Rat-liver lysosomal sialidase. Solubilization, substrate specificity and comparison with the cytosolic sialidase

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity. The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60 000 +/- 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides , those containing (alpha 2----3)sialyl linkages being better substrates than those with (alpha 2----6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin. In contrast to the intralysosomal sialidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60 000. It also hydrolyzed gangliosides but not submaxillary mucin.     

Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates.

A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).     

Human placental sialidase: partial purification and characterization

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Purification and characterization of cytosolic sialidase from rat liver

Sialidase has been purified from rat liver cytosol 83,000-fold by sequential chromatography on DEAE-cellulose, CM-cellulose, Blue-Sepharose, Sephadex G-200, and heparin-Sepharose. When subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the purified cytosolic sialidase moved as a single protein band with Mr = 43,000, a value similar to that obtained by sucrose density gradient centrifugation. The purified enzyme was active toward all of the sialooligosaccharides, sialoglycoproteins, and gangliosides tested except for submaxillary mucins and GM1 and GM2 gangliosides. Those substrates possessing alpha 2----3 sialyl linkage were hydrolyzed much faster than those with alpha 2----6 or alpha 2----8 linkage. The optimum pH was 6.5 for sialyllactose and 6.0 for orosomucoid and mixed brain gangliosides. The activity toward sialyllactose was lost progressively with the progress of purification but restored by addition of proteins such as bovine serum albumin. In contrast, neither reduction by purification nor restoration by albumin was observed for the activity toward orosomucoid. When mixed gangliosides were the substrate, bile acids were required for activity and this requirement became almost absolute after the enzyme had been purified extensively. Intracellular distribution study showed that about 15% of the neutral sialidase activity was in the microsomes. The enzyme could be released by 0.5 M NaCl; the released enzyme was indistinguishable from the cytosolic sialidase in properties.     

Studies on plasma membranes

Summary Plasma membranes and lysosomes were isolated from rat liver and assayed for sialidase activity with gangliosides and sialyllactose as substrates. Plasma membrane and lysosomal activities differed in substrate preference, heat stability, inhibition by Cu²⁺,K _m values and pH dependence. It is concluded that plasma membranes and lysosomes contain different sialidases. Hepatoma plasma membranes also exhibited sialidase activity towards the two substrates.     

Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules

Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain)

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.     

Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli

Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.     

Neuraminidase deficiency in the cherry red spot-myoclonus syndrome

Two patients with the cherry red spot-myoclonus syndrome excreted excessive quantities of sialylated oligosaccharides in urine. Skin fibroblasts from both patients had a severe deficiency of neuraminidase activity using α-L-N-acetylneuraminosyl-(2→6′) lactose and a sialylhexasaccharide from their urine as substrates. A less severe deficiency was found using 2-(3′ methoxyphenyl)-N-acetylneuraminic acid, fetuin, and α-L-N-acetylneuraminosyl-(2→3′) lactose as substrates. I propose that the primary enzyme defect in this autosomal recessive disorder is a deficiency of lysosomal neuraminidase.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.     

Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside

A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver.

The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.     

Enzymatic properties of sialidase from the ovary of the starfish, Asterina pectinifera

A sialidase [EC 3.2.1 18] was isolated and highly purified from the ovary of the starfish, Asterina pectinifera, and its enzymatic properties were compared with those of human placental sialidase. The final preparation gave one broad protein band corresponding to sialidase activity on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 360 000 by HPLC on Sigma Chrome GFC-1300 and Sephadex G-150 column chromatography, and 55 000 by SDS-PAGE, suggesting the presence of a hexamer in the native protein. The optimum pH was between 3.0 and 4.0, and the enzyme liberated sialyl residues from the following compounds: α(2-3) and α(2-6) sialyllactose, colominic acid, fetuin, transferrin, gangliosides GM₃, GD_1a and GD_1b. The enzyme was strongly inhibited by 4-aminophenyl and methyl thio-glycosides of sialic acid, but not by those glycosides of 5-amino sialic acid or sialic acid methyl ester. The enzyme was also highly inhibited by sulfated glucan and glycosaminoglycans. The substrate specificity and the effects of inhibitors on starfish sialidase were very similar to those of human placental sialidase.     

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.     

Enzymatic properties of neuraminidases from Arthrobacter ureafaciens.

Neuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51,000 and 39,000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneuraminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53 degrees C, were stable between pH 6.0 and 9.0, and were thermostable up to 50 degrees C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid of Hg2+. Both neuraminidases I and II were able to hydrolyze the alpha-ketosidic linkage of N-glycolylneuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate substantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid. Remarkable differences were observed between neuraminidases I and II as regards substrate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neuraminidase II 143-fold. Although neuraminidases I and II were able to hydrolyze (alpha,2-3), (alpha,2-6), and (alpha,2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-alpha,2-6-lactose was greater than that of the alpha,2-3-isomer.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of GM2 synthase, UDP-N-acetylgalactosamine: GM3 beta-N-acetylgalactosaminyltransferase from rat liver

A UDP-N-acetylgalactosamine:ganglioside GM3 beta-N-acetylgalactosaminyltransferase which catalyzes the conversion of ganglioside GM3 to GM2 has been purified over 6300-fold from a Triton X-100 extract of rat liver particulate fractions by hydrophobic chromatography and affinity chromatography on GM3-acid-Sepharose. The purified enzyme has two identical subunits of 64,000 daltons. The enzyme has a pH optimum of pH 6.7-6.9 and requires divalent cations such as Mn2+ and Ni2+. In studies on substrate specificity GM3 containing N-acetylneuraminic acid (GM3(NeuAc] and GM3 containing N-glycolylneuraminic acid were both good acceptors for the purified enzyme. The plots of the activity of transferase as a function of GM3(NeuAc) showed sigmoidal relationships. The oligosaccharide of GM3, sialyllactose, was also a good acceptor, which indicates that the preferred acceptor substrate has the possible structure NeuAc alpha 2- or NeuGc alpha 2-3 Gal beta 1-4Glc-OR.