Sialyltransferase Reaction Research Articles

ChemInform Abstract: Studies on the Precise Chemical Synthesis of Human Glycoproteins

AbstractReview: 52 refs.

Studies on the Precise Chemical Synthesis of Human Glycoproteins

Abstract Our research group demonstrated the semi-synthesis of complex-type biantennary sialyloligosaccharide-library by means of branch-specific glycosidase and several sialyltransferase reactions toward complex-type H2N-Asn-(biantennary compex-type sialyloligosaccharide)-COOH isolated from egg yolk at over a gram scale. This methodology yielded 35 kinds of homogeneous complex-type oligosaccharides. Using this oligosaccharide library, an efficient Boc solid phase peptide synthesis (SPPS) of acid labile sialylglycopeptide-α-thioester was demonstrated. Our research group showed that the sialoside in which the carboxylic acid is protected with a phenacyl group is stable under strong acidic conditions, and this critical finding enabled us to demonstrate the first Boc-SPPS for the synthesis of a sialylglycopeptide-α-thioester. Using both a synthetic method of sialylglycopeptide-α-thioester and native chemical ligation (NCL), our research group synthesized several glycoproteins such as chemokine MCP-3, three kinds of erythropoietin analogues, and interferon-ß. Our research group also demonstrated a unique synthesis that was an intentional synthesis of homogeneous misfolded glycoproteins bearing M9-high-mannose-type oligosaccharide. This unique synthetic misfolded-glycoprotein is useful for the purpose of uncovering the mechanism of molecular recognition in glycoprotein quality control (GQC) in the endoplasmic reticulum (ER). This account discusses the function of the oligosaccharides of glycoproteins based on our research findings.

Enhanced Bacterial α(2,6)-Sialyltransferase Reaction through an Inhibition of Its Inherent Sialidase Activity by Dephosphorylation of Cytidine-5'-Monophosphate.

Bacterial α(2,6)-sialyltransferases (STs) from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP) generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP), which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6)-ST from P. leiognathi JT-SHIZ-145 (P145-ST), the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6)-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling.

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmSTWT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmSTE271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmSTE271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.

Unique Self‐Anhydride Formation in the Degradation of Cytidine‐5′‐monophosphosialic Acid (CMP‐Neu5Ac) and Cytidine‐5′‐diphosphosialic Acid (CDP‐Neu5Ac) and its Application in CMP‐sialic Acid Analogue Synthesis

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.

Trans-sialidase activity of Photobacterium damsela 2,6-sialyltransferase and its application in the synthesis of sialosides

Trans-sialidases catalyze the transfer of a sialic acid from one sialoside to an acceptor to form a new sialoside. alpha2,3-Trans-sialidase activity was initially discovered in the parasitic protozoan Trypanosoma cruzi, and more recently was found in a multifunctional Pasteurella multocida sialyltransferase PmST1. alpha2,8-Trans-sialidase activity was also described for a multifunctional Campylobacter jejuni sialyltransferase CstII. We report here the discovery of the alpha2,6-trans-sialidase activity of a previously reported recombinant truncated bacterial alpha2,6-sialyltransferase from Photobacterium damsela (Delta15Pd2,6ST). This is the first time that the alpha2,6-trans-sialidase activity has ever been identified. Kinetic studies indicate that Delta15Pd2,6ST-catalyzed trans-sialidase reaction follows a ping-pong bi-bi reaction mechanism. Cytidine 5'-monophosphate, the product of sialyltransferase reactions, is not required by the trans-sialidase activity of the enzyme but enhances the trans-sialidase activity modestly as a non-essential activator. Using chemically synthesized Neu5AcalphapNP and LacbetaMU, alpha2,6-linked sialoside Neu5Acalpha2,6LacbetaMU has been obtained in one-step in high yield using the trans-sialidase activity of Delta15Pd2,6ST. In addition to the alpha2,6-trans-sialidase activity, Delta15Pd2,6ST also has alpha2,6-sialidase activity. The multifunctionality is thus a common feature of many bacterial sialyltransferases.

Bacterial CMP-sialic acid synthetases: production, properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell-cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5'-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.

