Purification and properties of swine kidney phosphoprotein phosphatase

Abstract

Phosphoprotein phosphatase was purified from swine kidney by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and Sepharose 413 columns containing covalently bound hexanediamine and polylysine. The enzyme was purified more than 20 000-fold and the homogeneous preparation had a specific activity of 2.8 μmol per min per mg of protein with saturating concentrations of ³²P-histone as the substrate. The phosphatase showed only a single protein band when examined by polyacrylamide gel electrophoresis and a single protein peak containing all of the enzymatic activity was observed during chromatography on Sephadex G-100 column. The molecular weight of the purified enzyme was determined to be 70 000±5 000 by exclusion chromatography on a calibrated Sephadex G-100 column. Similar values were obtained by sucrose density centrifugation, 70 000 ± 5000, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 70 000 ± 3 000. The purified enzyme catalyzed the dephosphorylation of the phosphorylated forms of glycogen synthase, phosphorylase, historic, phosphofructokinase, Type 11 regulatory subunit of cyclic AMP-dependent protein kinase, casein and protamine. The apparent Km values for these substrates were 3.6 μM, 2.8 μM, 66 μM, 3.3 μM, 8.0 μM, 6.6 μM and 100 μM, respectively. The enzyme did not hydrolyze low molecular weight phosphate esters such as glucose 6-phosphate, glycerol phosphate, adenosine nucleotides and inorganic pyrophosphate. The activity of the enzyme towards a phosphorylated protein substrate was competitively inhibited by the addition of other substrates. These results suggest that swine kidney contains a phosphoprotein phosphatase with a rather broad substrate specificity for a number of endogenous and exogenous phosphoprotein substrates.