Nuclear phosphoprotein phosphatase from calf liver

Abstract

Calf liver nuclear phosphoprotein phosphate (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been purified approx. 850-fold. The enzyme has a mol. wt. of 34 000 as determined by SDS-polyacrylamide gel electrophoresis. The purified enzyme has a pH optimum between 7.0 and 7.5 with phosphosphorylase, phosphohistones f 1 and f 2b, and phosphoprotamine as substrates. The enzyme activity towards these substrates follows the order, phosphophosphorylase > phosphohistone f 1 > phosphohistone f 2b > phopshoprotamine. The K m values toward phosphophosphorylase and phosphohistone f 1 are 17 and 28 μM phosphate, respectively. Dephosphorylated histone f 1 and orthophosphate are competitive inhibitors of the enzyme with respective K i values of 11 μM and 4.1. mM. NaCl and divalent metal ions inhibit the enzyme but CaCl 2 is slightly stimulatory. It appears that metal ion inhibition occurs at two sites, one on the enzyme and the other on the substrate. The enzyme is also inhibited by NaF and EDTA. Nucleotides bearing the pyrophosphate structure are potent inhibitors of the enzyme while mononucleotides are slightly inhibitory. DNA and other polyions also inhibit the enzyme. The enzyme appears to require free sulfhydryl groups for activity since it is inhibited by N- ethylmaleimide and p- hydroxymercuribenzoate ; the latter inhibition can be reversed by mercaptoethanol and dithiothreitol.