Reversible Sialylation: Synthesis of Cytidine 5‘-Monophospho-N-acetylneuraminic Acid from Cytidine 5‘-Monophosphate with α2,3-Sialyl O-Glycan-, Glycolipid-, and Macromolecule-Based Donors Yields Diverse Sialylated Products†

Sialyltransferases transfer sialic acid from cytidine 5‘-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer α2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcα2,3Galβ1,3GalNAcα unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5‘-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming α2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5‘-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5‘-CMP had a wide optimum range (pH 5.2−7.2 and 4.8−6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5‘-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5‘-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5‘-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.

Chemoenzymatic synthesis of diverse asparagine-linked alpha-(2,3)-sialyloligosaccharides.

Partial sialyl transfer reaction by alpha-(2,3)-sialyltransferase toward (Gal-beta-1,4-GlcNAc-beta-1,2-Man-alpha-1,6/1,3-)(2)Man-beta-1,4-GlcNAc-beta-1,4-GlcNAc-beta-1-asparagine-Fmoc 1 was examined to obtain mono-alpha-(2,3)-sialyloligosaccharides and then branch-specific exo-glycosidase digestion (beta-D-galactosidase, N -acetyl-beta-D-glucosaminidase and alpha-D-mannosidase) toward the asialo-branch was performed to obtain diverse asparagine-linked complex type alpha-(2,3)-sialyloligosaccharides. In addition, two kinds of disialyloligosaccharides in which the sialyl linkage was a mixture of alpha-(2,3)- and alpha-(2,6)-types were also specifically prepared by an additional alpha-(2,6)-sialyltransferase reaction toward mono-alpha-(2,3)-sialyloligosaccharides thus obtained.

Characterization of a (2 → 6)-sialyltransferase reaction intermediates: Use of alternative substrates to unmask kinetic isotope effects

This chapter illustrates the way in which the rival explanations can be addressed by employing a poorer alternative substrate to unmask intrinsic kinetic isotope effects and to reveal significant mechanistic details about sialyltransferase catalysis. Enzyme action on a preferred substrate is often characterized by rate processes that lead to high catalytic efficiency but also prevent a thorough kinetic analysis of the reaction mechanism. This limitation frequently can be obviated by the use of alternative substrates that exhibit lower rates of catalysis and often unmask kinetic features that are indiscernible with the natural substrate. In the case of α(2→6)- sialyltransferase, the alternative donor substrate uridine monophosphate glycoside of N-acetylneuraminic acid (UMP-NeuAc) provides the opportunity to probe the mechanism in greater detail than achieved with cytidine monophosphate glycoside of N-acetylneuraminic acid (CMP-NeuAc). The specificity of CMP-NeuAc synthase for cytidine triphosphate (CTP) posed a synthetic challenge for the preparation of UMP-NeuAc isotopomers by direct enzymatic means from uridine triphosphate (UTP) and labeled N-acetylneuraminic acid (NeuAc). This was overcome by use of diazotization and subsequent hydrolysis to convert radioisotopically labeled CMP-NeuAc isotopomers directly to the corresponding UMP-NeuAc isotopomers, thereby greatly facilitating analysis of kinetic isotope effects.

A new fluorescent assay for sialyltransferase

A new fluorescent assay for the sialyltransferase reaction was established. After incubation of the sialyltransferase reaction, the sialyloligosaccharide obtained was treated by acid hydrolysis, and then the NeuAc that was released was labeled with 1,2-diamino-4,5-methylenedioxibenzene. The fluorescent-labeled NeuAc could be estimated by HPLC (excitation: 373 nm; emission: 448 nm) and a Lineweaver–Burk plot could be plotted with the data from this analysis.

Control of Bisecting GlcNAc Addition to N-Linked Sugar Chains

In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between beta-1,4-GalT (UDP-galactose:N-acetylglucosamine beta-1, 4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:beta-d-mannoside beta-1, 4-N-acetylglucosaminyltransferase) was examined. We isolated a beta-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed beta-1,4-GalT-I or GnT-III. In the beta-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where beta-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the beta-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular beta-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that beta-1, 4-GalT competes with GnT-III for substrate in the cells.

