CA2+ REGULATION OF SARCOPLASMIC RETICULAR PROTEIN PHOSPHATASE-ACTIVITY

Isolated rabbit skeletal muscle sarcoplasmic reticulum contains Ca2+-dependent protein kinase and protein phosphatase activities which may regulate the Ca2+ transport ATPase [Hörl, W. H., & Heilmeyer, L. M. G., Jr. (1978) Biochemistry 17, 766-772; Hörl, W. H., Jennissen, H. P., & Heilmeyer, L. M. G„ Jr. (1978) Biochemistry 17, 759-766]. Addition of ATP-Mg2+ to the concentrated suspension of sarcoplasmic reticulum in the presence of 10μM Ca2+ inhibits the endogenous protein phosphatase activity to 74-90%. This inhibition can be reversed by reducing the free Ca2+ concentration with ethylene glycol bis(2-aminoethyl ether)-N,-N.N-tetraacetic acid (EGTA) or by high dilution of the membrane suspension. Kinetically, 10 μM Ca2+ increases the Km' for troponin about 10-fold without any change in Vmax. Dilution of the membranes in the presence of Ca2+ (10μM) leads to an approximately sevenfold decrease in the Km' for phosphorylase a and an approximately fourfold increase in Kmax; in the absence of Ca2+ (10 nM) no change in Km is observed and the VmiX increases approximately two- to threefold. Membranes without the ATP-Mg2+ step during preparation do not show the Ca2+-induced protein phosphatase inhibition; however, after preincubation with the catalytic subunit of the cyclic AMP dependent protein kinase and ATP-Mg2+, the Ca2+-dependent inhibition of the endogenous protein phosphatase reappears. Centrifugation experiments show that the association of the protein phosphatase to the membranes is Ca2+ and protein concentration dependent. In the presence of 10 μM Ca2+ and at a protein concentration higher than 5 mg/mL, approximately half the amount of phosphatase activity remains in the supernatant in comparison to 10 nM Ca2+. © 1979, American Chemical Society. All rights reserved.