Structure of the disaccharide chain of galactosyl-N-acetylgalactosaminyl-protein synthesized in vitro.

In order to obtain a [14C]galactosyl-N-acetylgalactosaminyl-protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N-acetylgalactosaminyl-protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [14C]galactosyl-N-acetylgalactosaminyl-protein product and as glycopeptides by proteolytic digestion of the glycoprotein. The reduced disaccharide consisted of equimolar amounts of galactose and N-acetylgalactosaminitol and was characterized by thin-layer chromatography, high-voltage electrophoresis and gas-liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [14C]galactose was linked to position C-3 on the N-acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [14C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was beta. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: alpha-D-N-acetylgalactosaminyl-protein beta 1 leads to 3 transferase activity.     

Studies on the latency of UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal and Golgi subfractions from rat liver

The activity of UDPgalactose-asialo-mucin galactosyltransferase (EC 2.4.1.74) in microsomal and Golig subfractions was stimulated 2.4-fold after disruption of the membrane permeability barrier by hypotonic incubation. In the presence of Triton X-100, galactose transfer to asialo-mucin was increased 12-fold in rough microsomes and 5-fold in smooth microsomes both with and without hypotonic incubation; while in the Golgi subfractions no stimulation by detergent was observed. These experiments indicate differences in enzyme-lipid or enzyme-protein interactions in microsomes and Golgi membranes. Furthermore, these results strongly support the conclusion that the UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal fractions is not due to contamination by Golgi vesicles but represents an enzyme activity endogenous to the endoplasmic reticulum.     

Identification of UDP-galactose: lactose (lactosylceramide) alpha-4 and beta-3 galactosyltransferases in human kidney

Two galactosyltransferases were identified in human kidney microsomes which both transfer galactose from UDP Gal to lactose as well as to lactosylceramide. Using a solubilized and a partially purified enzyme preparation sufficient product could be obtained for detailed structural analysis. The trisaccharide products were isolated by gel permeation chromatography and separated by preparative high performance thin layer chromatography. The anomeric configuration of the transferred galactose was determined by specific glycosidase digestion and the linkage was identified by methylation and gas-liquid-chromatography. The glycolipid products were not separated but analyzed directly, before and after alpha or beta galactosidase digestion, by methylation, hydrolysis and thin layer chromatography. Into both acceptor substrates galactose was incorporated in alpha 1-4 (30%) and beta 1-3 (70%) linkages. The alpha 1-4 galactosyltransferase is responsible for the synthesis of the Pk antigen Gal alpha 1-4 Gal beta 1-4 Glc-ceramide in human kidney. The beta 1-3 galactosyltransferase has not previously been identified.     

Roles of active site tryptophans in substrate binding and catalysis by alpha-1,3 galactosyltransferase

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in alpha-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314, and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of alpha-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High-resolution crystallographic structures of the Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of k(cat) changes in modulating glycosyltransferase specificity.     

Studies on uridine diphosphate-galactose pyrophosphatase and uridine diphosphate-galactose: glycoprotein galactosyltransferase activities in microsomal membranes.

Rat liver microsomes showed very active uridine diphosphate-galactose pyrophosphatase activity leading to the hydrolysis of uridine diphosphate-galactose into galactose1-phosphate and finally into galactose. The activity was observed in presence of buffers with wide ranges of pH. Different concentrations of divalent cations, such as Mn²⁺, Mg²⁺, and Ca²⁺ had no significant effect on the enzyme activity. A number of nucleotides and their derivatives inhibited the pyrophosphatase activity. Of these, different concentrations of uridine monophosphate, cytidine 5′-phosphate and cytidine 5′-diphosphate have slight or no effect; cytidine 5′-triphosphate, adenosine 5′-triphosphate, guanosine 5′-triphosphate, cytidine 5′-diphosphate-glucose and guanosine 5′-diphosphate-glucose showed strong inhibitory effect whereas cytidine 5′-diphosphate-choline showed a moderate effect on the pyrophosphatase. All these nucleotides also showed variable stimulatory effects on uridine diphosphate-galactose:glycoprotein galactosyltransferase activity in the microsomes which could be partly related to their inhibitory effects on uridine diphosphate-galactose pyrophosphatase. Among them uridine monophosphate, cytidine 5′-phosphate, and cytidine 5′-diphosphate stimulated galactosyltransferase activity without showing appreciable inhibition of pyrophosphatase, cytidine 5′-diphosphate-choline, although did not inhibit pyrophosphatase as effectively as cytidine 5′-triphosphate, guanosine 5′-triphosphate, adenosine 5′-triphosphate, cytidine 5′-diphosphate-glucose, and guanosine 5′-diphosphate-glucose but stimulated galactosyltransferase activity as well as those. The fact that cytidine 5′-diphosphate-choline stimulated galactosyltransferase more effectively than cytidine 5′-phosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate suggested an additional role of the choline moiety in the system. It has been also shown that cytidine 5′-diphosphate-choline can affect the saturation of galactosyltransferase enzyme at a much lower concentration of uridine diphosphate-galactose. Most of the pyrophosphatase and galactosyltransferase activities were solubilized by deoxycholate and the membrane pellets remaining after solubilization still retained some galactosyltransferase activity which was stimulated by cytidine 5′-diphosphate-choline. In different membrane fractions a concerted effect of both uridine diphosphate-galactose pyrophosphatase and glycoprotein:galactosyltransferase enzymes on the substrate uridine diphosphate-galactose is indicated and their eventual controlling effects on the glycopolymer synthesis in vitro or in vivo need careful evaluation.     

