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Detergent Influence on Rat‐Liver Galactosyltransferase Activities towards Different Acceptors
Abstract
1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high‐ and low‐molecular‐weight acceptors. Transfer to N ‐acetylglucosamine was measured after the separation of the reaction product N ‐acetyllactosamine from all other radioactive molecules (including galactose) on an ion‐exchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.
2. Both enzymic activities were enriched 60–80‐fold (compared with the post‐nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 95%), presumably because both compete for the same enzyme.
3. The transferase activities were enhanced by the nonionic detergent Triton X‐100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the w/v concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation: Half‐maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50% of the fully activated enzyme could still be sedimented (1 h, 100000 × g ).
4. Galactose transfer to the high‐molecular‐weight acceptor required a higher Triton/phospholipid ratio for half‐maximal activation than did the transfer to the monosaccharide N ‐acetylglucosamine (1 mg/mg compared with 0.5 mg/mg). The degree of activation maximally achieved was much higher with the protein acceptor (400%) than with the sugar (150%). Because both activities are probably due to the same enzyme, it is suggested that these differences in activation reflect properties of the membrane rather than the enzyme, e.g . the presence of a tight diffusion barrier for ovomucoid and the breakdown of this barrier by the detergent.
... The activity of this trans-Golgi enzyme was measured by a technique developed by (Bretz and Staubli, 1977). This enzyme catalyses the addition of galactose onto Nlinked oligosaccharides bearing the GlcNAc2-Man3-GlcNAc2 structure ( Fig. 1.4 ...
A matrix that binds medial Golgi enzymes can be isolated as a detergent insoluble complex. Some components of this complex were identified as cytoskeletal proteins, e.g. cytokeratins and actin. To further analyse this matrix, Golgi membranes were extracted in detergent and salt followed by dialysis and fractionation on a linear sucrose density gradient. An oligomer was identified with an apparent molecular weight of 2x106D as assessed by size-exclusion chromatography. It contained two medial Golgi enzymes, α-1,3-1,6-mannosidase II (Mann II) and β-1,2-N-acetylglucosaminyltransfer-ase I (NAGT I), along with various other proteins. Some of the components of the oligomer were identified as being proteins homologous to the lectins, ERGIC53/p58 and VIP36, and members of the p24 family of putative cargo receptors. The isolation of the oligomer and the identification of some of its components represent a step closer to the elucidation of the mechanisms of enzyme retention and the structural maintenance of the Golgi apparatus. The early Golgi t-SNARE, syntaxin 5, is thought to provide targeting specificity for both COPI and COPII vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway. Two forms of syntaxin 5 were shown to be generated from the same mRNA by alternative initiation of translation. The short form (35kD) corresponds to the published sequence (Bennett et al. (1993), Cell, 74, 863-73). The longer form (42kD) is novel, with an N-terminal, cytoplasmic extension, containing a predicted type II ER targeting signal. When grafted onto a reporter molecule, this signal localised the construct to the ER as assessed by immuno-fluorescence microscopy. This signal could function to retrieve syntaxin 5 from later Golgi compartments but several lines of evidence, including the absence of this longer form from the yeast homologue, Sed5p, point to a function unique to higher organisms.
... Measurement of galactosyltransferase. (Method adapted fromBretz and Staubli, 1977). 80/Lil of assay mix (40mM sodium cacodylate pH 6.6, 17.5mg/ml ovomucoid, 40mM 2mercapto-ethanol, 0.2mM UDP-galactose, 0.2% triton X-100,2mM ATP, 40mM MnClj and 0.5)LtCi/ml [^H]-UDP-galactose) was incubated with 20jLil sample for 30 minutes at 37 " C. The reaction was stopped with 1ml of ice cold 1% phosphotungstic acid/0.5M ...
Antigen processing for presentation by antigen presenting cells (APCs) expressing class II major histocompatibility complex molecules (MHC II) involves partial digestion of antigen, binding of antigen-derived peptides to MHC II, and intracellular trafficking of both antigen and MHC II. In this thesis, two major aspects of these intracellular events have been studied. A flow cytometric assay was developed to measure fluid-phase endocytosis by dendritic cells, potent APCs which have been shown to have poor lysosomal function, and which have been suggested to have a low level of endocytic activity. The assay used a two compartment model to measure the separate activities of early endosomes and late endosomes. Comparison of dendritic cells with different B lymphocyte APCs showed that endocytic traffic through late endosomes, some of which are thought to be related to the site of peptide loading, was similar in dendritic cells and B cells. Thus, the low endocytic activity reported elsewhere was not detected in this project. Organelles containing MHC II were studied in a cell-free system using membranes from disrupted APCs. These organelles were studied by density gradient centrifugation and separated from total membrane by immuno-isolation on magnetic beads. Density gradient centrifugation demonstrated that organelles containing newly synthesized MHC II had a marginally higher density than early endosomes. Immuno-isolated MHC II was found to co-isolate with endocytic markers, in particular markers of early endosomes. This indicates that MHC II is widely distributed throughout the endocytic pathway, including early endosomes, even though it is not widely thought that antigen binds MHC II in early endosomes. In addition, immuno-isolated MHC II was enriched for newly synthesized molecules, which indicates that the organelles where peptide loading is thought to occur were immuno-isolated. In initial experiments, immuno-isolated newly synthesised MHC II was used to perform some of the biochemical events of antigen processing.
... This rrgnj-Golgi marker was measured by a technique developed by Bretz and Staubli (1977). This enzyme catalyses the addition of galactose onto N-linked oligosaccharides bearing the GlcNAc2 -Man3-GlcNAc2 structure. ...
Purified rat-liver Golgi stacks were extracted in a buffer containing 2% (w/v) TX-100, 50mM MOPS pH7.0, 0.1mM MgCl2, 1mM DTT and 10% (w/v) sucrose, and centrifuged to produce an insoluble pellet which contained the majority of three medial-Golgi enzymes, mannosidase II and N-acetylglucosaminyltransferases I and II. Proteins from other regions of the Golgi stack were mostly solubilised. A further extraction of this pellet in the TX-100 buffer containing 150mM NaCl led to complete solubilisation of these medial-Golgi enzymes. After the salt extraction, a second insoluble pellet was produced which was termed the Golgi matrix. The salt-solubilised medial-enzymes could rebind the matrix upon dialysis, while an enzyme from the trans-Golgi could not. Scatchard analysis revealed that rebinding was saturable and occurred with a high affinity, suggesting that the matrix contained a fixed number of receptors which specifically bound the medial-enzymes. This suggested that the enzyme insolubility in detergent was due to their interaction with the matrix. Digestion of intact Golgi membranes with proteinase K greatly increased the detergent-solubility to the medial-enzymes, suggesting that components of the matrix were present on the cytoplasmic, intercistemal face of the Golgi membranes. This topological orientation suggested that the matrix might play a role in stacking the Golgi cisternae. Binding of the enzymes did not, however, occur via their cytoplasmic or membrane-spanning domains, suggesting that the matrix is a complex structure, containing components on both sides of the Golgi membrane. Because of its topology and its binding capacity for medial-enzymes, the matrix may function in aiding the retention of Golgi proteins, maintaining the flattened cisternal morphology or in connecting adjacent cisternae to form the characteristic Golgi stack.
... The vesicle pellets were found at the 30/ 50% sucrose interface and were resuspended. Protein content was analyzed either by SDS–PAGE followed by semiquantitative Western blotting using specific antibodies, or by enzymatic activity using assays specific for NAGT I (Bretz and Staubli, 1977) and GalT (Vischer and Hughes, 1981). ...
