Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.53 (sialidase)
 2,694 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The total pellet from pig forebrain (from which the cytosolic sialidase was completely washed out) was treated with phosphatidylinositol phospholipase C (PIPLC) and centrifuged at high speed. The supernatant contained sialidase and 5'-nucleotidase activities. The greatest liberation of sialidase was obtained after incubation for 20 min with PIPLC at 37 degrees C using pH 6.0 and a ratio between PIPLC (as units) and protein of 1.6. Under these conditions, the release of sialidase, 5'-nucleotidase, and protein was 22, 50, and 18.5%, respectively. On treatment with PIPLC, a purified preparation of pig brain neuronal (synaptosomal) membranes released 28% of its sialidase whereas a purified preparation of pig brain lysosomes did not liberate any sialidase activity. The pH optimum of sialidase present in the supernatant obtained after PIPLC treatment of the total pellet was 4.2, the same as that of the enzyme embedded in the membrane. When this supernatant was subjected to ammonium sulfate fractionation, 88% of its sialidase, having a pH optimum of 4.2, was recovered in the fraction precipitated between 20 and 45% of salt saturation and subsequently dialyzed. Ammonium sulfate treatment caused the appearance of a second sialidase activity, having a pH optimum of 6.6 and behaving on fractionation similarly to the pH 4.2 sialidase. The Km and Vmax values of pH 4.2 and pH 6.6 sialidase were similar (1.48 x 10(-4) and 0.98 x 10(-4) M for Km and 1.6 and 1.4 mU/mg of protein for Vmax, respectively), whereas the stability on standing at 4 degrees C or exposure to freezing and thawing cycles was greater for pH 4.2 sialidase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solubilization of the membrane-bound sialidase from pig brain by treatment with bacterial phosphatidylinositol phospholipase C. 221 10

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.
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PMID:Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues. 239 43

Sialidase activity was assayed in homogenized rabbit alveolar macrophages using a fluorogenic substrate: sodium 4-methylumbelliferyl-alpha-D-neuraminate. After differential centrifugation one acid-active enzyme (optimum pH 4.2) was detected in the 16,000 X g pellet that contained lysosomes, mitochondria and peroxisomes. A second activity, with an optimum pH of 5.4, was found in the cytosolic fraction. The acid-active sialidase accounted for more than 95% of the total sialidase activity in crude homogenate. When alveolar macrophages were collected from rabbits stimulated with bacillus Calmette-Guerin (BCG), the acid-active sialidase specific activity was increased 2.5-fold whereas other lysosomal enzymes such as N-acetylglucosaminidase and beta-galactosidase were stable. The cytosolic sialidase activity did not change.
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PMID:Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin. 354 51

Using 4-methylumbelliferyl-N-acetylneuraminic acid as substrate, the cytosolic fractions of various rat tissues were assayed for sialidase activity. The activity was about 16 times greater in skeletal muscle than in liver, and the enzymes from the two sources were identical in chromatographic behavior, pH optimum, and substrate specificity. Apparently the same enzyme was found to be distributed in brain, heart, stomach, intestine, and testis, but not in lung and spleen. Sialidase was also present in kidney cytosol at a high level, but the enzyme resembled liver lysosomal sialidase rather than liver cytosolic sialidase in substrate specificity.
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PMID:Cytosolic sialidases of rat tissues with special reference to skeletal muscle enzyme. 381 55

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.
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PMID:Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes. 396 41

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.
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PMID:Purification and characterization of cytosolic sialidase from rat liver. 399 46

The subcellular distribution of sialidase in rat hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene was studied by using sialyllactose as a substrate in the pH range of 4.0-7.0. As found in rat liver, the activity was recovered largely in the mitochondrial/lysosomal fraction with an optimal pH of 4.5 and in the cytosolic fraction with an optimal pH of 6.0, although hepatoma lysosomal (acidic) sialidase was also distributed in the microsomal fraction. The lysosomal and cytosolic sialidases of the hepatoma were indistinguishable from the corresponding enzymes of liver in chromatographic behavior, kinetics and substrate specificity. The levels of lysosomal and cytosolic sialidase activities in liver and hepatomas were then studied in the pellet and supernatant fractions, respectively, obtained by centrifuging the postnuclear supernatant at 105,000g for 1 hr. All the hepatomas tested, one primary and three transplanted, showed higher lysosomal sialidase and lower cytosolic sialidase activities as compared with liver. Quantitative changes similar to those seen in hepatomas were observed in regenerating liver after partial hepatectomy.
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PMID:Sialidase of rat hepatomas: qualitative and quantitative comparison with rat liver sialidase. 652 22

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, M(r), 180 kDa) was resolved into a catalytic unit (M(r) 30 kDa), active but very liable upon storage at 4 degrees C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2 x 10(-5) and 1.6 x 10(-5) M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any beta-D-galactosidase, beta-D-N- acetylglucosaminidase, alpha-L-fucosidase, alpha-D-glucosidase and carboxypeptidase activities.
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PMID:Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units. 794 53

We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.
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PMID:Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase. 825 70

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and N-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu2+ (1 mM) and thiol-modifying 4-hydroxymercuribenzoate (50 microM) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac alpha-s-(2-6)Gal beta(1-4)Glc beta(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.
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PMID:Differential effect of various inhibitors on four types of rat sialidase. 835 26


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