a modified activation mechanism. The sequence further strengthens the hypothesis that
the chymotrypsin cleavage specificity developed seperately in the vertebrates and invertebrates.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
A family of approximately 10 trypsin genes was detected in a rat genomic library by hybridization and in vivo recombination techniques using cloned rat pancreatic trypsin I and II cDNAs as probes. Two separate clones containing the entire trypsin I gene and most of the trypsin II gene were sequenced. Four introns split the trypsin I coding sequence. The positions of the first three introns of the trypsin II gene are identical with those in the trypsin I gene (the fourth intron was not present in the trypsin II clone). The coding regions of the two genes are 88% homologous; the 5'-noncoding regions are 92% homologous, whereas the 3'-noncoding regions share 66% identity. In contrast, the proximal 5'-flanking regions from -1 to -500 which may contain the elements controlling gene expression are less than 30% conserved overall, but segments of approximately 70% homology can be discerned in this region. Some of these sequences are homologous to sequences found in the chymotrypsin and elastase genes. More distal upstream sequences (-500 to -2500) and the intervening sequences show no evident sequence homology (less than 20%). Unique sequences containing homopolymeric purine/pyrimidine repeats are found 2.5 kilobases upstream from the start of transcription of the trypsin I gene and within the second and third introns of the trypsin II gene. The nucleotide homologies as well as the similarities of intron positions of the two trypsin genes to those of other serine protease genes clearly support an evolutionary relationship between members of this gene family.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.
Trypsin (Tr) and chymotrypsin (Ch) have similar tertiary structures, yet Tr cleaves peptides at arginine and lysine residues
and Ch prefers large hydrophobic residues. Although replacement of the S1 binding site of Tr with the analogous residues of
Ch is sufficient to transfer Ch specificity for ester hydrolysis, specificity for amide hydrolysis is not transferred. Trypsin
is converted to a Ch-like protease when the binding pocket alterations are further modified by exchange of the Ch surface
loops 185 through 188 and 221 through 225 for the analogous Tr loops. These loops are not structural components of either
the S1 binding site or the extended substrate binding sites. This mutant enzyme is equivalent to Ch in its catalytic rate,
but its substrate binding is impaired. Like Ch, this mutant utilizes extended substrate binding to accelerate catalysis, and
substrate discrimination occurs during the acylation step rather than in substrate binding.
It appears that nature has experimented extensively with various modes of gene duplication in fish. We witness extreme diversity in the genome size as well as in the degree of gene duplication among fish. There are those that have increased their genome size exclusively by repeated tandem duplication of chromosomal segments. They show no proportional increase in the number of functional gene loci. In this group, the trend for progressive elimination of excessive genetic redundancy appears to have started in modern times.There is another group which went through tetraploid evolution. These fish are endowed with twice the number of gene loci for many of the gene products.
Polyploidy has long been recognized to have played an important role in the evolution of plant species (Lewis, 1980). However, polyploidy has only recently been recognized as a potentially important process in the evolution of vertebrates (Ohno, 1974; Fisher et al.,1980; Bogart, 1980; Schultz, 1980). Mammals and birds generally possess more DNA per cell than do fish and other chordates. This observation and the existence of many duplicated gene loci have led Ohno and co-workers (Ohno,1967, 1970a, 1974; Ohno et al., 1968) to propose that genome doubling has taken place at least once in the evolution of vertebrates. One tetraploid event apparently took place about 500 million years (Myr) ago in a common ancestor of all vertebrates. Fisher et al. (1980) describe isozyme studies that are consistent with this idea. Other tetraploid events may have taken place in major lineages of vertebrate evolution, possibly including a genome doubling in a reptilian ancestor of mammals (Ohno, 1967; Comings, 1972).
