title: Purification and characterization of a membrane-bound sialidase from pig liver.
abstract: A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.
authors.affiliation: Department of Clinical Pharmacology and Therapeutics, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: K
authors.initials: K
authors.lastname: Ikeda
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Saito
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009439:Neuraminidase