Upon addition of GTPS to in vitro budding reactions, COP I vesicles form but retain their coat, making them easy to isolate and analyze. We have developed an in vitro budding assay that reconstitutes the formation of COP I-derived vesicles under conditions where GTP hydrolysis can occur. Once formed, vesicles are uncoated and appear functional as they fuse readily with acceptor membranes. Electron microscopy shows a homogeneous population of uncoated vesicles that contain the medial/trans Golgi enzyme 1,2-mannosidase II. Biochemical quantitation of vesicles reveals that resident Golgi enzymes are up to 10-fold more concentrated than in donor membranes, but vesicles formed in the presence of GTPS show an average density of resident Golgi enzymes similar to that seen in donor membranes. We show that the sorting process is mediated by the small GTPase arf-1 as addition of a dominant, hydrolysis-deficient arf-1 Q71L mutant produced results similar to that of GTPS. Strikingly, the average density of the anterograde cargo protein, polymeric IgA receptor, in COP I-derived vesicles was similar to that found in starting membranes and was independent of GTP hydrolysis. We conclude that hydrolysis of GTP bound to arf-1 promotes selective segregation and concentration of Golgi resident enzymes into COP I vesicles.
... Nearly identical activity of PhyA was determined from methods 1 and 3. Whereas methods 1 and 2 gave only 22% difference in PhyA activity, these two methods produced nearly a 3-fold disparity for AppA2. Overall, the pH (22 ), the type of buffers (28 ), and the inclusion of ancillary chemicals such as the detergents Triton X-100 and BSA (29,30) each accounted for approximately one-third of the variations of AppA2 (Figure 4). The salt ions in solution can interact with the ionized basic and acidic amino acid side chains of the enzyme because of different ion sizes and electromagnetic strengths (31,32). ...
Aspergillus niger PhyA and Escherichia coli AppA2 are increasingly used in animal feed for phosphorus nutrition and environmental protection. The objective of this study was to determine the impacts of assay conditions on activity estimates of these two phytases and to compare their biochemical characteristics at a pH similar to the stomach environment. The activities of the unpurified AppA2 were more variable than those of PhyA with three commonly used phytase activity assays. The variations associated with AppA2 were accounted for by buffer, pH, and the inclusion of Triton X-100 and BSA by approximately one-third each. At the commonly observed stomach pH of 3.5, the purified AppA2 had a lower affinity to phytate (a higher K(m)), but greater V(max), k(cat), and k(cat)/K(m) than those of PhyA. In summary, differences between AppA2 and PhyA in responses to activity assay conditions and in inherent kinetic properties should be considered in interpreting their feeding efficacy.
... The proteins were then precipitated and processed for electrophoresis and fluorography as described above. The unlabeled fractions were analyzed for the following marker activities: galactosyl transferase for the Golgi apparatus (Bretz and Staubli, 1977) ; choline phosphotransferase for the ER (Percy et al ., 1991 ) ; alkaline phosphodiesterase for the plasma membrane (Storrie and Madden, 1990 ) ;,6-hexosaminidase for lysosomes (Storrie and Madden, 1990) ; and succinic dehydrogenase for mitochondria (Pennington, 1961) . The cis-Golgi compartment was assayed by Western blotting with 125 1-protein A using an antibody to p58, a protein which is a marker for this compartment (Saraste et al ., 1987) . ...
Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.
Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.
When intact rat liver Golgi vesicles were incubated with [acetyl-3H]acetyl coenzyme A, radioactivity was incorporated into the vesicles in a manner dependent upon temperature, time, protein, and acetyl-CoA concentration. The vesicles concentrated the label 121-fold relative to the medium within 20 min, suggesting an active transport mechanism operating in intact vesicles, and incorporated more than 50% of this label into acid-insoluble materials. This was supported by the finding that incorporation was markedly reduced by Triton X-100 at levels above its critical micellar concentration. While the intravesicular low molecular weight fraction was predominantly free acetate, acetate ions themselves were not permeant to the vesicles. Double-label experiments suggested that the transport process involved the entire acetyl-CoA molecule. This was further supported by the fact that coenzyme ASH, palmitoyl-CoA and butyryl-CoA were markedly inhibitory. Incorporation was optimal at 22 degrees C at pH 7.0, and was moderately stimulated by ATP. However, compounds known to abolish proton gradients or to inhibit the Golgi proton pump had no effect. The apparent Km for the utilization process was 0.61 microM with a Vmax of 21.3 pmol/mg of protein/min. Oligomycin and 4,4'-diisothiocyanostilbene-2,2'disulfonic acid were inhibitory, whereas CMP-NeuAc, UDP-GlcNAc, adenosine 3'-phosphate, 5'-phosphosulfate, atractylosides, tunicamycin, 2'5'-ADP, and 3',5'-ADP were not, showing that this transport process is distinct from other nucleotide transporters previously described in rat liver Golgi. 75-85% of the radioactivity incorporated was shown to be in O-acetylated sialic acids, by neuraminidase release, purification, and high pressure liquid chromatography. The majority of the neuraminidase-resistant radioactivity was released by alkaline hydroxylamine as [3H]acetylhydroxamate, but a significant fraction was resistant to this treatment. The nature of the non-sialic acid radioactivity remains unknown. The existence of this transport mechanism provides yet another level at which the O-acetylation of sialic acids could be regulated.
Amyloid precursor protein (APP) is a single transmembrane protein that undergoes sequential proteolysis to generate multiple peptides, including the amyloid β -peptide (A β) - the major component of the senile plaques that are diagnostic hallmarks of Alzheimer’s disease (AD).
A number of in vitro models have been developed to study the absorption of drugs across the intestinal epithelium (Osiescka et al., 1985). The majority of these models do not have the morphological and functional properties of normal adult human epithelial cells. Recently, a human adenocarcinoma cell line (Caco-2) that reproducibly displays properties of differentiated distal ileal epithelial cells has been characterised (Wilson et al., 1989; Hidalgo et al., 1989). When cultured on permeable supports (nitrocellulose filters), in chambers, Caco-2 cells form a confluent monolayer which is morphologically polar with a well developed brush border at their apical surface. The monolayers are functional in the specific and unidirectional transport of taurocholic acid, (Wilson et al., 1989), and the active transport of phenylalanine (Hidalgo and Borchardt, 1988).
Myeloperoxidase (MPO), stored in azurophil granules of neutrophils, is critical for an optimal oxygen-dependent microbicidal
activity of these cells. Pro-MPO goes through a stepwise proteolytic trimming with elimination of an amino-terminal propeptide
to yield one heavy and one light polypeptide chain. The propeptide of MPO may have a role in retention and folding of the
nascent protein into its tertiary structure or in targeting of pro-MPO for processing and storage in granules. A propeptide-deleted
pro-MPO mutant (MPOΔpro) was constructed to determine if deletion of the propeptide interferes with processing and targeting
after transfection to the myeloid 32D cell line. Transfection of full-length cDNA for human MPO results in normal processing
and targeting of MPO to cytoplasmic dense organelles. Although the efficiency of incorporation was lower for MPOΔpro, both
pro-MPO and MPOΔpro showed heme incorporation indicating that the propeptide is not critical for this process. Deletion of
the propeptide results in synthesis of a protein that lacks processing into mature two-chain forms but rather is degraded
intracellularly or secreted. The finding of continued degradation of MPOΔpro in the presence of lysosomotrophic agents or
brefeldin A rules out that the observed degradation takes place after transfer to granules. Intracellular pro-MPO has high
mannose oligosaccharide side chains, whereas stored mature MPO was found to have both high mannose and complex oligosaccharide
side chains as judged by only partial sensitivity to endoglycosidase H. The propeptide may normally interfere with the generation
of certain complex oligosaccharide chain(s) supported by the finding of high mannose side chains in secreted pro-MPO and lack
of them in MPOΔpro that contained complex oligosaccharide side chains only. In conclusion, elimination of the propeptide of
pro-MPO blocks the maturation process and abolishes accumulation of the final product in granules suggesting a critical role
of the propeptide for late processing of pro-MPO and targeting for storage in granules.