Elastase II and trypsin mRNAs were cloned 1n form of their cDNAs from pancreas of strain A/J mice, and their complete nucleotide
sequences were determined. The elastase II mRNA 1s 912 nucleotides long and encodes a protein of 271 amino adds. The cloned
trypsln mRNA species 1s 814 nucleotides long and encodes a protein of 246 amino acids. The elastase II gene, which exists
as a single copy in the haploid mouse genome, measures 11.2 kb from cap to poly(A) site and 1s interrupted by at least seven
introns. Between 5 and 10 trypsin genes exist in the mouse genome. Five different trypsln genes, two of which are closely
linked in a tail-to-tail manner, were studied in detail. They vary in size between 3.4 and 4.0kb, and all are Interrupted
by four Introns. DNA sequence comparison of the elastase II, trypsin and Amy-2a α-amylase genes reveals a conserved 13 nucleotide motif 1n their 5′-flanking regions. The differential accumulation of the
elastase II and trypsin mRNAs in the cytoplasm of the acinar pancreatic cell is regulated predominantly at the transcriptional
level.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (Km,app) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the Km,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (Vmax/Km,app) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The Km,app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the Km,app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. Vmax/Km,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show that Greenland cod trypsin is a better catalyst than bovine trypsin at low temperatures and that catalysis by the fish trypsin is less responsive to temperature change than is catalysis by bovine trypsin.
Protease activities in the digestive tract and carotenoid levels in flesh, ovaries and plasma were determined in Atlantic salmon (Salmo salar) in their third sea year during the period from early March until December. Protease activities in the digestive tissues changed significantly during the maturation process: the fish have higher enzyme activities in early maturation and lower in the later stages until maturity. The trypsin-like isozymes were studied by IEF electrophoresis during maturation, and variations observed were possibly genetic. The level of astaxanthin in flesh and ovaries decreased significantly during sexual maturation, but the total amount in the ovaries continued to increase. The covariance between astaxanthin in the plasma and trypsin-like activities in the digestive tissues was significant, and the level of astaxanthin in the plasma was also influenced by time and stage of sexual maturation.
1. The proteolytic activities of the proteases extracted from the digestive tracts of Atlantic salmon and rainbow trout show similar characteristics in the optimum temperatures. In Atlantic salmon the optimum temperature of the stomach extract is 37.5°C and of the pyloric caeca and the small intestinal extracts 52.5°C. 2. The extract from the large intestine shows two optimum temperatures of 45 and 52.5°C. 3. A similar pattern was observed in the extracts from the digestive tract of rainbow trout except for the stomach extract, where the optimum temperature is 35°C. 4. The proteolytic activities of the intestine are very pronounced in the area between the small and large intestines. 5. The characteristics of isoelectrofocusing (IEF) electrophoresis of tryptic-like enzymes are different between these two salmonids. 6. Genetic variation within species in tryptic-like enzymes is observed on the IEF electrophoresis of both Atlantic salmon and rainbow trout.
Trypsin-like isozyme patterns of 580 Atlantic salmon fry were studied by isoelectrofocussing. Three common isozymes designated TRP-1, TRP-2 and TRP-3 were observed, with the variant TRP-2 ( 92), and two variants possibly of the TRP-1 system. The distributions of genotype variants of the TRP-2 system within families seemed to be in accordance with a model assuming Mendelian inheritance. Clear association between fish size and genotypes was observed, and the fish of designated genotype TRP-2 ( 92/92) had an average weight significantly higher than those of the other genotypes. This covariation indicates a usefulness of the variants for improving genetic resources for future breeding programmes.
The growth rates of three different year-classes (1984, 1985 and 1987) of Atlantic salmon (Salmo salar) were studied using trypsin-like isozyme pattern in the pyloric caeca as a biological genetic marker. The fish with the homozygote variant TRP-2(92/92) and with the heterozygote variant TRP-2(92/100) had higher average weight than those with the genotypes which did not possess the variant TRP-2(92). The difference in weight between the two groups (with and without TRP-2(92) variant) was significant from smolt until the end of the experiment in autumn of the first sea-year (1984 year-class) and until spring in the second sea-year (1985 year-class). The difference was about 30%, and decreased to 10% at maturation. The specific growth rate was critical during the first sea-year. The fish with the allele TRP-2(92) showed the advantage of faster growth in the first sea-year, especially during winter time. With the covariation of significant difference in weight (until the autumn in the second sea-year) among different families, higher production of food fish of at least 20% could be possible at the time of slaughter during the second sea-year with the use of a selective breeding programme based either on trypsin-like isozyme pattern and family or on the isozyme pattern of individuals. The effects of selection by using the variant TRP-2(92) could be observed within the period of the generation selected.
