Rabbit skeletal muscle protein phosphatase(s). Identity of phosphorylase and synthase phosphatase and interconversion to the ATP-Mg-dependent enzyme form.

A high molecular weight protein phosphatase was purified from rabbit skeletal muscle displaying activity toward phosphorylase Q and synthase b isolated from the same tissue. The extensively purified enzyme was shown to have a M, = 260,000 by gel filtration and dissociated in sucrose density gradient centrifugations into a M, = 70,000 species. The 70,000-dalton enzyme form, resulting from preparative sucrose density gradient centrifugations, was shown to be homogeneous on gel electrophoresis in sodium dodecyl sulfate and exhibited both phosphorylase and synthase phosphatase activities identical with the high molecular weight species. Treatment of the M, = 260,000 phosphatase with urea or 2-mercaptoethanol completely converted the enzyme to the 70,000-dalton form, with no change in either one of its activities. The same treatments performed in the presence of trypsin produced lower molecular weight forms (Mr = 32,000 to 38,000), again without activity changes. With each step of purification of the protein phosphatase, more and more inactive ATP-Mg-dependent phosphatase (Fc) (Yang, S.-D., Vandenheede, J. R., Goris, J., and Merlevede, W. (1980) J. Biol. Chem 255, 11759-11767; Vandenheede, J. R., Yang, S.-D., Goris, J., and Merlevede, W. (1980) J. Biol. Chen. 255, 1176811774; Cold Spring Harbor Conf: Cell Proliferation 8, in press) was formed, and at any stage of purity the active enzyme slowly reverted to the inactive species upon storage at 4 “C. The FA (activator protein) and ATPMg-mediated activation of this FC was reversed when the ATP-Mg was removed from the reaction mixture. However, limited proteolysis of either the reactivated enzyme or the purified active phosphatase species prevented the reversal to the inactive Fc form. Micromolar amounts of Mn2+ can produce up to 50% of the potential phosphatase activity in Fc, thus partially mimicking the FA and ATP-Mg-mediated activation. A possible mechanism of activation by FA and ATP-Mg is discussed, with ATP-Mg as the physiological metal ion donor to the inactive Fc enzyme, through a proteinprotein interaction with the activating factor FA.