UDP-N-acetylglucosamine-glycoproteinN-acetylglucosaminyltransferase in regenerating rat liver

The assay condition for N-acetylglucosaminyltransferase activities in rat liver microsomal fraction was developed. The enzyme activities towards endogenous acceptors within 48 h after partial hepatectomy were lower than in controls, exceeding the control level by 96 h, and then higher than in controls up to 240 h after the operation. The changes in N-acetylglucosaminyltransferase activities towards exogenous acceptor (UPD-2-acetamido-2-deoxy-D-glucose: glycoprotein 2-acetamido-2-deoxy-D-glucosyltransferase, EC 2.4.1.51) were consistent with those in the enzyme activities towards endogenous acceptors at 144 h, but not at 48 h, after the operation. The contents of protein and the levels of protein-bound hexosamine in the liver microsomes were decreased at early period of liver regeneration.These results suggest that the acceptor capacity of liver microsomal proteins is diminished during first 48 h of the regeneration. This may be responsible for the decreased transfer of the amino sugar to nascent glycoproteins. However, the enzyme activity was enhanced at 144 h and the level of endogenous acceptors may increase.     

Alterations in N-linked oligosaccharides of glycoproteins during rat liver regeneration

[3H]Mannose-labeled glycopeptides in the slices after partial hepatectomy were characterized by column chromatography using Sephadex G-50, DE-52 and Con A-Sepharose, and further by digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H. They contained both 'complex type' and 'high-mannose type' oligosaccharides. A higher proportion of 'complex type' oligosaccharides was contained in regenerating liver 24 h after partial hepatectomy than in control. This tendency was increased gradually with time and was most pronounced at 144 h. In our previous studies, the activities of microsomal N-acetylglucosaminyltransferase towards endogenous and exogenous acceptors at 144 h after partial hepatectomy were shown to exceed most prominently that in control. No differences in the oligosaccharides were observed at 240 h when the deficit of liver had been restored. The oligosaccharides of glycopeptides in the incubation media were mostly 'complex type' and the differences between regenerating liver and control were observed only at 144 h. These results suggest that oligosaccharide processing of glycoproteins is regulated at the transfer step of peripheral N-acetylglucosamine to core oligosaccharides 144 h after partial hepatectomy, and that these alterations in oligosaccharides of glycoproteins may be related to hypertrophy and hyperplasia of hepatic cells in liver regeneration.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: II. Measurement of enzyme activities utilizing microsomal glycoproteins as exogenous acceptors

Microsomal sialyltransferase was assayed in chicken liver and hepatoma Mc-29 utilizing liver and hepatoma microsomal glycoprotein fractions, treated with Triton X-100, as exogenous acceptors. In a homologous assay system containing enzyme and acceptor from one and the same tissue no quantitative dependence of enzyme activity was revealed with increasing amount of the acceptor. In mixed experiments in which liver enzyme activity was tested towards hepatoma acceptor glycoproteins, a gradual drop in sialyltransferase activity occurred with increasing quantities of the acceptor. This effect seems to be a consequence of the presence of some inhibitor in the microsomal fractions from the hepatoma cells.     

The presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes (author's transl)

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.     

Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions

The ability of Co2+ to substitute for Mn2+ in exogenous and endogenous galactosyltransferase reactions was tested. Exogenous transfer was measured towards different high and low molecular weight galactose acceptors using galactosyltransferase from the following sources: crude serum, the serum enzyme partially purified by affinity chromatography and a pure enzyme preparation from milk. Endogenous transfer was estimated in preparations from human urinary bladder tumor cells and from rat liver microsomal fractions. The results show that Co2+ is able to substitute for Mn2+ in some exogenous and endogenous galactosyltransferase reactions. This ability seems to depend on the molecular structure of the galactose acceptor as well as on the nature of the enzyme.     

Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates.

Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.     

A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioure-idoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low Km values and Vmax values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver alpha 2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.     