1.1. Packed cell volume (PCV) and plasma catecholamines, cortisol and glucose were determined in blood samples obtained from race horses, after competition.2.2. All these parameters were higher than basais but catecholamines increased 6–7 times while cortisol was enhanced only by 25%.3.3. The increase of glucose and PCV was related to the rise of catecholamines.4.4. In samples withdrawn after overnight stress caused by change of horseshoes, it was not possible to find values from basal values significantly different.5.5. Influence of nervous temperament was observed only in the higher hematocrit found in basal conditions, particularly in females.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
Information compiled by automatic Edman degradation of Streptomyces griseus trypsin coupled with previous data has permitted the assignment of the first 36 residues at the NH2 terminus of the protein. Cyanogen bromide cleavage at the three methionine residues followed or preceded by reduction and aminoethylation resulted in the production of four fragments, Cnl to Cn4, which were separated by gel filtration on Sephadex G-50 or G-75. Fragments CN4 (15 RESIDUES) AND Cn3 (5 residues) were shown to be derived from the NH2 terminus of the protein while Cn2 (47 residues and devoid of homoserine) was from the COOH terminus. The arrangement of the fragments was thus Cn4-Cn3-Cn1-Cn2. Automatic Edman degradation in the sequenator coupled with peptides derived from alpha-lytic protease and chymotryptic digestion and from the peptic and tryptic peptides previously elucidated have permitted the sequence determination of fragments Cn1 and Cn2 and therefore of the whole protein. These studies show that extensive regions of identity or similarity exist between Streptomyces griseus trypsin and bovine trypsin. These include the NH2-terminal four residues, the sequences near histidine-57 (chymotrypsinogen A numbering system), aspartic acid-102, aspartic acid-189, and serine-195, the regions of the three disulfide bridges, and the COOH-terminal end (residues 225-229) of the proteins. When aligned to maximize homology the identity of residues is 34%. This identity is increased to 54% when only those residues classified as internal by Stroud et al. (Stroud, R. M., Kay, L. M., and Dickerson, R. E. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 125) are considered. These results indicate that the folding of the polypeptide chains of the two enzymes is very similar and are in agreement with the very similar enzymic, chemical, and physical properties of the two enzymes.
The amino acid sequence of pancreatic trypsin from the spiny Pacific dogfish (Squalus acanthias) has been determined and compared with the sequences of bovine and porcine trypsin. Dogfish trypsin contains one less amino acid residue (222) than the other two enzymes. Two-thirds of the residues in corresponding positions in dogfish and bovine trypsin are identical and the sequences ofall three enzymes are homologous. Of the 223 amino acid residues of bovine trypsin, 77 are replaced without significant changes in function. Seven replacements, all conservative, occur in the interior of the protein; the remainder are on the surface. All residues known to be components of the active site of bovine trypsin are present in corresponding positions in dogfish trypsin. Comparison of the three enzymes suggests calcium binding sites in dogfish trypsin. A corrected sequence of bovine trypsin identifies residue 67 as Asn and residues 84-87 as Ser-Asn-Thr-Leu.
A comparative kinetic analysis of Pacific salmon and bovine trypsins revealed that the former hydrolyzes p-nitroanilide-N,L-benzoyl-D,L-arginine (BApNA) with a far greater efficiency in comparison with bovine trypsin due to the decrease in Km. The inhibition constants for the BApNA hydrolysis by bovine and salmon trypsin with glycine, beta-alanine, L-lysine, L-arginine and benzamidine were determined. With an increase in the length of the hydrocarbon chain in the inhibitor molecule (i.e., in the order of glycine-beta-alanine-L-lysine) the inhibiting effect increased both with salmon and bovine trypsins. The Ki values for benzamidine and L-arginine appeared to be by one order of magnitude higher with salmon trypsin than with bovine trypsin. L-arginine was a much more effective inhibitor compared to L-lysine when both salmon and bovine trypsins were used.
Two similar, but distinct, cDNAs for Atlantic salmon serum albumin have been isolated from the same salmon liver. Comparison between the asSA-1 and asSA-2 sequences reveals 1% overall sequence difference.
A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.
