Components of purified sarcolemma from porcine skeletal muscle

Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabainsensitive p-nitrophenylphosphatase, [³H]ouabain binding, and (Na⁺ + K⁺)-ATPase activities indicated that 65-76% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both cAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca²⁺-ATPase activity (0.13 μmol P_i mg^-1 min^-1 at 37 °C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a M_r 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, N_i. An endogenous ADP-ribosyl-transferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous cAMP-dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate K _{1 2} for Ca²⁺ of 0.5 μm, and an approximate K _{1 2} for calmodulin of 0.1 μm. ¹²⁵I-Calmodulin affinity labeling of the sarcolemma, using dithiobis(succinimidyl propionate), demonstrated the presence of M_r 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinasecatalyzed phosphorylation.