Expression of N-acetylglucosaminyltransferase III in hepatic nodules during rat liver carcinogenesis promoted by orotic acid

The activity of N-acetylglucosaminyltransferase III, which adds a "bisecting" GlcNAc in beta 1,4 linkage to the beta-linked Man of the core of Asn-linked oligosaccharides (Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242), was determined in hepatic nodules of rats initiated by administration of a single dose of carcinogen 1,2-dimethylhydrazine.2HCl (100 mg/kg, intraperitoneal) 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. N-Acetylglucosaminyltransferase III was assayed using glycopeptide GlcNAc beta 1,2Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1, 4GlcNAc beta 1,4GlcNAc-Asn as substrate and, as enzyme sources, microsomal membranes of the hepatic nodules, surrounding liver, regenerating liver, and age- and sex-matched control liver. The nodules had significant N-acetylglucosaminyltransferase III activity (0.78-2.18 nmol GlcNAc transferred/h/mg of protein), while the surrounding liver, the regenerating liver (24 h after partial hepatectomy), and the control liver had negligible activity (0.02-0.03 nmol/h/mg of protein). Product isolated from a large scale N-acetylglucosaminyltransferase III incubation with hepatic nodules as enzyme source showed the presence of the bisecting GlcNAc residue by 500 MHz proton NMR spectroscopy. Concomitant with the appearance of N-acetylglucosaminyltransferase III activity in the preneoplastic nodules, the activities of N-acetylglucosaminyltransferase I and II were decreased in these membranes when compared to those from surrounding liver, regenerating liver, and control liver. These results suggest that N-acetylglucosaminyltransferase III is induced at the preneoplastic stage in liver carcinogenesis promoted by orotic acid and are consistent with the reported presence of bisecting GlcNAc residues in the Asn-linked oligosaccharides of rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (Kobata, A., and Yamashita, K. (1984) Pure Appl. Chem. 56, 821-832).     

Properties and regional distribution of cerebral CMP-N-acetylneuraminic acid: Glycoprotein sialyltransferase

Glycoprotein sialyltransferase was studied in the rat brain and in the frontal grey cortex and corpus callosum of the calf brain. Activities were measured with endogenous acceptors as well as with desialized α₁-acid glycoprotein as an exogenous acceptor. The enzyme was characterized by means of its pH optimum, K_m values and requirements for detergent and cations. The properties of the rat and calf brain enzymes appeared to be very similar. Substrate specificity studies indicate that more than one glycoprotein sialyltransferase reaction may occur in brain. The regional distribution of the enzyme in the calf brain was rather uniform. From this it was concluded that glycoprotein sialyltransferase, at least for the greater part, is localized in membranes other than those of the synaptic complexes, and occurs in both neurons and glia cells. The regional distribution of the amounts of endogenous glycoprotein acceptor sites, which could be calculated from the sialyltransferase activities, showed a striking correlation with that of the protein-bound sialic acid, but not with the sialyltransferase activity. The role of these endogenous glycoprotein acceptors in cerebral sialoglycoprotein biosynthesis is discussed.     

Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30-month-old rats following a necrogenic dose of thioacetamide

The influence of aging on the mechanisms of liver injury and regeneration was studied in a model of hepatotoxicity induced in 2-, 6-, 12-, 18- and 30-month-old rats by a sublethal dose of thioacetamide (500 mg/kg body weight), a soft nucleophilic and hepatotoxic compound metabolized by the hepatic microsomal FAD monooxygenase system. Samples-blood and hepatocytes-were obtained at 0, 12, 24, 48, 72 and 96 h following thioacetamide intoxication. Parameters of liver injury in serum (NADPH-isocitrate dehydrogenase (ICDH) activity) indicate that the severity of injury was significantly higher in the adult groups (6 and 12 months old) when compared either with the youngest (2 months old) or oldest (18 and 30 months old) groups. Parameters related to biotransformation, such as microsomal FAD monooxygenase, followed mainly the same pattern of age-dependent changes as those observed for injury. The profile of glutathione-S-transferase activity showed an initial induction parallel to liver injury and opposite to the levels of reduced glutathione and protein -SH groups. Enzyme activities and gene expression of the systems involved in the cell endogenous antioxidant defense, such as Mn- and Cu,Zn-superoxide dismutases (SOD), catalase and glutathione peroxidase (GPX) showed significant age-dependent changes that can be summarized as follows: an increase in all enzyme activities and gene expression and a decreased ability to restore the initial activities following 96 h of thioacetamide. We conclude, first, that the gene expression and activity of the enzymes involved in the intracellular antioxidant defense system increased with aging, which can be considered a consequence of the enhanced oxidative state of the cell (decreased in GSH level); and second, that the lower and delayed response in the aged groups significantly influenced the restoration towards normal of GSH and the antioxidant enzyme activities.     

