Characterization of Cardiac Sarcoplasmic Reticulum ATP‐ADP Phosphate Exchange and Phosphorylation of the Calcium Transport Adenosine Triphosphatase

Abstract

The terminal phosphate of (.gamma.-32P)ATP was rapidly incorporated into [dog] cardiac sarcoplasmic reticulum membranes (0.7-1.3 .mu.mol/g protein) in the presence of Ca and Mg. Cardiac sarcoplasmic reticulum membranes catalized an ATP-ADP phosphate exchange in the presence of Ca and Mg. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange was reached at an ionized Ca concentration of about 0.3 .mu.M. The Hill coefficients were 1.3. Transphosphorylation and ATP-ADP phosphate exchange required Mg and were maximally activated at Mg concentrations close to or equal to the ATP concentration. The phosphoprotein level was reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of Ca-dependent ATP splitting declined, while the rate of the Ca-dependent ATP-ADP phosphate exchange increased when the ADP/ATP ratio was varied from 0.1-1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remained constant. Phosphoprotein formation and ATP-ADP phosphate exchange were not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and ouabain, while both activities were reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. The isolated phosphoprotein was acid stable. The trichloroacetic acid denatured 32P-labeled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. Polyacrylamide gel electrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrated that the 32P-incorporation occurs into a protein of about 100,000 MW. The phosphoprotein represents a phosphorylated intermediate of the Ca-dependent ATPase which formation occurs as an early step in the reaction sequence of Ca-translocation by cardiac sarcoplasmic reticulum similar to that in skeletal muscle.