Analogues of the mycobacterial arabinogalactan linkage disaccharide as cell wall biosynthesis inhibitors

The mycobacterial arabinogalactan linkage disaccharide [alpha-L-Rha-(1-->3)-alpha-D-GlcNAc] provides a basis for the design of new antitubercular drugs, since it supports a key skeletal structure in the bacterial cell wall. A series of analogues of the linker was synthesized by coupling appropriate thiorhamnosyl donors modified at their 4-positions, with an N-acetyl glucosamine acceptor. In a cell-free enzyme inhibition assay, three analogues inhibited the activity of the galactosyltransferase that adds a Galf residue to the linkage disaccharide. Although the compounds were modest inhibitors, these data confirm the viability of this approach to anti-mycobacterial agents. It is especially significant that the three effective compounds are modified at the site of the acceptor atom in the natural substrate.     

Multiple forms of glycosyltransferases in Golgi complex membrane fractions

The distribution of multiple forms of galactosyltransferase (EC 2.4.1.22) and sialyltransferase (EC 2.4.99.1) from the microsomes and Golgi complex membrane fractions of rat liver was investigated. Three fractions of Golgi membranes, namely GF1, GF2, and GF3, differing in their morphology and marker enzyme activity, were obtained. A simultaneous increase of glycosyltransferases under study was observed in fractions GF3 less than GF2 less than GF1. Using isoelectrofocusing, the presence of at least 6-8 forms of galactosyl- and sialyltransferases in the microsomes and Golgi fraction was revealed. The distribution patterns of multiple forms along the pH gradient for each membrane fraction were found to be identical. However, the ratios of highly active and low active forms were specific for each fraction. The similarity of multiple form spectra for galactosyl-and sialyltransferase suggest their tight functional interaction and a possible "en block" packing of membrane glycosyltransferases.     

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.     

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.     

Biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine

The biosynthesis of galactosyl-beta 1,3-N-acetylglucosamine has been demonstrated using membrane preparations from pig trachea. Unlike the UDP-galactose:2-acetamido-2-deoxy-D-glucose 4 beta-galactosyltransferase, which is inhibited by high levels of N-acetylglucosamine, the UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase shows no inhibition at 200 mM N-acetylglucosamine. About 80% of the total disaccharide synthesized at 200 mM N-acetylglucosamine was base-labile suggesting the 1,3-linkage, alpha-Lactalbumin inhibits galactose incorporation into galactosyl-beta 1,4-N-acetylglucosamine but has little or no effect on the activity of the 1,3-galactosyltransferase. Escherichia coli beta-galactosidase readily hydrolyzed the base-stable product, but not the base-labile component. The apparent 1,3-linked disaccharide was reduced with NaBH4 and was isolated by Bio-Gel P-2 column chromatography. Methylation analysis by gas chromatography/mass spectrometry showed tetramethyl galactose and a 3-substituted N-acetylglucosaminitol. Neither the beta 1,4 nor the beta 1,3 disaccharide was hydrolyzed by green coffee bean alpha-galactosidase. Both disaccharides were readily hydrolyzed by bovine testes beta-galactosidase. This is the first report on the galactosyltransferase which catalyzes the synthesis of the galactosyl-beta 1,3-N-acetylglucosamine linkage such as found in the Type I chain of human blood group substances. A tissue survey in rats showed only rat intestine to have readily detectable UDP-galactose: N-acetylglucosamine 3 beta-galactosyltransferase activity. The intestinal membrane fraction like the tracheal enzyme catalyzes the synthesis of two disaccharides as judged by base treatment, and these appear to be the beta 1,3 and beta 1,4 isomers of galactosyl-N-acetylglucosamine.     

The Pathway of Golgi Cluster Formation in Okadaic Acid-Treated Cells

Using stereology and immunoelectron microscopy we examined the pathway of Golgi duster formation during treatment with the phosphatase inhibitor okadaic acid. During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesicles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall reduction in membrane the total amount of immunolabeling for galactosyltransferase over the Golgi clusters of a typical cell was maintained, indicating that galactosyltransferase had been retained in Golgi membranes. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Golgi cisternae increased 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cisternae was selective. Careful evaluation of immunolabeled clusters showed that most of the galactosyltransferase labeling was located over complex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both connected to disassembling cisternae and within forming Golgi clusters, indicating that they were intermediates in cluster formation. We also investigated the role of vesicular transport in cluster formation. During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane depletion and stack disassembly. Taken together, our results indicate that during Golgi cluster formation, membrane leaves the Golgi but galactosyltransferase is retained within a tubular reticulum which is a direct descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus.     

