The proteins of rabbit skeletal muscle sarcoplasmic reticulum

The proteins of the sarcoplasmic reticulum isolated from rabbit skeletal muscle by the method of Martonosi [J. Biol. Chem. (1968) 243, 71] have been shown by electrophoresis and other physical methods to be still aggregated when solubilized in either Triton X-100 or in water by the method of prior solubilization in 80% phenol. Total solubilization was achieved in sodium dodecyl sulfate and electrophoretic analysis in the presence of detergent showed the presence of a major protein A with a molecular weight close to 100,000. The incorporation of ³²P into this protein in the sarcoplasmic reticulum membranes indicated that it contained the Ca²⁺-dependent ATPase. The amino acid composition of protein A isolated by electrophoresis was identical to that of the whole sarcoplasmic reticulum protein. Analysis of solubilized sarcoplasmic reticulum or of protein A by electrophoresis in phenol:urea:acetic acid acrylamide gels or of reduced and carboxymethylated protein A in SDS-containing gels showed that it comprised two proteins, A1 and A2, both with molecular weights close to 100,000. Reduction of the two or three disulfide bonds in A1 and A2 did not affect their molecular weight. Cross-linking of protein A within the membrane was achieved with dimethylsuberimidate suggesting that a sizeable fraction of these protein molecules lie in very close proximity to each other in the sarcoplasmic reticulum. The carbohydrate content of the sarcoplasmic reticulum is also reported.