Two closely related genes encoding growth hormone were isolated from Atlantic salmon by genomic cloning. From one of these genes a total of 6500 nucleotides were determined including 3900 nucleotides in exons and introns and about 600 and 2000 nucleotides in 5' and 3' flanking regions. The gene is organized in six exons and encodes a polypeptide of 210 amino acids including a 22 amino acids signal sequence. The promoter region contains a typical TATA box 21 nucleotides upstream from the transcription start site. At the 3' end, three putative poly(A) signal sequences are present. The last two are within a 121 nt inverted repeat.
A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.
The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.
The crystal structure of bovine pancreatic β-trypsin (BPT) has been determined from a novel orthorhombic crystal form which contains substantially more solvent (filling 57% of the volume of the unit cell) than previously determined orthorhombic (44%) and trigonal (37%) BPT structures. The native and benzamidine-inhibited crystal structures of BPT in ammonium sulphate at pH 5.3 have been determined for the new form by molecular replacement techniques. The structures have been refined at 1·5 Å resolution with final R-values of 16·7% and 16·9%, respectively.
A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.
We have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, S2, and S3, that encode kallikrein and kallikrein-like serine proteases. The four enzymes share between 74% and 86% amino acid sequence identity and are identical in length with the exception of single two amino acid deletions in the S2 and S3 enzymes. The PS enzyme appears to be a true tissue kallikrein. The S1 enzyme shares 86% amino acid sequence homology with the PS enzyme and retains key amino acid residues thought to be primary determinants of kallikrein cleavage specificity. The S2 enzyme is rat submaxillary tonin. The amino acid sequence of the S3 enzyme is identical with tonin at 84% of its amino acid positions and retains the same amino acid substitutions at positions likely to determine substrate cleavage preferences.
Streptomyces griseus trypsin (SGT) is a bacterial serine proteinase that is more homologous to mammalian than to other bacterial enzymes. The structure of SGT has been solved primarily by molecular replacement, though some low-resolution phase information was supplied by heavy-atom derivatives. The mammalian pancreatic serine proteinases bovine trypsin (BT) and alpha-chymotrypsin (CHT) were used as molecular replacement models. Because these proteins have low homology with SGT compared to the majority of other successful replacement models, new strategies were required for molecular replacement to succeed. The model of SGT has been refined at 1.7 A resolution to a final R-factor of 0.161 (1 A = 0.1 nm); the correlation coefficient between all observed and calculated structure factor amplitudes is 0.908. Solvent molecules located in the crystal structure play an important role in stabilizing buried charged and polar groups. An additional contribution to stability can be seen in the fact that the majority of the charged side-chains are involved in ionic interactions, sometimes linking the two domains of SGT. A comparison of SGT with BT shows that the greatest similarities are in the active-site and substrate-binding regions, consistent with their similar substrate specificities. The modeling of complexes of SGT with two inhibitors of BT, pancreatic trypsin inhibitor (PTI) and the third domain of Japanese quail ovomucoid (OMJPQ3), helps to explain why PTI inhibits SGT but OMJPQ3 does not. Like BT, but unlike other bacterial serine proteinases of known structure, SGT has a buried N terminus. SGT has also a well-defined Ca2+-binding site, but this site differs in location from that of BT.
1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.
A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.
The primary structures of porcine and human pancreatic elastase II precursors were elucidated by molecular cloning and cDNA sequence analysis. The sequences of the cDNAs cloned from a human pancreatic cDNA library indicate that at least two elastases II are expressed in this tissue. These two human elastases II have been designated elastases IIA and IIB. All the cDNA sequences obtained, including porcine elastase II cDNA, reveal that elastase II is synthesized as a preproenzyme of 269 amino acids, including a predicted signal peptide of 16 amino acids and a predicted activation peptide of 12 amino acids. Human elastase IIA contains the published amino-terminal sequence (16 residues) for human pancreatic proelastase II, whereas elastase IIB shares 50% homology with the published 16-residue sequence. However, there is 90% homology between the overall amino acid sequences of elastases IIA and IIB. Blot hybridization analysis of the poly-adenylated RNAs isolated from various human tissues demonstrates that the human elastase II mRNAs are specifically detected in the pancreas.
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
Reinvestigation of the amino acid sequence of bovine chymotrypsinogen A suggests that the amino acid sequence at the N-terminus of the B-chain (residues 16-19) is -Ile-Val-Asn-Gly- rather than -Ile-Val-Gly-Asp- and that Ser-215 should be deleted.