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A genetic variant of β-glucuronidase in Drosophila melanogaster

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S D Langley
S D Langley
S D Langley
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S D Wilson
S D Wilson
S D Wilson
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A S Gross
A S Gross
A S Gross
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Cynthia Warner
Cynthia Warner
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Abstract

The beta-glucuronidase activity of Drosophila melanogaster exists as two chromatographically separable forms, both of which are glycoproteins. Form I is membrane-bound in vivo, has a pI of 8.0-8.5, and can be irreversibly inactivated either by incubation at 55 degrees C for 20 min or by incubation at 37 degrees C in the presence of 6 M urea. Form II exists both membrane-bound as well as membrane-free, has a pI of 4.5, and is resistant to the conditions which inactivate form I. The two forms are similar in Km and Vmax for the artificial substrate 4-methylumbelliferyl-beta-D-glucuronide and both forms are precipitated by antibody to form II. A natural genetic variant, beta-GluL1, completely lacks from I beta-glucuronidase. This variant behaves in a co-dominant fashion for the determination of the presence of form I and has been localized to the extreme distal portion of chromosome 3R. Other data indicate that at least one genetic determinant for the amount of form II is also localized to this portion of chromosome 3R.
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A genetic variant of β-glucuronidase in Drosophila melanogast
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... This revealed existence of a gene of unassigned function (CG15117) that has high amino-acid sequence similarity with that of the CG2135. This finding is consistent with an older report, where two chromatographically separable forms of β-GUS were shown to exist in Drosophila (Langley et al., 1983). Unlike lysosomal hydrolases, CG15117 lacked the N-terminal ERtargeting signal peptide, suggesting it to be a non-lysosomal enzyme that plays some non-conventional role (Fig. S4A,B). ...
Neuromuscular degeneration and locomotor deficit in a Drosophila model of mucopolysaccharidosis VII is attenuated by treatment with resveratrol
Article
Full-text available
  • Nov 2018
Mucopolysaccharidosis VII (MPS VII) is a recessively inherited lysosomal storage disorder caused by β-glucuronidase enzyme deficiency. The disease is characterized by widespread accumulation of non-degraded or partially degraded glycosaminoglycans, leading to cellular and multiple tissue dysfunctions. The patients exhibit diverse clinical symptoms, and eventually succumb to premature death. The only possible remedy is the recently approved enzyme replacement therapy, which is an expensive, invasive and lifelong treatment procedure. Small-molecule therapeutics for MPS VII have so far remained elusive primarily due to lack of molecular insights into the disease pathogenesis and unavailability of a suitable animal model that can be used for rapid drug screening. To address these issues, we developed a Drosophila model of MPS VII by knocking out the CG2135 gene, the fly β-glucuronidase orthologue. The CG2135−/− fly recapitulated cardinal features of MPS VII, such as reduced lifespan, progressive motor impairment and neuropathological abnormalities. Loss of dopaminergic neurons and muscle degeneration due to extensive apoptosis was implicated as the basis of locomotor deficit in this fly. Such hitherto unknown mechanistic links have considerably advanced our understanding of the MPS VII pathophysiology and warrant leveraging this genetically tractable model for deeper enquiry about the disease progression. We were also prompted to test whether phenotypic abnormalities in the CG2135−/− fly can be attenuated by resveratrol, a natural polyphenol with potential health benefits. Indeed, resveratrol treatment significantly ameliorated neuromuscular pathology and restored normal motor function in the CG2135−/− fly. This intriguing finding merits further preclinical studies for developing an alternative therapy for MPS VII. This article has an associated First Person interview with the first author of the paper.
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... Thus it seems unlikely that the probiotic effect we report here would be the consequence of the hydrolysis of endogenous β-glucuronides, as suggested by Gilissen et al. (1998), resulting in the release of -glucuronate that is an additional source of dietary carbohydrates. GUS activity was reported in Drosophila melanogaster (Langley et al., 1983) and in Musca domestica (Levvy & Marsh, 1959), but never investigated in aphids. Ingested GUS, however, could directly interfere with the aphid metabolism, enhancing the glycosaminoglycans degradation. ...