Properties of membrane-bound sialyltransferase from rana temporaria liver]

It was demonstrated that microsomal membranes from frog liver contain at least two different sialyltransferases involved in the synthesis of alpha 2 leads to 3 and alpha 2 leads to 6 oligosaccharide isomers. Studies on acceptor specificity of the sialyltransferase system with respect to low molecular acceptors revealed its similarity to the mammalian sialyltransferase system. However, sharp distinctions were observed in sialylation of mammalian glycoproteins. It was assumed that that the disaccharide unit of the acceptor oligosaccharide chain is a structural element, which in necessary but not sufficient for glycoprotein recognition by sialyltransferases.     

Regulation of rat-liver glycoprotein: N-acetylneuraminic acid transferase activity by pyrimidine nucleotides.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N-acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N-Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide-sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.     

Modified assay-procedure for guanosine diphosphate-L-fucose: 2-acetamido-2-deoxy-beta-D-glucopyranoside-(1 leads to 4)-alpha-L-fucosyltransferase with the aid of synthetic phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopy-ranosyl -beta-D-glucopyranoside as a reference compound

Starting from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-beta-D-glucopyranoside (1), chemical syntheses were developed for phenyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside (4) and phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopyranosyl -beta-D-glucopyranoside (8). Thin-layer chromatography in the solvent system 6:4:1:5 (v/v) 2-propanol-ethyl acetate-ammonium hydroxide-water clearly separated the synthetic trisaccharide 8 (RF 0.69) from synthetic disaccharide 4 (RF 0.78), fucose (RF 0.56), and GDP-fucose (which remained at the origin). Based upon this observation, a modified method for the determination of GDP-L-fucose: N-acetylglucosaminide-(1 leads to 4)-alpha-L-fucosyltransferase was developed that employed the synthetic disaccharide 4 as an acceptor, and compound 8 as an authentic reference-compound. This modified assay-procedure can simultaneously monitor possible competing reactions which may interfere with determination of alpha-(1 leads to 4)-L-fucosyltransferase activity; these include phosphorylase and alpha-L-fucosidase activities, and incorporation of alpha-L-[14C]-fucose into endogenous acceptors of enzyme preparations. Thus, the modified assay-procedure should facilitate determination of alpha-(1 leads to 4)-L-fucosyltransferase.     

Transfer of glucose from UDP-glucose in microsomal membranes of rat hepatocytes (author's transl)]

Microsomal preparations from rat liver mediate transfer of glucosyl units from UDP-glucose to three different kinds of acceptors: an endogenous glycoprotein, exogenous glycogen and collagen. Both glucosyl transferases work at acidic pH, 6.5 for transfer on endogeneous acceptor and glycogen and at pH 5.5 for transfer on collagen. None of these enzymes require divalent cations for activity. While transfers on endogenous acceptor and glycogen are inhibited by the presence of a non-ionic detergent, Triton X-100, the transfer on collagen is activated by the same detergent. Glycogen-synthase activity requires glucose 6-phosphate at an optimal concentration of 1 mM. The Km values for UDP-glucose are respectively: 0.5 mM, 0.33 mM, and 1 mM for transfer on endogenous acceptor, glycogen and collagen. Characterisation of the product indicates that a protein-bound alpha1-4 glucan is formed when no primer is added. Enzymatic and acidic hydrolyses of radioactive glycogen and collagen show only glucose as a radioactive sugar identified by thin layer chromatography on cellulose. Pre-treatment of microsomal membranes by alpha-amylase demonstrates that glucosyltransferases are not adsorbed on endogenous glycogen and seem to be really membranous enzymes.     

Glucose-1-phosphotransferase and N-acetylglucosamine-1-phosphotransferase have distinct acceptor specificities

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha Glc-1-P from UDP-Glc to endoglycosidase H-sensitive oligosaccharides on acceptor glycoproteins. We have previously demonstrated that Glc-phosphotransferase was specific for UDP-Glc as its nucleotide sugar substrate and thus appeared to be distinct from UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase), an enzyme specific for lysosomally destined acceptor glycoproteins. Here, sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs of endogenous acceptor glycoproteins in embryonic chick neural retina homogenates labeled by the presence of [beta-32P]UDP-Glc were shown to be distinct from those labeled by [beta-32P]UDP-GlcNAc, indicating that the two enzymatic activities recognize different populations of endogenous glycoproteins. To further probe the acceptor specificities of these enzymes, three glycoproteins known to be exogenous acceptors for GlcNAc-phosphotransferase were included in assays for Glc-phosphotransferase from retinal homogenates. Cathepsin D and beta-N-acetylhexosaminidase had no significant effects on phosphoglucose incorporation. Uteroferrin, an acid phosphatase, had a pronounced inhibitory effect on incorporation from UDP-Glc, and subsequent experiments suggested that phosphorylation of the Glc-phosphotransferase or another protein may be necessary for maximal activity to be seen. Also, I-cells, which have previously been shown to possess no GlcNAc-phosphotransferase activity, and control human fibroblasts were assayed for both Glc-phosphotransferase and GlcNAc-phosphotransferase. GlcNAc-phosphotransferase activity was observed only in control cells, whereas Glc-phosphotransferase was observed in both I-cells and controls at similar specific activities.     

Studies on the hyperplasia (''regeneration'') of the rat liver following partial hepatectomy. Changes in lipid peroxidation and general biochemical aspects.

Using the experimental model of partial hepatectomy in the rat, we have examined the relationship between cell division and lipid peroxidation activity. In rats entrained to a regime of 12 h light/12 h dark and with a fixed 8 h feeding period in the dark phase, partial hepatectomy is followed by a rapid regeneration of liver mass with cycles of synchronized cell division at 24 h intervals. The latter phenomenon is indicated in this study by pulses of thymidine kinase activity having maxima at 24 h, 48 h and 72 h after partial hepatectomy. Microsomes prepared from regenerating livers show changes in lipid peroxidation activity (induced by NADPH/ADP/iron or by ascorbate/iron), which is significantly decreased relative to that in microsomes from sham-operated controls, again at 24 h, 48 h and 72 h after the operation. This phenomenon has been investigated with regard to possible underlying changes in the content of microsomal fatty acids, the microsomal enzymes NADPH:cytochrome c reductase and cytochrome P-450, and the physiological microsomal antioxidant alpha-tocopherol. The cycles of decreased lipid peroxidation activity are apparently due, at least in part, to changes in microsomal alpha-tocopherol content that are closely associated in time with thymidine kinase activity.     

Sialylation in vitro of purified human liver beta-D-N-acetylhexosaminidase

In order to study structure-function relationships of lysosomal enzymes, human liver beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) has been purified by an extraction/affinity chromatography/ion-exchange procedure. The isoenzymes A and B, native as well as neuraminidase-treated, were incubated with a partially purified preparation of bovine colostrum sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1). Native beta-N-acetylhexosaminidases were found to be poor acceptors for the sialyltransferase used. However, incorporation of sialic acid into neuraminidase-treated beta-N-acetylhexosaminidase A and B amounted to a 58 to 72% saturation of the theoretical acceptor sites, respectively. The acceptor specificity of the sialyltransferase suggests that Gal beta(1 leads to 4)-GlcNAc units may be present on at least part of the beta-N-acetylhexosaminidase A and B molecules. However, oligomannosidic-type chains may also occur on the lysosomal enzyme, as shown by sugar composition of the enzyme. The presence and/or amount of sialic acid residues does not appear to affect the kinetic properties of beta-N-acetylhexosaminidase A and B towards 4-methylumbelliferyl glycoside substrate.     

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

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-acetylglucosaminyltransferase 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. Fucosyltransferase 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 differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, 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.     

Membrane glycoprotein transferases: Time courses of activity changes in a synchronized cell population and enzyme activity half-lives in an asynchronous population

The time course of activity changes of five membrane glycoprotein transferases: the glycoprotein:galactosyl, the fetuin:fucosyl, the PSM:fucosyl, the fetuin:N-acetylglucosaminyl, and the polypeptide: N-acetylgalactosaminyl was determined using defined acceptors and conditions in synchronized L5178Y cells. In addition, time course of activity changes of these transferases was determined for the endogenous acceptors present in the cell extract. Each of the membrane glycoprotein transferases was an S peak enzyme in the L5178Y cell. Activity with endogenous acceptors, in general, was constant throughout the cell cycle indicating that acceptor availability may, in part, control glycoprotein synthesis in vivo.