Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: I. ISOLATION AND CHARACTERIZATION

Abstract

Abstract An orthophosphate-repressible extracellular alkaline phosphatase has been isolated from cultures of Micrococcus sodonensis. The enzyme is pure as judged by molecular sieve chromatography, disc gel electrophoresis, and ultracentrifugation, and possesses the following properties: (a) molecular weight, 80,000; (b) pH optimum, pH 9 to 9.5; (c) competitive inhibitors, orthophosphate, arsenite, and arsenate; (d) substrate specificity, hydrolyzes a wide variety of phosphorylated compounds including phosphomonoesters, nucleotide mono-, di-, and triphosphates, and inorganic pyrophosphate. Enzymatic activity is dependent upon the presence of free sulfhydryl group(s); derivatization of the enzyme with 1 eq of p-chloromercuribenzoate results in the loss of approximately 75% of its catalytic activity. Activity of the enzyme exhibits an absolute requirement for divalent cation with the following relative reactivations of ethylenediaminetetraacetate-inactivated enzyme: Ca++, 90; Mn++, 22; Co++, 15; Sr++, 13; and Cu++, Mg++, Zn++, l1. The Km for Ca++ was estimated to be 1.5 x 10-5 m and the Kdissociation = 1.5 x 10-5 m as determined by equilibrium dialysis. Assuming a molecular weight of 80,000, the Ca++ content of the enzyme was estimated to be 8 g atoms per mole. Removal of Ca++ from the enzyme resulted in a marked increase in susceptibility of the enzyme to inactivation by heat and proteolytic digestion.