We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.
Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N-acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.
TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.
ABSTRACT In previous studies we have shown that 1251-labeled prolactin is taken up by a receptor-dependent process and concentrated in an intact form in Golgi elements from female rat liver (J . Biol . Chem., 1979, 254:209-214) . In this study we have examined the effect of colchicine on this uptake process into Golgi elements. Colchicine [25 iumol (10 mg)/100 gm body wt] was injected intraperitoneally in adult female rats, and hepatic Golgi fractions were prepared at 1, 2, and 3 h postinjection . The enzyme recoveries and morphological appearance of fractions from colchicine-treated and control (alcohol alone) animals were similar. At times >1 h after colchicine there was a marked (>60%) inhibition of uptake of 1251-ovine prolactin (' 25 1-oPRL) into Golgi light and intermediate fractions but no inhibition of uptake into Golgi heavy and plasmalemma elements. At times from 2 to 45 min postinjection, 125 1-oPRL was extracted from Golgi elements and found to be largely intact as judged by rebinding to receptors. The inhibitory effect of colchicine was seen at doses ranging from 0.25 Umol to 25 ttmol/100 g body wt. Vincristine also inhibited 1251-oPRL uptake into the Golgi light and intermediate fractions but lumicolchicine had no inhibitory effect. There was a smaller effect of colchicine both at early (1 h) and later (3 h) times on the extent and pattern of 1251-insulin uptake . Colchicine treatment did not produce a significant change in lactogen receptor levels in the Golgi fractions. These results demonstrate that colchicine treatment inhibited the transfer of prolactin into Golgi vesicular elements. The much smaller effect on insulin uptake suggests that there may be differences in the manner in which the two hormones are handled in the course of internalization .
This chapter describes some simple protocols derived from several earlier methods for obtaining highly purified Golgi stacks from rat liver and for determining their relative purity over the homogenate. Centrifugation is carried out using an L8-70M or Optima LE-80K ultracentrifuge and either a SW-28 rotor containing ultraclear tubes or a SW-41 rotor. It is very important to be as accurate as possible when mixing various components and to check the refractive index of each buffer using a refractometer. Aspirate and discard the lipid at the top and the cytosol and collect Golgi fractions that accumulate at the 0.5/0.86 M interface with a plastic Pasteur pipette. This protocol ensures large amounts of Golgi membranes with minimal contamination by other membranes. The relative purification of the Golgi stacks can be assessed by measuring the increase in specific activity of the Golgi enzyme ?-l,4-galactosyltransferase (GAIT) over that of the whole liver homogenate. The yields of Golgi membranes can be calculated from the ratio between the total GalT activity in the Golgi fractions and that of the homogenate.
We have studiedglycosyltransferase activitiesin hu man lymphocytes stimulatedwith the plant mitogensE phytohemagglutinin or concanavalin A (ConA) and have comparedthe resultswithactivitiesfoundin restinglym phocytes.Comparedto resting lymphocytes,Con A-stim ulated lymphocytespossessan enhancedcapacityto transferthe sugars,siabicacid, galactose,andN-acetyb glucosaminefromtheir respectivenucleotidedonorsto both endogenouscellular acceptorsand added exoge nous glycoproteinacceptors.The enhanced glycosyl transferaseactivityinducedby ConA is not inhibitedby puromycin despiteeffectiveinhibitionof de novoprotein synthesis,indicating that synthesis of newglycosyltrans ferase enzymesis not necessaryfor the observedin creasesin glycosylation activity.Bycontrastto the find ingsin ConA-stimulated cells,the corresponding glyco syltransferaseactivitiesof E-phytohemagglutinin-stimu bated lymphocytes donotdifferfromthoseof unstimulated lymphocytes.These data indicatethat individualplant lectinshavedifferenteffectson the biosynthesis of corn plexsaccharides byculturedhumanlymphocytes.
The study of Golgi structure and function has been greatly facilitated by the purification of this membrane organelle from cellular homogenates. Purified Golgi membranes have been used in a variety of cell‐free assays to investigate sugar modifications, vesicle transport, Golgi structure formation and Golgi–cytoskeleton interactions. Golgi membranes can be purified from cells and tissues using a number of different methods, with liver as a preferred source. Highly purified Golgi stacks can be obtained after two sequential density gradient centrifugations of rat liver homogenate. The relative purity of the prepared Golgi stacks is assessed by measuring the increase in activity of a Golgi enzyme, β‐1,4‐galactosyltransferase (GalT), over that of the total liver homogenate. A typical preparation can yield milligrams of Golgi membranes that are purified 80‐ to 100‐fold over the homogenate and 60–70% in stacks, which provides abundant material for both structural and functional studies of this organelle.
Key Concepts
The Golgi apparatus is an essential membrane‐bound organelle in the centre of the secretory pathway in almost all eukaryotic cells.
The primary function of the Golgi is to modify and package proteins and lipids into transport carriers and send them to the proper locations.
Golgi can be separated from other membranous organelles by density gradient centrifugation.
Glycosyl transferases are used as marker enzymes to monitor the yield and purity of the purified Golgi apparatus during Golgi preparation.
Purified Golgi membranes can be used in a variety of cell‐free assays to investigate sugar modifications, vesicle transport, Golgi structure formation and Golgi–cytoskeleton interactions.
Galactosyltransferase (GAL Tase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chtoroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine F9 carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.
The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer’s disease phenotype
are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected
cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but
previous studies could not differentiate between the full-length presenilin proteins and their fragments. We carried out subcellular
fractionation of cells stably transfected with PS1 or PS2 to determine the localization of full-length presenilins and their
fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were
localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon 9 (ΔE9), we observed only
full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the ΔE9
holoprotein, apparently due to decreased degradation within the ER. Our results suggest that that full-length presenilin proteins
are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported
to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.
The effects of carbon tetrachloride and 1,2-dichloroethane (1,2DCE) on the recovery of slices of rat liver from cellular swelling in vitro were studied. Slices took up water during pre-incubation at 1 degrees C, then cellular volume and ultrastructure were rapidly restored during subsequent incubation at 38 degrees C. Ouabain (2 mm) inhibited water extrusion by less than 50%, while inducing formation of peri-canalicular vesicles, apparently derived from the Golgi apparatus. Neither CCl(4) nor 1,2DCE (up to 10 mm) affected the initial extrusion of water at 38 degrees C in the absence of ouabain, but renewed swelling occurred after 60 min with either agent; this was associated with loss of membrane selectivity and some histological damage. By contrast, 1,2DCE inhibited water extrusion in the presence of ouabain after less intensive exposure, for example with 5 mm-1,2DCE for 60 min or 10 mm for 15-30 min. With ouabain present, 1,2DCE (10 mm) caused marked swelling of the endoplasmic reticulum, reduced the peri-canalicular vesicles seen with ouabain alone and reduced the formation of canalicular microvilli. Both CCl(4) and 1,2DCE inhibited the ATP-dependent accumulation of Cl(-) by isolated vesicles of the Golgi apparatus, The delayed swelling of hepatocytes at high concentrations of 1,2DCE and CCl(4) in the absence of ouabain is probably a non-specific consequence of membrane damage. By contrast, 1,2DCE specifically inhibits the ouabain-resistant extrusion of water, possibly by interfering with a postulated mechanism for the exocytotic expulsion of water.