Appican, the proteoglycan form of the amyloid precursor protein, contains chondroitin sulfate E in the repeating disaccharide region and 4-O-sulfated galactose in the linkage region

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.     

A water-soluble analogue of glucosaminylphosphatidylinositol distinguishes two activities that palmitoylate inositol on GPI anchors

2-Palmitoylation of the inositol residue occurs during biosynthesis of glycosylphosphatidylinositol (GPI) anchors, but the enzymology of this step has been enigmatic. With endogenously synthesized glucosamine-PI (GlcN-PI; a GPI intermediate), a CoA-dependent palmitoyl-CoA-independent acyltransfer activity (AT-1) has been reported in rodent preparations. In contrast, a palmitoyl-CoA-dependent GlcN-PI acyltransferase activity (AT-2) was reported in both rodent and yeast preparations with a novel water-soluble dioctanoyl GlcN-PI analogue, GlcN-PI(C8). We report that AT-1, as well as AT-2, can be detected in rodent microsomes with GlcN-PI(C8), thus demonstrating the coexistence of these activities in a single membrane preparation and the general utility of GlcN-PI(C8) for studying the GPI pathway. Unexpectedly, AT-2 was peripherally associated with microsomes, a property atypical for GPI biosynthetic enzymes.     

B3LYP/6-311++G* * study of structure and spin-spin coupling constant in heparin disaccharide

Structures of heparin disaccharide have been analyzed by DFT using the B3LYP/6-311++G( * *) method. The optimized geometries of two forms of this disaccharide, differing in the conformation ((1)C(4) and (2)S(0)) of the IdoA2S residue, confirmed considerable influences of the sulfate and the carboxylate groups upon the pyranose ring geometries. The computed energies showed that disaccharide having the (1)C(4) form of the IdoA2S residue is more stable than that with the (2)S(0) form. Interatomic distances, bond and torsion angles showed that interconversion of the IdoA2S residue results in geometry changes in the GlcN,6S residue as well. Three-bond proton-proton and proton-carbon spin-spin coupling constants computed for both forms agree with the experimental data and indicate that only two chair forms contribute to the conformational equilibrium in disaccharide. Influences of the charged groups upon the magnitudes of spin-spin coupling constants are also discussed.     

Receptor-mediated endocytosis of enterokinase by rat liver: Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30–45 kDa range.     

Receptor-mediated endocytosis of enterokinase by rat liver. Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Preparation and galactosyltransferase acceptor activity of derivatives of antifreeze glycoproteins of an Antarctic fish.

A series of 12 closely related glycoproteins containing alpha-linked N-acetyl-D-galactosamine (GalNAc) as the sole carbohydrate moiety have been prepared by degradation of the antifreeze glycoproteins from the serum of the Antarctic fish Trematomus borchgrevinki. The polypeptide moieties of these glycoproteins contain substitutions in the normal -Ala-Ala-Thr- repeating tripeptide sequence which introduce alterations in the amount of alpha-helical structure and the density of acceptor sites, and theoretically also in the amount of rigidity, polarity, and hydrophobicity of the polypeptide. Of these alterations only density of acceptor sites has a statistically significant effect on the ability of the GalNAc alpha leads to Thr moiety to act as a substrate for galactosyltransferase (EC 2.4.1.22) activity solubilized from rat liver microsomes. This result suggests that in the biosynthesis of rat liver glycoproteins these structural features of the polypeptide moiety of glycoproteins are not part of the substrate specificity of the galactosyltransferase activity that transfers the second monosaccharide. Hence, these structural features do not play a major role in determining the structure of the threonine-linked oligosaccharide after its synthesis has been initiated.     

Enzymic transfer of α-L-arabinopyranosyl residues to exogenous 1,4-linked β-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-β-L-arabinopyranose

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.     

Enzymatic synthesis of 3-O-methyl-4-O-β- -galactopyranosyl- -glucose (3-O-methyl-lactose); a potential agent for the assessment of intestinal lactase activity

Enzymatic synthesis of 3-O-methyl-4-O-β- -galactopyranosyl- -glucose (3-O-methyl-lactose) has been attempted using both galactosyltransferase and galactosidase activities. The transferase-catalysed reaction produces exclusively the desired product in β-1,4-glycosidic linkage whereas the galactosidase-catalysed reactions predominantly form a 1,6-linked disaccharide. With galactosidase, in order to change the regioselectivity, blocking of the 6-position of 3-O-methyl- -glucose and anomeric modification of the acceptor structure were investigated. Although acetylation of the 6-position of 3-O-methyl glucose catalysed by lipase was successful, the synthesis of the desired disaccharide did not occur.