title: Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus.
abstract: Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.
authors.affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University.
authors.forename: H
authors.initials: H
authors.lastname: Itami
authors.forename: Y
authors.initials: Y
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Shimamoto
authors.forename: H
authors.initials: H
authors.lastname: Hama
authors.forename: M
authors.initials: M
authors.lastname: Tsuda
authors.forename: T
authors.initials: T
authors.lastname: Tsuchiya
mesh_terms: D015720:5'-Nucleotidase; D000838:Anions / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008566:Membranes / Q000201:enzymology; D009708:Nucleotidases / Q000032:analysis / Q000302:isolation & purification*; D010743:Phospholipids / Q000494:pharmacology; D013268:Stimulation, Chemical; D014736:Vibrio parahaemolyticus / Q000201:enzymology*
chemical_list: D000838:Anions; D002412:Cations; D010743:Phospholipids; D009708:Nucleotidases; D015720:5'-Nucleotidase