In vitro studies of skeletal muscle membranes characterization of a phosphorylated intermediate of sarcolemmal ( [formula omitted

Abstract

The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating 32P from [γ− 32P]ATP. The membrane protein phosphorylation requires Mg 2+. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation. The Mg 2+-dependent 32P labeling was significantly enhanced by Na +. The rate of Na + -stimulated 32P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K + reduced the Na + -stimulated 32P-incorporation but enhanced the 32P i release. This inhibitory effect of K + on Na + -stimulated 32P incorporation was prevented by the cardiac glycoside, ouabain. The Na + -dependent 32P labeling showed substrate dependency and the Na + site was saturable. The apparent K m for ATP was 2 · 10−5 M. The optimum pH for 32P labeling was between 7 and 8. Na + -dependent membrane phosphorylation showed a direct relationship with the (Na + + K +ATPase activity. The high turnover rate of 32P intermediate (12 000 min −1) suggested its functional significance in the overall transport ATPase reaction sequence. The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 32P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of 32P incorporation was also observed into a 25 000 dalton subunit and Ca 2+ slightly enhanced the phosphorylation of this component. The size ( M r 108 000 ) and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na + + K +ATPase preparations reported so far.