UDPgalactosyltransferase activity (UDPgalactose:mucopolysaccharide galactosyltransferase, EC 2.4.1.74) was measured in a well-characterized fraction of Golgi membranes in the presence of UDPgalactose and exogenous acceptor sites. Substrate saturation for 0.05 mg Golgi protein was achieved at a concentration of 4.6 mM UDPgalactose. Desialylated mucin proved to be the most suitable acceptor protein. Access to galactose acceptor sites was not rate limiting for the reaction when 20 mg of asialo-mucin/ml of incubation mixture was used. With these concentrations of substrates the use of nucleotides to inhibit pyrophosphatases and of detergents to perturb the membrane structure was not necessary and proved, in fact, to be inhibitory to galactose transfer. UDPgalactosyl:asialo-mucin transferase activity in Golgi membranes was 230 nmol galactose transferred/mg Golgi protein per 30 min.
Following rapid isolation, it has been found that Golgi apparatus from ethanol-intoxicated animals contain high levels of galactosyltransferase but also detectable glucose-6-phosphatase and microsomal esterase, as well as 5'-nucleotidase activity. In experiments carried out in parallel on littermate animals but without intoxication, similar recoveries and specific activities of the four enzymes were observed. Morphologic analysis of Golgi fractions isolated from control animals demonstrated no striking morphologic difference to those from the ethanol-intoxicated animals. Indeed, using galloyl glucose-lead staining techniques to mark the lipoprotein particles in situ, it was found that all Golgi apparatus of hepatocytes from control animals were marked by very low density lipoprotein particles. It is therefore concluded that within the limits of the present analyses, Golgi fractions isolated from control animals are as valid as those isolated from ethanol-intoxicated rats.
Lectin-resistant mutants with specific defects in glycosylation have been selected from the mouse lymphoma cell line, BW5147 (Thy-1+). The quantitative expression of cell surface glycoproteins on the mutant cells has been studied. The results show that some glycosylation defects that confer resistance to the cytotoxic effects of concanavalin A block the expression of Thy-1 glycoprotein on the cell surface. However, some changes in the oligosaccharides of Thy-1 glycoprotein generated by glycosylation defects found in PHAR mutant cells and restricted to the termini of complex-type oligosaccharides have no effect on the ability of Thy-1 to reach the cell surface. No glycosylation defects were found that interfered with the expression of either gp 69, 71 or H-2 on the surface of the mutant cells. It is concluded that aberrant biosynthesis of Thy-1 oligosaccharides can interfere with its expression on the cell surface, but that specific changes in oligosaccharide structure are necessary to block transport to the cell surface and integration into the plasma membrane.
We have studied glycosyltransferase activities in human lymphocytes stimulated with the plant mitogens E-phytohemagglutinin or concanavalin A (Con A) and have compared the results with activities found in resting lymphocytes. Compared to resting lymphocytes, Con A-stimulated lymphocytes possess an enhanced capacity to transfer the sugars, sialic acid, galactose, and N-acetyl-glucosamine from their respective nucleotide donors to both endogenous cellular acceptors and added exogenous glycoprotein acceptors. The enhanced glycosyltransferase activity induced by Con A is not inhibited by puromycin despite effective inhibition of de novo protein synthesis, indicating that synthesis of new glycosyltransferase enzymes is not necessary for the observed increases in glycosylation activity. By contrast to the findings in Con A-stimulated cells, the corresponding glycosyltransferase activities of E-phytohemagglutinin-stimulated lymphocytes do not differ from those of unstimulated lymphocytes. These data indicate that individual plant lectins have different effects on the biosynthesis of complex saccharides by cultured human lymphocytes.
Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors.
We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the α subunit of Gs (Gsα) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900–7903], three membrane-bound forms of Gsα were identified through ratezonal centrifugation in sucrose density gradients, Gsα-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gsα in the high-density portion of the gradient (1.13–1.16 g/cm3) is identical with that of β-adrenergic-receptor binding, Na/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09–1.11 g/cm3), contains a much lower total amount of Gsα and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH–cytochrome-c reductase and glucose-6-phosphatase.
The identity of the third pool of Gsα located at the top of the gradient (1.06–1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of Gsα is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100000 ×g proves its particulate (membrane) character. The light membrane form of Gsα subunit is functionally active in an adenylate cyclase complementation assay using cyc− membranes devoid of Gsα.
Overall, our data indicates that a substantial portion of Gsα is localized in membrane pools other than plasma membrane.
Insulin binding to its plasma membrane receptor stimulates a cascade of protein kinases and phosphatases which ultimately affects multiple processes in the membrane, cytosol, and nucleus of the cell, including transcription of specific genes. To gain insight into the relationship between the kinase cascade and the mechanism of insulin-induced nuclear events, we have studied the effect of insulin on the phosphorylation of DNA-binding nuclear proteins in differentiated NIH-3T3-F442A adipocytes. Insulin induced the phosphorylation of seven DNA-binding proteins: pp34, pp40, pp48, pp62, pp64, pp66, and pp72. The half-maximal response was observed at 10-30 min and reached its maximum at 60 min. The insulin-induced phosphorylation of each of these proteins was dose-dependent with ED50S of 2-10 nM. The phosphorylation of pp62, pp64, and pp72 took place on serine residues. On the basis of immunoprecipitation and immunoblotting experiments with anti-lamin antibodies, we found that the insulin-induced DNA-binding phosphoproteins pp62, pp64, pp66, and possibly pp48 were related to lamins A and C. The ED50 for insulin-stimulated lamin phosphorylation was approximately 10 nM, and phosphorylation was half-maximal at 30 min. The insulin-dependent phosphorylation of lamins and other DNA-binding proteins (pp34, pp40, and pp72) may play a mediatory role in the long-term effects of insulin.
Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.
To investigate the identity of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in myeloid cells, we have developed a method that yields subcellular fractions highly enriched in Ins(1,4,5)P3 binding. HL-60 cells were disrupted by nitrogen cavitation, and subcellular fractions were obtained by differential centrifugation, followed by Percoll- and sucrose-density-gradient separations. A subcellular fraction enriched 26-fold in Ins(1,4,5)P3-binding sites was obtained. This fraction showed no enrichment in plasma-membrane markers and only a comparatively moderate enrichment (7-fold) in endoplasmic-reticulum markers. The ratio between specific enrichment of Ins(1,4,5)P3 binding and endoplasmic-reticulum markers in the different fractions varied over 50-fold, from less than 0.1 to greater than 5. The purified Ins(1,4,5)P3-binding fraction was enriched to a similar extent (27-fold) in the putative intravesicular Ca(2+)-storage protein calreticulin. Our results favour the concept of a distinct Ins(1,4,5)P3-binding, calreticulin-containing compartment (i.e. the calciosome) in HL-60 cells.
Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)
Galactosyltransferase (GALTase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chloroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine PC carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.
In the present studies, we investigated the activity of tyrosylprotein sulfotransferase (TPST) in the Golgi apparatus of PC12 cells and the regulation of this enzyme by 2-chloroadenosine, an adenosine receptor agonist. Studies employing continuous sucrose gradient and trypsinization of the membranes demonstrate that TPST is located on the luminal side of Golgi apparatus in PC12 cells. Treatment of PC12 cells with 2-chloroadenosine results in a dose-dependent decrease of TPST activity which is observable as early as 3 h after initiation of treatment, maximizes at 24-48 h with continuous exposure, and is readily reversible upon removal of the drug. While forskolin, an agent that directly increases intracellular cAMP, has no effect on TPST activity, 2-chloroadenosine equally suppressed the enzyme activity in both the wild type and a protein kinase A-deficient mutant strain of PC12 cells, indicating that such regulation of TPST activity by 2-chloroadenosine was independent of cAMP-dependent protein phosphorylation. This effect of 2-chloroadenosine can be potentiated by an adenosine uptake blocker dipyridamole but cannot be elicited by other adenosine A1 or A2 receptor agonists, further suggesting that TPST activity in PC12 cells is regulated by 2-chloroadenosine via a novel membrane receptor. Incubation of the cells with cyclo heximide, a protein synthesis inhibitor, also led to a time- and dose-dependent suppression of TPST activity. At concentrations of cycloheximide that produced maximal inhibition (approximately 50%), cotreatment with 2-chloroadenosine did not lead to a further decrease of the TPST activity. These results suggest that the sensitivity of TPST activity to be controlled by protein synthesis provides a mechanism for regulation of its activity by 2-chloroadenosine.
Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucos-aminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyl-transferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acce9tor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.
We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles. They support a role for Cl- that depends on its uptake as a counter ion for H+ and suggest that it may also stimulate proton transport by a more direct effect on a component of the transport system.
Renal uptake and degradation of cytochrome c and lysozyme were investigated, using preparations that were labelled by means of covalent coupling of either protein to iodinated tyramine-cellobiose. Following proteolytic digestion, the label remains 'trapped' within intracellular organelles. Within 15 min after intravenous injection, 43% of the [125I]tyramine-cellobiose-cytochrome c and 29% of the [131I]tyramine-cellobiose-lysozyme were recovered in the kidneys. Isopycnic sucrose-gradient fractionation indicates that the two proteins initially exhibit closely similar intracellular distributions, being associated with vesicles of an equilibrium density slightly lower than that of plasma membranes. However, within 5 min after injection, the two proteins exhibit distinctly different distribution profiles. The [125I]tyramine-cellobiose-cytochrome c is localized predominantly in the lysosomal fraction of the gradient. The [131I]tyramine-cellobiose-lysozyme is also translocated to the lysosomal fraction, but at a much lower rate. For both proteins, the rates of intracellular degradation correlate with their rates of translocation. The observed difference in their kinetics of intracellular movement suggests that the two proteins are translocated at different rates into transport vesicles.
It has been reported previously that some complex-type Asn-linked oligosaccharides contained in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal galactosyl residues. We report here that extracts from S. mansoni adult male and female worms contain a beta 1,4-galactosyltransferase activity that transfers galactose from the donor substrate UDP-galactose to the acceptor substrate N-acetylglucosamine in a beta 1,4-linkage position to form the disaccharide Gal beta 1,4GlcNAc. In this respect the schistosome-derived activity is similar to that commonly found in mammalian tissues. The kinetic properties, however, of the common beta 1,4-galactosyltransferase activity in mammalian tissues are dramatically altered in the presence of the modifier protein alpha-lactalbumin, whereas the beta 1,4-galactosyltransferase activities in adult male and female schistosomes are not altered by this modifier. Overall, our results demonstrate that adult schistosomes contain a beta 1,4-galactosyltransferase activity and that it is unlike that commonly found in mammalian tissues.
Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of ‘peripheral‐type’ benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density‐gradient centrifugation, one peak (ϱ= 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (ϱ= 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer‐membrane location, the density of the non‐mitochondrial acceptor was insensitive to sonication.
The non‐mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some type of plasma membranes, as jugged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction.
Both acceptors were shown to be peripheral‐type high‐affinity acceptors as judged by ligand specificities and by photoaffinity labelling.
By means of a monoclonal antibody (BH3), we have identified a 57-kD protein (p57) that in interphase is restricted largely to the perinuclear region of the cell. Double label immunofluorescence microscopy suggests localization of p57 to the Golgi complex and associated membranous structures. Protease protection experiments and chemical extractability indicate that p57 is a peripheral membrane protein exposed to the cytoplasm. p57 displays unique behavior during mitosis. At the end of G2 or in early prophase, p57 leaves the perinuclear region and accumulates very rapidly within the nucleus, at a time when the nuclear envelope is still intact and before nuclear lamina disassembly. This relocation of p57 coincides with its hyperphosphorylation on serine and threonine residues. After nuclear envelope breakdown p57 becomes uniformly distributed throughout the mitotic cytoplasm until in late telophase when it returns to its perinuclear location and is once again excluded from the nucleus. The behavior of p57 during mitosis suggests that it may play a role in the cellular reorganization evident during mitotic prophase.
Protein-tyrosine kinases, either in the form of growth-factor receptors or as the polypeptide products of oncogenes, appear to be important in the regulation of cell growth and transformation. A major question, however, is how their substrates mediate changes in gene expression (reviewed in refs 6-8). Because binding of proteins to specific DNA sequences represents the most direct mechanism for regulating transcription, we have investigated the possibility that some DNA-binding proteins may be substrates of protein-tyrosine kinases. Here, we present evidence for nuclear phosphotyrosyl-proteins in murine fibroblasts transformed by the v-abl protein-tyrosine kinase. Furthermore, we have found that these proteins are not significantly phosphorylated in normal NIH 3T3 cells. Finally, using affinity competition chromatography with bacterial and mouse DNA, we have demonstrated that some of these proteins preferentially bind to mouse DNA. The identification of phosphotyrosyl-proteins with selective DNA-binding properties suggests a possible mechanism through which protein-tyrosine kinases may effect changes in gene transcription.
The distribution of nonmitochondrial Ca2+ pumping sites and the site of action of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) were studied in subcellular fractions of human neutrophils. In homogenates, two different Ca2+ pools could be observed: a mitochondrial Ca2+ pool and a nonmitochondrial, ATP-dependent, Ins 1,4,5-P3-responsive Ca2+ pool. When the homogenate was separated into microsomes, primary granules, and secondary granules, the nonmitochondrial Ca2+ pumping and the Ins 1,4,5-P3-induced Ca2+ release occurred only in the microsomal fraction. In a gradient developed to separate different microsomal organelles, maximal Ca2+ pumping activity occurred in fractions of low densities. Correlations between Ca2+ uptake and organelle markers were negative for the endoplasmic reticulum (r = -0.49) and positive for plasma membrane (r = 0.47), Golgi (r = 0.62), and endosomes (r = 0.96). Because the Ca2+ pumping organelles in these fractions were insensitive to micromolar vanadate and digitonin treatment, they are unlikely to be plasma membrane vesicles. We conclude first that microsomal fractions of human neutrophils contain organelles that lower the ambient free Ca2+ concentration and respond to Ins 1,4,5-P3. Second, granules are not involved in intracellular Ca2+ regulation in neutrophils. Third, nonendoplasmic reticulum organelles, such as endosomes, Golgi elements, or yet undefined specialized structures, play a major role in intracellular Ca2+ homeostasis in human neutrophils.
Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], arising from hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], is proposed as the link between membrane-receptor activation and mobilization of Ca2+ from intracellular sites in hormone-secreting cells. The location of Ins(1,4,5)P3-sensitive membranes was investigated in cultured neonatal beta-cells. Membranes were obtained after lysis of cells attached to positively charged Sephadex. After lysis the presence of the enzyme markers 5'-nucleotidase, glucose-6-phosphatase, NADH-cytochrome c reductase, UDP-galactosyltransferase and succinate dehydrogenase indicated the mixed nature of the preparation. After sonication, however, UDP-galactosyltransferase and succinate dehydrogenase activities were undetectable, but 4.8% of total cellular glucose-6-phosphatase and 3.4% of total cellular NADH-cytochrome c reductase remained with 5'-nucleotidase in the preparation, indicating endoplasmic-reticulum association. ATP-dependent 45Ca2+ accumulation was shown in this preparation (410 +/- 24 pmol/mg of protein at 150 nM free Ca2+) and was inhibited by vanadate (100 microM). Ca2+ release was effected by Ins(1,4,5)P3, with half-maximal release at 0.5 +/- 0.14 microM-Ins(1,4,5)P3, t1/2 11.2 +/- 1.1 s. GTP- and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG)-promoted release of 45Ca2+ was demonstrated in this preparation, but the kinetics of release (half-maximal Ca2+ release at 5.4 +/- 0.7 microM, with t1/2 77.3 +/- 6.9 s, and at 51.1 +/- 4.2 microM, with t1/2 19.0 +/- 2.2 s, for GTP and p[NH]ppG respectively), and the ability of neomycin sulphate to block p[NH]ppG-induced release only, are indicative of separate release mechanisms after treatment with these agents. A close association between plasma membrane and elements of the endoplasmic reticulum is indicated in this model, providing a possible mechanism for local alterations in free Ca2+ in the sub-plasma-membrane region.
