Title

Studies Of The Dephosphorylation Of Phosphorylase Kinase

Date of Award

1982

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

Abstract

In this work the dephosphorylation of phosphorylase kinase by four rabbit skeletal muscle protein phosphatases was studied. The four enzymes used were protein phosphatases C-I, C-II, H-I and H-II. Phosphatases C-I, C-II and H-II were obtained as homogeneous preparations using procedures previously developed. Phosphatase H-I was purified 700-fold from rabbit skeletal muscle for the purposes of this study.Phosphatases C-I and H-I were relatively specific for removal of the (beta)-subunit phosphate of phosphorylase kinase, this occurring at rates approximately 100 times more rapidly than the removal of the (alpha)-subunit phosphate. In contrast, phosphatases C-II and H-II displayed a relative specificity for removal of the (alpha)-subunit phosphate, although this occurred at rates only 3-5 times the rate of the removal of (beta)-subunit phosphate. Studies of crude tissue extracts revealed that phosphatase H-I is the major phosphorylase phosphatase and (beta)-subunit phosphatase activity, while phosphatase H-II is a major (alpha)-subunit phosphatase activity in rabbit skeletal muscle. (alpha)-subunit phosphorylation does not affect (beta)-subunit dephosphorylation, and (beta)-subunit dephosphorylation and not (alpha)-subunit dephosphorylation is correlated with activity changes in phosphorylase kinase phosphorylated by cAMP-dependent protein kinase.An examination of the hypothesis that protein phosphatases C-I and C-II represent the catalytic subunits of protein phosphatases H-I and H-II respectively, was carried out. Protein phosphatase C-II (M(,r) = 34,000) was not a subunit of protein phosphatase H-II (subunits = M(,r) 70,000 and 35,000), but after trypsin treatment H-II was converted to a form with a subunit identical in size to phosphatase C-II. Dissociation of phosphatase H-I by various procedures did not reveal evidence for a simple subunit-holoenzyme relationship, but suggested that phosphatase C-I may be a catalytic sub-fragment of H-I since species of higher molecular weight than M(,r) = 35,000 were observed.(Portions of this work have been published (M. K. Ganapathi, S. R. Silberman, H. Paris and E. Y. C. Lee, J. Biol. Chem., 256, 3213-3217, 1981; E. Y. C. Lee, S. R. Silberman, M. K. Ganapathi, H. Paris and S. Petrovic, in Cold Spring Harbor Conferences on Cell Proliferation, Vol. 8: Protein Phosphorylation, pp. 425-439, 1981.)

Keywords

Chemistry, Biochemistry

Link to Full Text

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