化学酵素法を利用する糖タンパク質糖鎖のシアリル化反応

This article presents a new synthetic method for the preparation of cytidine-5′-monophospho-neuraminic acid analogues (CMP-NeuAc analogues) and the application of this reaction in combination with sialyltransferases. Several CMP-NeuAc analogues have been synthesized in moderate yields. The sialyltransferase reaction has further been applied to glycoprotein immobilization, in which a CMP-NeuAc was attached to a solid support at the 9″ position and the ability of this imbobilized molecule to effect a transfer reaction was confirmed by an assay using an asialoglycoprotein as the acceptor. Another aspect is a chemoenzymatic synthesis of modified sialyloligosaccharide. 9-Deoxy-9-fluoro- [3-13C] -NeuAc-α-2, 6- [U-13C] -Gal-β-1, 4-GlcNAc sequence was synthesized at the nonreducing end of an intact glycoprotein (ovalbumin) and its conformational properties were analyzed by NMR spectroscopy. The conformational properties of sialylgalactoside on this protein turned out to be similar to those of NeuAc-α-2, 6-Gal-β-1, 4-GlcNAc-β-1- O (CH2) 8COOMe.

Enzymic glycosphingolipid synthesis on polymer supports. III. Synthesis of GM3, its analog [NeuNAcalpha(2-3)Galbeta(1-4)Glcbeta(1-3)Cer] and their lyso-derivatives.

Two water-soluble polymers, carrying 0.24 meq g(-1) of lactosyl-beta(1-1)-sphingosine (7) and 0.13 meq g(-1) of lactosyl-beta(1-3)-sphingosine (8) were prepared. The polymers served as acceptors in the alpha-(2-3)-sialyltransferase reaction (up to 55.3 and 38.5% transfer yields, respectively). Subsequent photolysis, released compounds 11 (lyso-GM3) and 12 (lyso-GM3 analog), respectively; acylation and chromatography afforded (5-acetamido-3,5-dideoxy-D-glycero-alpha-galacto-2-nonulopyranosyloni c acid)-(2-3)-beta-D-galactopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-1)-(2 S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (13, GM3) and (5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylo nic acid)-(2-3)-beta-D-galactopyranosyl-(1-4)-beta-D-glucopyranosyl-(1-3)-(2 S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (14, GM3 analogue), respectively, thus presenting a route to glycosphingolipids possessing the unusual glycosyl-beta(1-3)-spingosine linkage.

Directed mutagenesis of the Trypanosoma cruzi trans-sialidase enzyme identifies two domains involved in its sialyltransferase activity.

Of the increasing number of sialidases found to be made by microorganisms, the trypanosome trans-sialidase is unique in its added ability to efficiently carry out a sialyltransferase reaction using preformed glycoconjugates. The enzyme is predicted to have a multidomain structure, with one domain containing sequence and expected structural features found in bacterial sialidases. The trans-sialidase is very similar in overall sequence to another trypanosome enzyme that has only sialidase activity. Hybrid expression constructs containing pieces of these trypanosome trans-sialidase and sialidase genes were used to determine which regions of trans-sialidase are required for sialyltransferase activity. Two domains were found to influence the enzymatic activity: the N-terminal catalytic domain, and a downstream domain that resembles an Fn3-like module.

Mechanism of polysialic acid chain elongation in Escherichia coli K1

Understanding the mechanisms of polysialic acid synthesis in Escherichia coli K1 requires a molecular description of the polymerase complex. Since the number of potential models explaining polysialic acid assembly would be constrained if only one sialyltransferase were required for this process, the phenotypes of a sialyltransferase null mutation generated by transposon mutagenesis were investigated. The chromosomal insertion mutation was mapped by Southern hybridization analysis and by complementation with plasmid subclones, demonstrating that sialyltransferase is encoded by neuS, a gene implicated previously as coding for the polymerase (Vimr et al., 1989). As expected, if only one gene encoded sialyltransferase, the null mutant had undetectable polymerase activity when assayed with endogenous or exogenous acceptors, and accumulated sugar nucleotide precursors intracellularly. Nested deletion analysis of neuS ruled out polarity effects of transposon insertion mutation and provided more precise mapping of the sialyltransferase structural gene. Maxicell analysis of the nested deletion set implicated a 34,000 molecular weight polypeptide as the neuS gene product. These results, together with biochemical characterization of sialyltransferase reaction products in the wild type, indicated that CMP-sialic acid is the probable sialosyl donor for polysialic acid elongation and that chain growth is by sequential addition of monomeric units.