Effects of potato plants expressing the NPTII-gus fusion marker genes on phhysiology and exploratory behaviour of the peach-potato aphid Myzus persicae.
Article
Full-text available
  • Feb 2003
Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII-gus gene construct and exhibiting different β-glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min-1.mg-1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach-potato aphid, Myzus persicae, under laboratory-controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4-choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non-transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non-transformed cultivar. It is concluded that the β-glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.
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... The human heparanase has a pH optimum between 3.5 and 5.0 (Levvy and Marsh, 1959; Dutton, 1980). In Caenorhabditis elegans, the GUS has its optimum activity at pH 5.0 (Sebastiano et al., 1986) and between pH 3.0 and 5.5 in Drosophila (Langley et al., 1983 ). In contrast, E. coli βglucuronidase belonging to family-2 glycosyl hydrolase has an optimal activity at pH of 7.0 (Jefferson, 1987). ...
Beta glucuronidase activity in early stages of rice seedlings and callus - A comparison with Escherichia coli beta glucuronidase expressed in the transgenic rice
Article
Full-text available
  • Aug 2013
We have chosen rice as a model crop to ascertain endogenous β-glucuronidase (family 79) activity and to differentiate the same from Escherichia coli based β-glucuronidase (family-2). The investigation dwells on characterizing endogenous β-glucuronidase (GUS) activity during the early stages of seed germination and from rice callus. Also, similar studies were made from homozygous transgenic rice line expressing E. coli GUS under the control of glyoxalase I promoter. Endogenous GUS activity was detected in plumules, shoots and calli of rice, nevertheless, showing differential response to pH. Further, endogenous GUS in rice was specifically inhibited by saccharic acid 1,4-lactone (SL) but, the E.coli β-glucuronidase remain unaffected, indicating distinct biochemical properties of family-2 and family-79 β-glucuronidase.
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... Thus it seems unlikely that the probiotic effect we report here would be the consequence of the hydrolysis of endogenous β-glucuronides, as suggested by Gilissen et al. (1998), resulting in the release of -glucuronate that is an additional source of dietary carbohydrates. GUS activity was reported in Drosophila melanogaster (Langley et al., 1983) and in Musca domestica (Levvy & Marsh, 1959), but never investigated in aphids. Ingested GUS, however, could directly interfere with the aphid metabolism, enhancing the glycosaminoglycans degradation. ...
Effects of potato plants expressing the nptII-gus fusion marker genes on reproduction, longevity, and host-finding of the peach-potato aphid, Myzus persicae
Article
  • Feb 2003
  • ENTOMOL EXP APPL
Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII-gus gene construct and exhibiting different β-glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min−1.mg−1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach-potato aphid, Myzus persicae, under laboratory-controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4-choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non-transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non-transformed cultivar. It is concluded that the β-glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.
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... Some novel GUS or GUS-like genes were also isolated and characterized from plants, such as Scutellaria baicalensis Georgi [45]. GUS-like enzymes were known to be present in microorganisms [2,3,46,47], animals [48,49] and plants [11,13,36,39,50], such as Scutellaria baicalensis [51], rye [52], and maize [53]. Sudan and his colleagues demonstrated that GUS is ubiquitously present in plants [36]. ...
A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants
Article
Full-text available
A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.
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... However, one of the problems associated with the use of the bacterial gus gene as a reporter is the presence of 'endogenous GUS' activity in some species. Endogenous GUS activity has been reported in various organisms including: 1bacterial species, such as the enterobacterium E. coli and Shigella, nonenterobacterial such as Bacteroides and Clostridium (Hawkesworth et al., 1971), Streptococcus, Staphylococcus, Corynebacterium (Levvy and Marsh, 1959), Alcaligenes (Jefferson, 1989) and many soil bacteria (Ritz et al., 1994); 2 -Some tissues of vertebrates such as kidney, liver and spleen (Kyle et al., 1992), human breast milk (Alonso et al., 1991;Ince et al., 1995); 3 -Invertebrates such as snails (Levvy and Marsh, 1959), nematodes (Jefferson, 1985;Sebastiano et al., 1986) and insects (Langley et al., 1983;Levvy and Marsh, 1959), housefly (Musca domestica) and various locusts (Levvy and Marsh, 1959) and 4 -Plant species. In plants, endogenous GUS activity was first described in Arabidopsis by Jefferson et al. (1987). ...
Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies
Article
Full-text available
  • Jul 2011
The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24°C, 38°C and 55°C) were investigated. To avoid this problem in microspores, two incubation temperatures (38°C and 55°C) at different periods after GUS assay (4, 24 and 48h) and in the presence of 1mM potassium ferricyanide and 1mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1mM potassium ferricyanide and 1mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55°C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2μM gibberellic acid.
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Beta-Glucuronidase Activity: Another Source of Ethyl Glucuronide
Article
  • Jun 2022
Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to ß-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs’ activity in the presence of ethanol (0–70% by volume) using different ß-D-glucuronides (phenolphthalein-ß-D-glucuronide, 4-nitrophenol-ß-D-glucuronide, morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide) as substrates. It was found that ß-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated ß-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tißues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism.
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The Structure and Metabolism of Carbohydrates
Chapter
  • Jan 1994
Compared with the variety of carbohydrates in plants, relatively few sugars or sugar derivatives are regularly found in animals either free or as components of more complex compounds. However, it is possible that sugars originating from plants in the diet are transiently retained in animals and distort the normal sugar spectrum. Approximately a dozen sugars and sugar derivatives are regularly found in animals: the pentoses d-ribose and 2-deoxy-d-ribose; the hexoses d-glucose, d-galactose, d-mannose, d-fructose and l-fucose; the uronic acids d-glucuronic acid and l-iduronic acid; and the hexosamines d-glucosamine and d-galactosamine. In addition, d-erythrose, d-ribulose, d-xylulose and d-sedoheptulose in the form of their phosphoric acid esters are intermediates of the pentose phosphate pathway.
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Understanding isothermal semicrystalline polymer drying: Mathematical models and experimental characterization
Article
  • Nov 1998
  • J POLYM SCI POL PHYS
The drying mechanism of semicrystalline poly(vinyl alcohol) (PVA) was investigated. PVA samples of various molecular weights were crystallized by annealing at temperatures slightly above the glass transition temperature of PVA, and swollen in water for different time periods. The water volume fraction in the sample was measured using a buoyancy technique. The samples were dried in air at constant temperatures, and the drying kinetics were investigated using thermogravimetric analysis. The change in degree of crystallinity of the swollen polymer during drying was measured by differential scanning calorimetry (DSC) as well as by Fourier transform infrared spectroscopy (FTIR). The degree of crystallinity of the samples increased during drying, which in turn was found to alter the drying rate. The drying kinetics were faster at higher temperatures, for lower molecular weights, and for lower degrees of crystallinity. A mathematical model was developed to predict drying rates of semicrystalline polymers by considering the crystallization kinetics during drying. The model predictions included the thickness of the polymer sample, the degree of crystallinity of the polymer, and the water weight loss as functions of drying time. Model predictions were found to agree reasonably well with the experimental results. (C) 1998 John Wiley & Sons, Inc.
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Review: Biosafety of E. coli β-glucuronidase (GUS) in plants
Article
  • May 1998
The -glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers
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Demonstration of a rat liver microsomal binding protein specific for beta-glucuronidase
Article
Full-text available
  • Jun 1979
A binding protein with apparent specificity for beta-glucuronidase has been partially purified from a Triton X-100 extract of rat liver microsomes by affinity chromatography on glucuronidase-Sepharose 2B. It appears that once removed from the membrane, this binding protein self-aggregates to form large macromolecular complexes. With the use of polyacrylamide gel electrophoretic and sucrose density gradient ultracentrifugation assays to monitor the conversion of glucuronidase tetramer to a very high molecular weight complex, it was shown that the binding activity is heatlabile and protease-sensitive. However, binding activity is not influenced by salts, carbohydrates, other proteins or glycoproteins, or by extensive periodate oxidation of beta-glucuronidase, nor does binding occur with any other protein tested. The binding protein does not discriminate against any form of beta-glucuronidase from any rat organ tested. However, the binding protein does show organ localization, being present in the liver and kidney but not the spleen. The possible relationship of this binding protein to egasyn, a membrane protein which stabilizes beta-glucuronidase in mouse liver endoplasmic reticulum, is discussed.