We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.
We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.
Here we describe results which show that recombinant lymphotoxin (rLT), like the T-lymphocyte derived differentiation inducing factor (DIF), inhibited the clonogenic growth of some myeloid leukemia cell lines by concentrations of 1 to 30 pmol/l. Wild type HL-60 cells were resistant at these concentrations but responded with differentiation into monocyte-like cells at higher concentrations. An antigenic relationship between DIF and LT was indicated because a neutralizing monoclonal anti-LT antibody bound to and neutralized both differentiation and growth inhibitory effects of DIF. An activity, which cochromatographed with DIF during all purification steps, competed with binding of both rLT and recombinant tumor necrosis factor (rTNF) to HL-60 cells. By use of radioiodinated ligand, 2100 binding sites for rLT were detected on HL-60 cells with a Kd of 330 pmol/l. At 37 degrees C bound ligand was transferred to lysosomes, followed by degradation. rTNF and rLT were shown to compete for binding sites on HL-60 cells. Receptors for both rLT and rTNF were downregulated by activators of protein kinase C such as phorbol diester or diacylglycerol; the number of cell surface receptors decreased while the Kd remained unchanged. Our observations demonstrate a functional and antigenic relationship between DIF and LT and indicate that TNF, LT and DIF share binding sites on myeloid leukemia cells that are downregulated by activation of protein kinase-C.
This study investigated the effects of vitamin A excess on hepatic galactosyltransferase (EC 2.4.1.13) activity in livers of rats achieved either by feeding of high levels of retinyl palmitate for 16 wk or gavaging with retinol in olive oil for 3 d. Both hypervitaminotic conditions were characterized by hepatic lipid accumulation. Golgi apparatus fractions were isolated and purity of the fractions was monitored by marker-enzyme analyses and electron microscopy. The quality of the fractions isolated from livers of rats receiving vitamin A excess was not different from that of fractions from control rats. An increase in fat-storing cells in liver, observed in vitamin A excess, coincided with the presence of a floating lipid layer present during isolation of the Golgi apparatus. Galactosyltransferase specific activity (with ovomucoid as acceptor) of Golgi apparatus of rats fed excess vitamin A was 27% of control with chronic feeding and 59% of control with administration by gavage. Activity of another luminally oriented protein, uridine 5'-diphosphate phosphatase, was increased under both in vivo regimens. Vitamin A content of Golgi apparatus, as determined by high performance liquid chromatography, correlated negatively with galactosyltransferase activity after both chronic and acute administration of excess vitamin A.
Treatment of bovine liver microsomes with a partially purified preparation of phospholipase A from Naja naja venom leads to activation of UDP-glucuronyltransferase with p-nitrophenol as glucuronyl acceptor. This stimulation of activity is due to a 6-fold increase in activity at Vmax. As activity at Vmax increases, there is a progressive decrease in binding affinity of the enzyme for both substrates, and although the enzyme remains stable at 23°, it becomes unstable at 37°. This unstable form of UDP-glucuronyltransferase decays to another stable form with a maximum activity 2.5-fold greater than that of untreated enzyme.
As with phospholipase A, treatment with phospholipase C also activates UDP-glucuronyltransferase, but to a lesser extent.
In addition to phospholipases other agents which can alter microsomal lipids also activate UDP-glucuronyltransferase. Triton X-100, sonication, and exposure to pH 9.8 increased activity at Vmax and had variable effects on the binding constants for UDP-glucuronic acid and p-nitrophenol. Maximal activation by these treatments was less than that obtained with phospholipase A; no two treatments had similar effects on all kinetic parameters of the enzyme. Nevertheless, additive effects could not be demonstrated.
Although Triton stimulated glucuronidation of p-nitrophenol by the enzyme, it was without effect on the reverse reaction. This fact plus the other data indicate that the activation of UDP-glucuronyltransferase in these experiments cannot be attributed to compartmentation of the enzyme but is due to phospholipid-induced alterations of enzyme conformation.
An active multiglycosyltransferase system involved in the transfer of N-acetylgalactosamine, N-acetylglucosamine, and galactose to various glycoprotein acceptors was found in the rat small intestinal mucosa. The present studies are concerned with the subcellular localization of these glycosyltransferases. Satisfactory isolation of smooth and rough surfaced microsomes from the intestinal mucosa was obtained by modifying the method of Dallner (Acta Pathol. Microbiol. Scand Suppl., 166, 1 (1963)). Kinetic studies, performed to establish optimum conditions for quantitative assays of the glycosyltransferases, were linear with respect to enzyme concentration and time. Smooth surfaced microsomes were the main submicrosomal loci for the glycosyltransferases; for example 95% of total microsomal polypeptidyl:N-acetylgalactosaminyltransferase activity was found in the smooth surfaced microsomes. The results of alkaline borohydride treatment and acid hydrolysis indicated that N-acetylgalactosamine was incorporated into the heat-treated rough surfaced microsomal acceptors primarily at the protein to carbohydrate linkages while in the case of the heat-treated smooth surfaced microsomal acceptors, this sugar was probably added to positions more distal in the polysaccharide moiety. Immunodiffusion showed that only the smooth surfaced microsomes contained a component immunologically identical with purified rat small intestinal mucin. These results suggest that both stepwise glycosylation and probably the attainment of an immunological identity of rat small intestinal mucin occur in the smooth surfaced microsomes.
The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6–7%
of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction
(GF1) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF3) is contaminated by endoplasmic reticulum membranes to the extent of ∼15% of its protein. The three fractions taken together
account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for ∼70% of the activity of the original
homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated
with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.
Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of ä-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of α-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order: Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 °C) the attachment of Mn2+ is not at thermo-dynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of α-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human α-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine α-lactalbumin with glucose, can be reasonably explained only by assuming that α-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that α-lactalbumin attaches to an enzyme Mn2+ UDP-galactose complex at thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of ä-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four α-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for α-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in α-lactalbumins.
Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.
Zonal centrifugation has been used to isolate a fraction from bovine liver which appears to be derived from the Golgi apparatus. Morphologically, the fraction consists mainly of sacs and tubular elements. Spherical inclusions, probably lipoproteins, are occasionally seen in negative stains of this material. The preparation is biochemically unique. UDP-galactose:N-acetyl glucosamine, galactosyl transferase activity is concentrated about 40-fold in this fraction compared to the homogenate. Rotenone- or antimycin-insensitive DPNH- or TPNH- cytochrome c reductase activities are 60-80% of the level of activities found in microsomes. Purified organelles from bovine liver such as plasma membranes, rough microsomes, mitochondria and nuclei have negligible levels of galactosyl transferase. Some activity is present in smooth microsomes but at a level compatible with the possible presence of Golgi membranes in this fraction. The Golgi fraction does not contain appreciable amounts of enzymes such as ATPase, 5'-nucleotidase, glycosidase, glucose-6-phosphatase, acid phosphatase, or succinate-cytochrome c reductase. Similar fractions isolated from bovine epididymis also have very high levels of galactosyl transferase. The fraction is heavily osmicated when incubated for long periods of time at elevated temperatures, a characteristic property of Golgi membranes.
The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6-7% of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction (GF(1)) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF(3)) is contaminated by endoplasmic reticulum membranes to the extent of approximately 15% of its protein. The three fractions taken together account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for approximately 70% of the activity of the original homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.