Effect of acute and chronic ethanol administration on rat liver alpha 2,6-sialyltransferase activity responsible for sialylation of serum transferrin.

The effect of a single administration and a 6-week treatment with ethanol on rat liver sialyltransferase activity towards asialoglycoproteins and N-acetyllactosamine (Gal beta 1,4GlcNAc) was studied. Since only the alpha 2,6-sialyltransferase is involved in the in vivo sialylation of transferrin, Gal beta 1,4GlcNAc was chosen as an acceptor and alpha 2,6-sialyl-N-acetyllactosamine was separated from the corresponding alpha 2,3-sialyl isomer present in the sialyltransferase reaction mixture by high-performance liquid chromatography. After a single ethanol administration there was a low (about 20%) but significant (p less than 0.005) reduction of sialyltransferase activity towards asialotransferrin as well as a reduced alpha 2,6-sialyltransferase activity towards N-acetyllactosamine. An opposite result was found in the chronically ethanol-treated rats: in these animals either the total or alpha 2,6-sialyltransferase activity was slightly higher than in control animals. Blood ethanol concentration was significantly high (3.3 +/- 1.2 mg/ml) only in the acute-treated animals, suggesting that the accumulation in the body of ethanol and/or its metabolites induces a reduction of liver alpha 2,6-sialyltransferase activity responsible for the transferrin sialylation. Current results are consistent with the finding (Stibler H, Hultcrantz R: Alcohol Clin Exp Res 11:468-473, 1987) that an enhanced level of hyposialylated transferrin isoforms is a marker of present but not previous alcohol abuse.

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C18 cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar GM3-ganglioside is simple and rapid relative to previously described methods. Recovery of GM3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (GM3 synthase; ST-1), the transfer of [14C] sialic acid from CMP-[14C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.

Glycosyltransferases of the human cervical epithelium II. Characterization of a CMP- N-acetylneuraminate: Galactosyl-glycoprotein sialyltransferase

GMP- N-Acetylneuraminate: galactosyl-glycoprotein sialyltransferase (CMP- N-acetylneuraminate: d-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity was identified in the human cervical epithelium. The enzyme has a pH optimum of 6.0, a temperature optimum of 28°C, and demonstrates a partial requirement for Triton X-100. Michaelis constants for asialofetuin and CMP- N-acetyl[ 14C]neuraminic acid are 0.64 · 10 −5 M (expressed as the concentration of terminal galactose residues) and 2.05 · 10 −5 M, respectively. Sialyltransferase demonstrated minimal affinity for the low molecular weight acceptors tested, and may have a requirement for a glycoprotein acceptor having a terminal N-acetyllactosamine (Galβ(1 → 4)GlcNAc) type structure. Cytidine nucleotides are potent inhibitors of the sialyltransferase reaction; CMP acts as a competitive inhibitor.

Sialyltransferases in young rat brain.

1. There are more glycolipid acceptor sites for NeuNAc than for glycoproteins in 11--15 day old rat cerebra. 2. The glycolipid acceptors appear to be almost exclusively Cer-Glc-Gal and GM1 ganglioside and each is a substrate for a different sialyltransferase. 3. The sialyltransferase(s) that acted on glycoprotein could be differentiated from the ones that acted on the glycolipids. 4. The apparent Km for CMP-NeuNAc was the same for all four of the sialyltransferase reactions studied. 5. Electron microscopic examination and marker enzyme studies on continuous sucrose gradient fractions found that most of the sialyltransferase activities appeared to be localized in smooth microsomal membrane and the Golgi complex derivatives and not associated with the synaptosomes.