Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.
The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology / Q000648:ultrastructure; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis*; D009132:Muscles / Q000201:enzymology / Q000648:ultrastructure*; D010740:Phospholipases / Q000494:pharmacology; D010743:Phospholipids / Q000032:analysis; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000648:ultrastructure*
D009124:Muscle Proteins; D010743:Phospholipids; D010740:Phospholipases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium