Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of uridine diphosphate-D-galactose:D-xylose galactosyltrans.

UDP-D-Galactose:D-xylose galactosyltransferase, a membrane-bound enzyme which catalyzes the second glycosyl transfer reaction in the biosynthesis of chondroitin sulfate chains, has been solubilized and partially purified from embryonic chick cartilage. Solubilization was effected by treatment of a particulate fraction of a homogenate (sedimenting between 10,000 and 100,000 times g) with the nonionic detergent Nonidet P-40 (0.5%) and KCl (0.5 M) or by the alkali-detergent method described previously (Helting, T. (1971) J. Biol. Chem. 246, 815-822). The applicability of the salt-detergent procedure as a general method for solubilization of membrane-bound glycosyltransferases was tested by assay of four other glycosyltransferases involved in chondroitin sulfate synthesis (UDP-D-xylose:core protein xylosyltransferase, UDP-D-galactose:4-O-beta-D-galactosyl-D-xylose galactosyltransferase, UDP-D-glucuronic acid: 3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine: (GlcUA-GalNAc-4-sulfate)4 N-acetylgalactosaminyltransferase). In each case, greater than 70% of the activity was solubilized and, on gel chromatography on Sephadex G-200, the enzymes appeared largely in included positions and partially separated from each other. After partial purification by gel chromatography on Sephadex G-200, UDP-D-galactose:D-xylose galactosyltransferase was purified further by chromatography on one of several affinity matrices, i.e. xylosylated core protein of cartilage proteoglycan coupled to CNBr-activated Sepharose, a core protein matrix saturated with UDP-D-xylose:core protein xylosyltransferase or UDP-D-xylose:core protein xylosyltransferase covalently bound to Sepharose. The specific activities of the enzyme preparations obtained by these procedures were approximately 1000-fold greater than that of the crude homogenate.     

Galactosyltransferase, pyrophosphatase and phosphatase activities in luminal plasma of the cauda epididymidis and in the rete testis fluid of some mammals

Galactosyltransferase activity was measured in the luminal plasma of the cauda epididymidis of mice, rats, rabbits, rams and boars, and in the rete testis fluid of rams and boars. The activities of nucleotide pyrophosphatase and alkaline phosphatase, which compete with galactosyltransferase for substrate, were also determined. In these species, galactosyltransferase activity in the luminal plasma of the cauda epididymidis was similar when the inhibitory effect of pyrophosphatase and phosphatase was minimized by assay conditions. However, under assay conditions that did not minimize the effect of these enzymes, the galactosyltransferase activities of these species were very different and were inversely correlated with the activities of pyrophosphatase and phosphatase. The ratio of galactosyltransferase activity to pyrophosphatase and phosphatase activity was much higher in the rete testis fluid than in the luminal plasma of the cauda epididymidis in both rams and boars. In rams, galactosyltransferase in the luminal plasma of the cauda epididymidis was more heat resistant than that in serum. These results suggest that there is a species difference in the availability of galactosyltransferase activity in the luminal plasma of the cauda epididymidis and that in some species, galactosyltransferase in the luminal fluid is unlikely to have any function. The results are also discussed with respect to the possible function of galactosyltransferase, pyrophosphatase and phosphatase in epididymal luminal plasma and rete testis fluid.     

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine)

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell‐wall xyloglucans and heteromannans. The full‐length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem‐specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)‐α‐xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum‐graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell‐culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP‐xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP‐galactose and the same cello‐oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α‐linkages. Analysis of the products of PrGT34B by MALDI‐TOF MS showed that up to three xylosyl residues were transferred from UDP‐xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.     

The Caenorhabditis elegans genes sqv-2 and sqv-6, which are required for vulval morphogenesis,encode glycosaminoglycan galactosyltransferase II and xylosyltransferase

In mutants defective in any of eight Caenorhabditis elegans sqv (squashed vulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of sqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAbeta1,3Galbeta1, 3Galbeta1,4Xylbeta-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.     

Stage- and tissue-specific expression of a β-1,4-galactosyltransferase in the embryonic epidermis

Changes in oligosaccharide structures of glycoconjugates have been observed, and are postulated to have key roles in embryonic development and differentiation. N-Acetylglucosamine (GlcNAc) beta-1,4-galactosyltransferase (beta4GalT) AKI showed different expression patterns in time and space, and different enzymatic activity from the other known family members. The epidermis of mouse embryo included a high level of AKI activities, which transferred galactose (Gal) to endogenous glycoprotein (molecular weight 130 kDa) (GP130). The maximum activity was for 13.5-d postcoitum embryos. Specific antibody against AKI inhibited 81% of GlcNAc betaGalT activities, which indicates that AKI represents the major part of the embryonic epidermis enzymes. AKI shows 2.2 times higher galactosyltransferase activity toward Gal-acceptor glucose with alpha-lactalbumin (alpha-LA) than toward GlcNAc without alpha-LA. AKI is also expressed in mouse melanoma and leukemia cell lines and in human basal cell carcinoma specimens. The GP130 Gal acceptor once galactosylated by AKI may be directly involved in epidermal differentiation and oncogenesis.     

Glycolipid glycosyltransferases in human embryonal carcinoma cells during retinoic acid induced differentiation

Retinoic acid induced differentiation of TERA-2-derived human embryonal carcinoma cells is accompanied by a dramatic reduction of extended globo-series glycolipids, including galactosyl globoside, sialylgalactosyl globoside, and globo-A antigen (each recognized by specific MoAbs). Associated with these glycolipid changes, the activities of two key enzymes, alpha 1----4 galactosyltransferase (for synthesis of globotriaosyl core structure) and beta 1----3 galactosyltransferase (for synthesis of galactosyl globoside), were found to be reduced 3- to 4-fold. The latter enzyme plays a key role in the synthesis of extended globo-series structures, and its characterization has not been reported previously. Therefore, its catalytic activity was studied in detail, including substrate specificity, detergent and phospholipid effects, pH and cation requirements, and apparent Km. During retinoic acid induced differentiation, a series of Lex glycolipid antigens (recognized by anti-SSEA-1 antibody) and their core structures (lacto-series type 2 chains) increase dramatically. In parallel with these changes in glycolipid expression, the activities of two key enzymes, beta 1----3 N-acetylglucosaminyltransferase (for extension of lacto-series type 2 chain) and alpha 1----3 fucosyltransferase (for synthesis of Lex structure), were found to increase by 4- and 2-fold, respectively. Similarly, an increase in the expression of several gangliosides (e.g., GD3 and GT3) during retinoic acid induced differentiation was mirrored by a 4-fold increase in the activity of alpha 2----3 sialyltransferase (for synthesis of ganglio core structure, GM3). The results suggest a coordinate regulation of key glycosyltransferases involved in core structure assembly and terminal chain modification.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes.

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialytransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60--80 micrograms WGA to the Golgi membrane, while only 40 micrograms of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialytransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20--30 min in the presence of 1 micromol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 X 10(-6) M from a Steck-Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 X 10(-5) M/mg of Golgi membrane protein.     

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.     

Catabolism of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium. The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria. The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue. These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R. pachyptila, because these nucleosides can be delivered to other parts of the worm. This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways. The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome. The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery. In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome. We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont.     

Several Galactosyltransferase Activities Are Associated with Mouse Chromosome 17

Indirect evidence suggests that some major histocompatibility complex (MHC) proteins are glycosyltransferases. No sequence or mapping information is available for transferases, although ganglioside variations in mice are linked to the H-2 complex on chromosome 17, and one galactosyltransferase activity on mouse sperm varies with T/t complex genotypes, also on chromosome 17. In the present experiments, diploid and trisomy 17 mouse embryos were assayed for four different galactosyltransferase activities. The same preparations were assayed for isocitrate dehydrogenase (Id-1, chromosome 1) and glyoxalase-1 (Glo-1, chromosome 17). Galactosyltransferase specific activities in trisomy 17 embryos are almost 1.5 times higher than in diploid embryos. The correlation between galactosyltransferase activities and chromosome 17 dosage indicates that the structural or regulatory gene for these enzymes are located on chromosome 17.     

Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

A cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated from a human melanoma line library. The new cDNA sequence encoded a type II membrane protein with 327 amino acid sequence and showed 38% homology to the Caenorhabditis elegans sqv-3 gene involved in the vulval invagination and oocyte development. Extracts from L cells transfected with the galactosyltransferase cDNA in an expression vector and a fusion protein with protein A exhibited marked galactosyltransferase activity specific for p-nitrophenyl-beta-D-xylopyranoside. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis of galactosyltransferase I-deficient Chinese hamster ovary mutant pgsB-761 cells. Analysis of the enzyme product by beta-galactosidase digestion, mass spectroscopy, and NMR spectroscopy revealed that the reaction product was formed via beta-1,4 linkage, indicating that the enzyme is galactosyltransferase I (UDP-galactose:O-beta-D-xylosylprotein 4-beta-D-galactosyltransferase, EC 2.4.1.133) involved in the synthesis of the glycosaminoglycan-protein linkage region of proteoglycans.     

Biosynthesis of chondroitin 4-sulfate-proteoglycan by a transplantable rat chondrosarcoma.

The activities and subcellular distribution of five glycosyltransferases involved in the biosynthesis of chondroitin sulfate by a transplantable rat chondrosarcoma were compared with the activities and distribution of the corresponding enzymes of normal embryonic rat and chick cartilage.Two important differences were found: 1) UDP-d-xylose:core protein β-d-xylosyltransferase was found in concentrations 10–15 times higher in the chondrosarcoma, and 2) all five glycosyltransferases were found to be more soluble in the chondrosarcoma. More than 90% of the xylosyltransferase activity could be extracted from the tumor without rupturing cells. This transferase exhibited optimal activity in solutions of 0.25 m KCl. The K_m for the exogenous protein acceptor obtained by Smith degradation of bovine chondroitin sulfate-proteoglycan was 300 μg/ml; the K_m for Ser-Gly-Gly, 30 mm. The activity of xylosyltransferase was maximal at pH 6.5 and was dependent upon the presence of Mg²⁺ or Mn²⁺. The K_m for UDP-xylose was 5 × 10^?5, m. In view of the extraordinarily high level of xylosyltransferase activity found in the chondrosarcoma the authenticity of the xylosyl transfer reaction was verified by chemical characterization of [¹⁴C]xylose-labeled products.     

Salvage synthesis of purine nucleotides by Helicobacter pylori

G.L. MENDZ, B.M. JIMENEZ, S.L. HAZELL, A.M. GERO AND W.J. O'SULLIVAN. 1994. The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.     

Effect of ovarian hormones on glycosyltransferase activities in the endometrium of ovariectomized rats.

Some of the properties of galactosyl- and sialyltransferases present in rat endometrial tissue were investigated. The enzyme activities were found to be partly membrane-bound and partly in soluble form. The galactose enzyme was also present in uterine secretions. The specific activities of both galactosyl- and sialyltransferases were greatly enhanced in endometrium of ovariectomized rats following 17 β-estradiol injections, although the enzyme activities in the liver remained unaffected. Mixing experiments with the homogenates of endometrium from control and estradiol-treated rats failed to suggest the presence of any “activator” or “inhibitor” of the enzymes. Diethylstilbesterol, estrone, and estriol also stimulated galactosyl- and sialyltransferase activities, whereas testosterone stimulated sialyltransferase only. Prolactin administration had no effect on either of the enzymes. The effect of estradiol on both enzymes was shown to be dose-dependent and the specific activities of the enzymes started to increase about 6 hr after hormone administration, reaching a peak around 48 h. Progesterone, on its own, had no effect on the galactosyltransferase in ovariectomized rat endometrium but effectively prevented the stimulatory effect of estradiol. When estradiol-primed rats were treated with progesterone, it was found that very small doses of progesterone resulted in decrease of galactosyltransferase activity. In such animals sialyltransferase activity was stimulated by a low concentration of progesterone which was followed by inhibition at higher concentrations. These effects of ovarian hormones on glycosyltransferase activities in endometrium are compatible with earlier reports on the effects of these hormones on glycoprotein and glycosaminoglycan levels in rabbit uterus after ovariectomy (7). Regulation of glycosyltransferase activities in endometrium induced by estradiol and progesterone may bear some relationship to the “receptive” state of the uterus for blastocyst implantation.     

Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

The major anthocyanins accumulated by an Afghan cultivar ofDaucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.Abbreviations CGT UDP-galactose: cyanidin galactosyltransferase - CGXT UDP-xylose: cyanidin 3-galactoside xylosyltrans-ferase - DEAE diethylaminoethyl This study was supported by a grant from the Deutsche Forschun-gsgemeinschaft and a fellowship (W.E.G.) from the Land Baden-Württemberg. The skilful technical assistance of Johannes Madlung is gratefully acknowledged.     

Inhibition of chondroitin and heparan sulfate biosynthesis in Chinese hamster ovary cell mutants defective in galactosyltransferase I

We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.     

Activities of prolyl hydroxylase, lysyl hydroxylase, collagen galactosyltransferase and collagen glucosyltransferase in the liver of rats with hepatic injury

The activities of four enzymes catalysing post-translational modifications of the collagen polypeptide chains were assayed in the livers of rats with experimental hepatic injury. The liver injury was induced by injecting carbon tetrachloride twice weekly, and assays of the enzymic activities were carried out 2 and 4 weeks after commencement of administration of carbon tetrachloride. The liver homogenates were preincubated with Triton X-100 before the assays, because such treatment was found to increase the activities of all four enzymes in the supernatants of liver homogenates. The activities of all four enzymes had increased by 2 weeks after commencement of carbon tetrachloride administration. No increase was found in the collagen content of the livers at this stage and thus an increase in all four enzyme activities preceded an increase in the collagen content of the liver. A further slight increase was found in three of the enzyme activities during the subsequent 2 weeks of the experiment, whereas no further increase was found in the collagen galactosyltransferase activity. A statistically significant correlation was found between all four enzyme activities, but the magnitude of the increases varied considerably. The largest increase was found in lysyl hydroxylase activity, and at 4 weeks the magnitude of this was about three times that of the collagen galactosyltransferase activity. The results thus indicate that the increased enzyme activities cannot be explained simply by an increase in the number of collagen-producing cells having similar enzyme activity patterns to those of the cells initially present in the liver.     

The effect of linoleic acid and benzyl alcohol on the activity of glycosyltransferases of rat liver golgi membranes and some soluble glycosyltransferases

The effects of the membrane perturbing reagents linoleic acid and benzyl alcohol on the activities of four rat liver Golgi membrane enzymes, N-acetylglucosaminyl-, N-acetylgalactosaminyl-, galactosyl-, and sialytransferases and several soluble glycosyltransferases, bovine milk galactosyl- and N-acetylglucosaminyltransferases and porcine submaxillary N-acetylgalactosaminyltransferases have been studied. In rat liver Golgi membranes, linoleic acid inhibited the activities of N-acetylgalactosaminyl- and galactosyltransferases by 50% or greater, sialyltransferase by 10–15%, and N-acetylglucosaminyltransferase not at all. The isolated bovine milk N-acetylglucosaminyltransferase and porcine submaxillary N-acetylgalactosylaminyltranferase were not inhibited but bovine milk galactosyltransferase was inhibited by 95% or greater. The inhibition by linoleic acid on Golgi membrane galactosyltransferase appears to be a direct effect of the reagent on the enzyme. Incorporation of bovine milk galactosyltransferase into liposomes formed from saturated phospholipids, DMPC, DPPC, and DSPC (dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine) prevented inhibition of the enzyme activity suggesting that the lipid formed a barrier which did not allow linoleic acid access to the enzyme. The water soluble benzyl alcohol was more effective in inhibiting enzymes of the isolated rat liver Golgi complex. All four glycosyltransferases were inhibited, the N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases by more than 95%. A higher concentration of benzyl alcohol was necessary to inhibit the galactosyltransferases than was required for the other Golgi enzymes. Benzyl alcohol also inhibited the isolated bovine milk N-acetylglucosaminyl- and galactosyltransferases 90% to 95%, respectively, but did not affect the isolated porcine submaxillary gland N-acetylgalactosaminyltransferase. Benzyl alcohol did not inhibit the milk galactosyltransferase incorporated into DMPC or DPPC liposomes but showed a complex effect on the activity of the enzyme incorporated into DSPC vesicles, a stimulation of activity at low concentrations followed by an inhibition. A lipid environment consisting of saturated lipids appears to present a barrier to inhibiting substances such as linoleic acid and benzyl alcohol, or lipid may stabilize the active conformation of the enzyme. The different effects of these reagents on four transferases of the Golgi complex suggest that the lipid environment around these enzymes may be different for each transferase.     

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.