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Articles

Phosphoprotein Phosphatase of Soybean Hypocotyls

PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES ¹, ^,2

Tokunori Mori³ and Joe L. Key

Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546, Department of Biochemistry, University of Georgia, Athens, Georgia 30601, Department of Botany, University of Georgia, Athens, Georgia 30601

A soybean histone-type protein kinase was used to prepare ³²P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.

The amounts of ³²P released from [³²P]histone H1 are proportionally recovered as [³²P]Pi, indicating that the dephosphorylation is due to the action of phosphoprotein phosphatase. The enzyme shows maximal activity at pH 7 to 8 and has a specific activity of 19 nanomoles of [³²P]Pi released from [³²P]histone H1 per minute per milligram of protein. The apparent K_m for phosphohistone H1 is 4.0 ± 0.4 micromolar. The estimated molecular weight of the enzyme is approximately 30,000 by gel filtration. The enzyme activity does not depend upon the addition of reducing agent and metal ion. Zn²⁺, Co²⁺, NaF, pyrophosphate, or ATP at 1 millimolar, however, inhibits the enzyme activity by about 70%. The enzyme activity is unaffected by cyclic nucleotides and plant growth substances but is inhibited by polyamines. All the phosphorylated histone species and protamine, not low molecular weight phosphoesters, act as competitive inhibitors for the dephosphorylation of [³²P]histone H1.

Besides its action on phosphohistone H1, the soybean enzyme also catalyzes the dephosphorylation of other phosphohistone species (H2A, H2B, H3, and H4), degraded phosphohistone H1, and possibly phosphorylated casein and phosvitin. All these results indicate that the enzyme is a nonspecific phosphoprotein phosphatase.

⁴ To whom correspondence should be addressed.

¹ This work was supported in part by a fund from the University of Kentucky Faculty Research Committee (to P. P. C. L.), by United States Public Health Service Grant CA 11624 from the National Cancer Institute (to J. L. K.) and by a grant from the Government Forest Experiment Station of Japan (to T. M.).

² This paper (No. 80-3-139) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.

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