A soybean histone-type protein kinase was used to prepare 32P-labeled histone Hi as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.13.16) phorylation and dephosphorylation has been suggested to be an important mechanism in the control of metabolic processes in higher plants (12,25). Randall and his colleagues (21) have shown that phosphorylation of the pyruvate dehydrogenase complex from broccoli mitochondria results in inactivation of the enzyme. Additionally, the nuclear (17), ribosomal (20, 24) and membrane proteins (1) of higher plants are also subject to modification by phosphorylation, although the biological significance of the phosphorylation is unknown. The phosphorylation of these proteins apparently is catalyzed by several protein kinases, including histone-type and casein (or phosvitin)-type enzymes (9,12,25). Although much work has been carried out on phosphorylation of proteins and demonstration of the activities of protein kinases, little attention has been paid to phosphoprotein phosphatase (EC 3.1.3.16) which catalyzes the protein dephosphorylation reaction. There apparently has been no characterization of the phosphatase from higher plants, except a brief mention of the presence of phosphohistone phosphatase activity in pea seedlings (16). Here, we describe the purification and properties of a phosphoprotein phosphatase from soybean hypocotyls. The enzyme is distinct from the phosphatases which catalyze the hydrolysis ofnonprotein low mol wt phosphoesters. Additionally, several lines of evidence indicate that the soybean enzyme is a nonspecific phosphoprotein phosphatase acting on phosphorylated histones, protamine, and, possibly, phosphorylated casein and phosvitin. MATERIALS AND METHODSUnless otherwise stated, the chemicals and biochemical compounds used in this study were obtained from the sources as indicated in the previous paper (12). [y-32PJATP was prepared according to the method of Schendel and Wells (22). All other chemicals were of reagent grade. Soybean seeds (Glycine max var. Wayne) were germinated in the dark and the mature hypocotyls were harvested as described (12).Preparation of 32P-labeled and Nonradioactive Phosphohistone Proteins. Lysine-rich histone HI and other histone species (H2A, H2B, H3, and H4) were purified from the total calfthymus histone mixture (Sigma II or IIA) by column chromatographies on BioGel P-60 and P-10 (12, 14). A histone-type protein kinase with high specificity for histone HI was partially purified from soybean hypocotyls as described (12); the kinase was used to phosphorylate histone H1. For the preparation of 32P-labeled histone species other than histone H1, a cAMP-dependent histone kinase from rat liver cytosol (4) was used. The liver kinase was partially purified by ammonium sulfate fractionation and DEAE-Sephadex A-25 column chromatography.To prepare a large quantity of 32P-labeled histone H1, the reaction mixture in a total volume of 10 ml contained 20 mM Tris-HCI (pH 8.
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