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Lysosomal and Microsomal Glucuronidase: Genetic Variant Alters Electrophoretic Mobility of Both Hydrolases
Article
  • Aug 1974
An electrophoretic variant of β-glucuronidase is present in certain inbred mouse lines. The variant simultaneously affects the mobility of the lysosomal and microsomal forms of the enzyme. The difference is inherited as a single gene mapping at the position of the structural gene on chromosome 5. This confirms that β-glucuronidase at both intracellular sites is coded by the same structural gene.
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Multiple forms of β-glucuronidase in rat liver lysosomes
Article
  • Jan 1975
Multiple forms of β-glucuronidase have been demonstrated using sucrose gradient and polyacrylamide gel isoelectric focusing techniques in 6 m urea. Microsomal β-glucuronidase, a membrane-bound enzyme, was solubilized from lysosome-free, Ca2+-precipitated microsomes by detergents and isolated by chromatography on columns of rabbit anti-rat preputial gland β-glucuronidase antibody bound to Sepharose. The enzyme has a pI of 6.7. Polyacrylamide gel isoelectric focusing resolves the microsomal enzyme into three components, each of which is protease sensitive. The protease-modified microsomal enzyme is very similar to several forms of β-glucuronidase in lysosomes. The lysosomal β-glucuronidase, isolated from osmotically shocked lysosomes, is very heterogeneous after isoelectric focusing over the range pI 5.4–6.0. The lysosomal enzyme can be resolved into 10–12 bands by polyacrylamide gel isoelectric focusing. The more acid forms of the lysosomal enzyme are neuraminidase sensitive, suggesting they may be sialoglycoproteins.
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Disc Electrophoresis ? II Method and Application to Human Serum Proteins
Article
S ummary The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
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Isoelectric Focusing in Polyacrylamide Gel
Article
  • Apr 1971
  • Biochim Biophys Acta Protein Struct
A rapid method for fractionating proteins by isoelectric focusing in small rods of polyacrylamide gel is described. An apparatus is described in which multiple samples can be fractionated in different pH gradients in less than6 h. Smooth and stable pH gradients were achieved by passing a current of 1 mA through high porosity gels containing 2% (w/v) ampholyte with solutions of 0.01 M H3PO4 and 0.02 M NaOH as anolyte and catholyte respectively. The pH gradients were highly reproducible, and the desired pH range occupied over 80% of the length of the gel. The advantages and limitations of this technique are discussed and examples are given of its application to the separations of several well-characterised proteins.
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The measurement of lysozyme activity and the ultraviolet inactivation of lysozyme Biochim
Article
  • Apr 1952
  • Biochim Biophys Acta
1. 1. A rapid and reproducible method for measurement of lysozyme activity is described. 2. 2. Using this method, the ultra-violet inactivation of lysozyme has been studied and found to conform to a first order reaction. 3. 3. The measured quantum yield for inactivation at 2537 A is 0.024 and remains unaltered over the pH range 3.6 to 12.0. A small effect of concentration on quantum yield is indicated. 4. 4. Inactivation is accompanied by photo-oxidation and other secondary processes, with no apparent liberation of free amino acids or peptides. 5. 5. The relation of molecular weight to quantum yield, as well as the other findings, are discussed.
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Purification and characterization of rat liver microsomal β-glucuronidase
Article
  • Aug 1976
  • Biochim Biophys Acta
Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.
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Properties of Two Molecular Forms of β‐Glucuronidase from the Mollusc Littorina littorea L.
Article
  • Feb 1979
The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.
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