The following trisaccharide unit terminates the oligosaccharide chains of several plasma glycoproteins: sialic acid → galactose → N-acetylglucosamine → oligosaccharide → protein. Previous studies showed that this trisaccharide unit was synthesized by the sequential action of three glycosyl-transferases isolated from goat colostrum; each glycosyltransferase catalyzed the transfer of a monosaccharide residue (N-acetylglucosamine, galactose, or sialic acid) from its nucleotide derivative to the appropriate glycoprotein acceptor.
The present studies are concerned with the intracellular location of these glycosyltransferases in rat liver. Kinetic studies were first performed to establish optimum conditions for determining quantitatively the particle-bound rat liver glycosyltransferases; these enzymes exhibited similar properties to the soluble, partially purified glycosyltransferases previously obtained from goat colostrum. The subcellular localization of the enzymes was then investigated by two techniques, differential centrifugation and discontinuous sucrose density gradient centrifugation. In these studies, the following were used as "markers" for various subcellular organelles: DNA, RNA, glucose 6-phosphatase, NADPH-cytochrome c reductase, acid phosphatase, 5'-nucleotidase, and glutamic dehydrogenase. The results indicated that the glycosyltransferases were all located in the same membranous subcellular component and that this component was different from organelles containing the above marker substances; the active subcellular particle was characterized by electron microscopy as being rich in Golgi apparatus. The apparent location of the glycosyltransferases in the Golgi apparatus suggests that these enzymes may be involved in terminating the synthesis of plasma glycoproteins by the liver during secretion, and may possibly be required for secretion of these proteins.
Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025-0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.
The galactosyl acceptor specificity of the highly purified A protein (galactosyltransferase) isolated from bovine milk was examined in the absence and presence of α-lactalbumin. α-Lactalbumin inhibits the transfer of galactose to N-acetylglucosamine but does not appreciably inhibit the transfer to oligomers of N-acetylglucosamine and other β-1,4 linked glycosides such as cellobiose, cellobiulose (glucosyl-β-1,4-fructose), β-d-methylglucose, glucosyl-β-1,4-mannose, indoxyl-β-d-glucose, and ovalbumin. α-Glycosides are poor substrates in the presence of α-lactalbumin but are not substrates in its absence. The biosynthesis of lactose and the formulation of the Gal-β-1,4-GlcNAc linkage in the carbohydrate side chain of glycoproteins are compatible and are carried out by the same galactosyltransferase.
This chapter discusses the synthesis of uridine diphosphate (UDP)-D-galactose from milk. The radioactive lactose labeled with 14C in the D-galactose moiety is prepared using UDP-D-galactose-14C as the D-galactose donor and D-glucose as the acceptor. Lactose labeled in the D-glucose moiety is obtained by using UDP-D-galactose and D-glucose-14C as subtrates with the same enzyme preparation. After incubation, the reaction mixture is passed through a column of a strong anion exchange resin. The unreacted negatively charged UDP-D-galactose is retained, while the neutral lactose formed passes through the column. The solution containing the lactose is collected, and its radioactivity is determined. Under the assay conditions described in the chapter, lactose is the sole 14C-labeled compound that passes through the enzyme assay column. The percentage of lactose synthesized is defined as the difference in counts per minute obtained from zero and five-minute incubation mixtures multiplied by 100 and divided by the total initial radioactivity. The reagents used, procedure followed, and the steps involved in the purification are also described in the chapter.
The separation of sugars has been achieved by the elution of their borate complexes from strong-base anion exchangers with boric-borate buffers. Disaccharides are readily separated from the monosaccharides and the components of hexose or pentose mixtures are easily separated. Hexose-pentose mixtures can be analyzed by the techniques presented. The results are consistent with current concepts of the structures of sugar-borate complexes and the reactions of free sugars in aqueous solutions.
A fractiion rich is membranes of the Golgi apparatus was isolated from rat liver by discontinuous density gradient centrifugation. Electron microscopic analysis of the fraction revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements. The Golgi-rich fraction contained over 90% of the UDP-galactose; galactosyltransferase, about 2% of the glucose-6-phosphatase and 12% of the AMP phosphohydrolase present in the post-nuclear supernatant of liver homogenates.
A method for the determination of phosphorus in amounts from 1.0 to 100 μg has been developed by reinvestigating and combining the desirable features of several previous methods. The method is fast, simple, and accurate, and the reagents and heteropoly blue color are stable.
While columns of anion-exchange resins in the borate form have been ysed previously for separation of mix-tures of carbohydrates as their aorafe complexes in aqueous solutions, the potentialities of the technique as a rapid method of quantitative analysis seem to have been overiooked until very recently.
1. At short incubation times, and under suitable osmotic conditions, the lactose synthesized by Golgi-derived vesicles of rat mammary gland is 85-90% particulate. Evidence is presented for its occlusion within the lumen ofthe vesicles. 2. Ovalbumin is used as a bulky active-site inhibitor to show that the active site of lactose synthase lies on the inner face of the Golgi membrane. 3. Phlorrhizin and phloretin inhibit lactose synthesis by such vesicles, indicating the presence of a glucose-transport system. 4. The relationship of this topography to the synthesis of N-acetylneuraminyl-lactose and to the secretion of milk sugars is discussed.
Glycoproteins have a wide distribution in nature and serve a vast number of functions. There are glycoprotein enzymes and hormones; glycoproteins are found in blood and secretions, in cell membranes, and in connective tissue. Of all the biologically occurring macromolecules the glycoproteins, which consist of carbohydrate moieties convalently linked to a polypeptide backbone, represent the most diverse group, ranging from substances in which the carbohydrate component represents less than 1% of the total weight to those in which it represents over 80% of the total. The proteoglycans, which are classified separately from other glycoproteins and include the chondroitin sulfates, dermatan sulfates, and heparin primarily carbohydrate in the form of numerous heteropolysaccharide chains attached to a polypeptide chain at closely spaced intervals. The sugars that commonly occur in glycoproteins include galactose, mannose, glucose. N-acetylglucosamine, N-acetylgalactosamine, sialic acids, fucose, and xylose. The proteoglycans also contain various uronic and sulfated amino sugars.
1. Three mechanical methods for disrupting membranes substantially stimulated two forward reactions and a reverse reaction of UDPglucuronyltransferase (EC 2.4.1.1.7) in guinea-pig liver microsomes. Stimulation of glucuronyltransferase was highly significant and at lease as extensive as that of nucleoside diphosphatase, reportedly a marker intracisternal enzyme. 2. Stimulation of glucuronyltransferase did not appear to be caused by induction of lipid peroxidation or by lipid hydrolysis during membrane disruption. 3. Addition of phospholipid dispersions failed to significantly re-constrain glucuronyltransferase stimulated by mechanical disruption and did not markedly inhibit the enzyme in untreated control microsomes. 4. Compartmentation appears at least as feasible an explanation for the latency of glucuronyltransferase in guinea-pig liver microsomes as is its conformational constraint by membrane phospholipids. Compartmentation of glucuronyltransferase is functionally attractive since it would ensure its effective interaction with nucleoside diphosphatase on the cisternal side of the endoplasmic reticulum. This would tend to make conjugation reactions unidirectional.
The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.
1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a 'high-speed' supernatant. The 'high-speed' supernatant was designated the 'saline wash' and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with alpha-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 x 10(-6) M. Alpha-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.
The Lowry protein assay is a sensitive but highly nonspecific procedure. The standard Lowry protein assay has been modified so that protein can be assayed in the presence of interfering chemicals. The method is based on the observation that in the presence of 125 μg/ml of Na-deoxycholate, bovine serum albumin (5—50 μg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid. Interference by sucrose, glycerol, Tris—HCl, Tricine, and EDTA can be eliminated. Protein samples containing carrier ampholytes can also be assayed provided their NaCl concentration is adjusted to 1 m.
