title: Kinetic studies on the membrane form of intestinal alkaline phosphatase.
abstract: We have purified different membrane and soluble forms of alkaline phosphatase from human placenta and bovine intestine. The enzymes will be used as markers in immunoconjugates and/or as model for membrane enzyme studies. The membrane form of alkaline phosphatase extracted from bovine intestine was purified on Q-Sepharose and on L-histidyldiazobenzyl-phosphonic acid-agarose columns to remove phosphodiesterase activity. The purified enzyme had a molecular mass of 61 kDa, Km of 1208 microM, and Vmax 240 mumol pNP/min when assayed in 1 M diethanolamine, 0.5 mM MgCl2 buffer, pH 9.8, containing 10 to 2250 microM of pNPP at 37 degrees C. In the present investigation we studied the effect of salts and inositol derivatives on this enzyme activity, which was found to depend on 0.5 mM Mg2+, and to be fully inhibited by 1.2 mM Hg2+. Vanadate (0.5 mM) and Zn2+ (0.5 mM) reduced the Km value by 43% and 84%, respectively. Inositol (2 mM) and inositol-2-monophosphate (2 mM) reduced the activity by 23% and 17%. Inositol-1-monophosphate (0.5 mM) and cyclic-inositol-(1:2)-monophosphate (0.5 mM) enhanced their Km value by at least 30% compared to p-nitrophenylphosphate.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, São Paulo, Brasil.
authors.forename: M H
authors.initials: MH
authors.lastname: Costa
authors.forename: A C
authors.initials: AC
authors.lastname: Arisi
authors.forename: C R
authors.initials: CR
authors.lastname: Matos
authors.forename: L V
authors.initials: LV
authors.lastname: Silva
authors.forename: I
authors.initials: I
authors.lastname: Raw
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D000469:Alkaline Phosphatase