Abstract
A mechanism of activation of the ATP.Mg-dependent protein phosphatase (FC.M) has been proposed (Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R., and Merlevede, W. (1984) J. Biol. Chem. 259, 5864-5870) in which a transient phosphorylation by the kinase FA of the modulator subunit (M) is the driving force for the transition of the inactive catalytic subunit (FC) into its active conformation. Incubation of FC.M with kinase FA and Mg2+ and adenosine 5'-(gamma-thio)triphosphate results in thiophosphorylation of M and also a conformational change in the phosphatase catalytic subunit; however, the enzyme remains inactive. Proteolysis of this inactive, thiophosphorylated complex causes proteolytic destruction of the modulator subunit and yields an active phosphorylase phosphatase species. Similar treatment of the native inactive enzyme does not yield active phosphatase. Evidence is presented, suggesting that a molecule of modulator is bound at an "inhibitory site" on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FA and ATP.Mg but does partially inhibit the expression of the phosphorylase phosphatase activity.
Highlights
From the Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205
Evidence is presented, suggesting that a molecule of modulatoris bound at an “inhibitory site” on the native enzyme. This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FAand ATP .Mg but does partially inhibit the expression of the phosphorylase phosphatase activity
A4g-The rabbit skeletal muscle ATP Mg-dependent protein phosphatase described in Ref. 5 is referred to as the native Fc.M enzyme
Summary
From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000Leuuen, Belgium. Incubation of Fc-M with kinase FAand Mg2+ and adenosine 5’-(ythio)triphosphate results in thiophosphorylation of M and a conformational change in the phosphatase catalytic subunit; the enzyme remains inactive. Evidence is presented, suggesting that a molecule of modulatoris bound at an “inhibitory site” on the native enzyme This modulator does not prevent the conformational change in the phosphatase catalytic subunit upon incubation with kinase FAand ATP .Mg but does partially inhibit the expression of the phosphorylase phosphatase activity. In the absence of exogeneously added modulator, only 1,200 units of phosphorylase phosphatase activity were measured after activation by kinase FA and ATP.Mg. The heat-stable modulator was purified to homogeneity fromrabbit skeletal muscle according to Ref. 12, except that, in the final purification step, the modulator was eluted from the blue Sepharose CL6B column wi;h a linear gradient from 0.1 to 0.3 M NaCl in buffer A ”. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed as in Ref. ll except that 10%acrylamide gels wereused
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