Addition of lysolecithin caused very marked activation of UDP-galactose:glycoprotein galactosyltransferase in rat liver microsomes and in Golgi-rich membranes. Lysolecithin activated galactosyltransferase when the enzyme was assayed both with endogenous acceptor and with exogenous proteins or monosaccharides as acceptors. Lactose synthetase activity in presence of α-lactalbumin was also stimulated by lysolecithin. Lecithin, lysophosphatidylethanolamine, lysophosphatidic acid, and glycerophosphorylcholine did not activate the enzyme, suggesting that both fatty acyl and phosphorylcholine groups of the lysolecithin molecule are required for the observed activation. The degree of activation was about the same when myristoyl-, palmitoyl-, oleoyl-, or stearoyllysolecithin were tested. The activation by lysolecithin was observed well within the physiological concentration of the lipid in the liver cell. Saturating amounts of Triton masked the effect of lysolecithin.Brief preincubation with phospholipase A activated the enzyme and generated lysolecithin in the membranes. Triton and lysolecithin activated the enzyme without any lag time, whereas phospholipase A activation was dependent on preincubation and also on an alkaline pH favorable for the hydrolysis of phospholipid. EDTA blocked the activation effect of phospholipase A but had no effect on activation by lysolecithin. Albumin and cholesterol opposed the effects of lysolecithin and phospholipase A on the enzyme. Two successive incubations of the microsomes with lysolecithin caused considerable release of the enzyme into the soluble fraction. The role of lysolecithin in the activation of the enzyme is probably related to the solubilization of the membrane and consequent enhanced interaction of the enzyme with substrate. Lysolecithin also activated N-acetylglucosaminyl- and sialyltransferase activities in microsomes. A possible role of lysolecithin is indicated in the regulation of glycosylation reactions in mammalian system.
A Golgi apparatus-rich fraction isolated from rat liver catalyzed the transfer of galactose from UDP-galactose to N-acetylglucosamine with the formation of N-acetylaminolactose as well as the transfer of glucosamine from UDP-N-acetyl- glucosamine to endogenous protein acceptors. Based on enzymatic and morphological criteria, Golgi apparatus fractions were estimated to contain at least 80% Golgi apparatus-derived material. Approximately half of the total glycosyl transferase activities of the original homogenates was recovered in the Golgi apparatus fraction. The glycosyl transferase activities of purified endoplasmic reticulum fractions were much lower than those of the Golgi apparatus. Plasma membrane fractions as well as the soluble supernatant fraction contained little or no activity.
1.1. A Golgi-rich fraction from rat liver has been isolated by the direct application of the zonal centrifugation method used previously to isolate Golgi-rich membranes from bovine liver.2.2. galactosyl transferase is concentrated about 100-fold in this fraction compared to the homogenate and appears to be a useful marker enzyme for this organelle in rat liver as well as beef liver. As in bovine liver Golgi, the fraction from rat liver is only slightly contaminated by other organelles as evidenced by low glucose-6-phosphatase, ATPase and acid phosphatase activities. Thiamine pyrophosphatase activity was found present in endoplasmic reticulum and plasma membranes as well as Golgi membranes from rat liver, and was therefore not a useful marker.3.3. Some species differences in the Golgi preparations exist. Rat liver Golgi contain little or no rotenone-insensitive NADH- or NADPH-cytochrome c reductase activity whereas bovine liver Golgi have significant levels of these activities. In addition, the Golgi fraction from rat liver appears to have a unique and characteristic protein profile after electrophoresis in polyacrylamide gels as compared with endoplasmic reticulum, plasma membranes and mitochondria. Bovine liver Golgi preparations, on the other hand, appear very similar to endoplasmic reticulum after electrophoresis on acrylamide gels.4.4. A major protein of band mobility 0.456 ± 0.008, relative to ribonuclease A, is present in rat liver Golgi preparations. This band is also characteristic of bovine liver Golgi preparations and may be due either to serum very low density lipoproteins or serum albumin, or both.5.5. Using the information obtained from this type of preparation, a simpler, one-step procedure was devised which increases the yield of Golgi membranes from rat liver to 0.15–0.3 mg Golgi protein per g as compared to 0.05 mg Golgi protein per g obtained by the zonal procedure.
Treatment of bovine liver microsomes with a partially purified preparation of phospholipase A from Naja naja venom leads to activation of UDP-glucuronyltransferase with p-nitrophenol as glucuronyl acceptor. This stimulation of activity is due to a 6-fold increase in activity at Vmax. As activity at Vmax increases, there is a progressive decrease in binding affinity of the enzyme for both substrates, and although the enzyme
remains stable at 23°, it becomes unstable at 37°. This unstable form of UDP-glucuronyltransferase decays to another stable
form with a maximum activity 2.5-fold greater than that of untreated enzyme.
As with phospholipase A, treatment with phospholipase C also activates UDP-glucuronyltransferase, but to a lesser extent.
In addition to phospholipases other agents which can alter microsomal lipids also activate UDP-glucuronyltransferase. Triton
X-100, sonication, and exposure to pH 9.8 increased activity at Vmax and had variable effects on the binding constants for UDP-glucuronic acid and p-nitrophenol. Maximal activation by these treatments was less than that obtained with phospholipase A; no two treatments had
similar effects on all kinetic parameters of the enzyme. Nevertheless, additive effects could not be demonstrated.
Although Triton stimulated glucuronidation of p-nitrophenol by the enzyme, it was without effect on the reverse reaction. This fact plus the other data indicate that the
activation of UDP-glucuronyltransferase in these experiments cannot be attributed to compartmentation of the enzyme but is
due to phospholipid-induced alterations of enzyme conformation.
1.1. The dissociation of the Semliki Forest viral membrane by the neutral detergent Triton X-100 was studiedusing analytical and preparative ultracentrifugation.2.2. The binding of Triton to the membrane started below the critical micelle concentration of Triton and increased thereafter with increased Triton concentration.3.3. Release of the nucleocapsids from the virus was observed when more than 0.2–0.4 mg of Triton was bound per mg membrane.4.4. When more than 1.6 mg of Triton was bound the membranes dissociated into small protein-lipid-detergent complexes.5.5. With still larger amounts of Triton present delipidation of the membrane protein took place.6.6. The nucleocapsids did not bind detectable amounts of detergent.
Rat serum was found to contain an enzymatic activity capable of catalyzing the transfer of galactose from UDP-galactose to ovalbumin, a glycoprotein whose carbohydrate complement does not include galactose. Other proteins examined could not serve as galactose acceptors, nor would ovalbumin accept the glycosyl groups of UDP-N-acetylglucosamine or GDP-mannose. The enzyme displayed an absolute requirement for Mn++, which could not be replaced by Mg++ or Ca++. The pH optimum for the galactosyltransferase is approximately 6 in Trismaleate buffer. The Km for UDP-galactose is 2.95 × 10−5. The activity is present in soluble form, and is stable to storage at −20° for 3 weeks. Acid hydrolysis releases all of the radioactivity of the labeled product as 14C-galactose. Mild alkaline hydrolysis releases less than 4% of the incorporated radioactivity, demonstrating that galactose is not attached to serine or threonine residues via O-glycosidic linkages.
Commercial ovomucoid has been freed from the usual contaminants, lysozyme, ovoinhibitor, and ovalbumin, by successive batch treatment with anion and cation exchangers. Only a few hours working time are required to obtain highly purified material. Purity and homogeneity have been demonstrated by gel electrophoresis, fluorescence, sedimentation-diffusion, sedimentation equilibrium, and absence of chymotrypsin inhibition.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
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Fleischer, B. & Fleischer, S. (1970) Biochim. Biophys. Acta,
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White, B. N. & Carubelli, R. (1974) Carhohydr. Res. 33, 366-
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