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JBC, Vol. 251, Issue 7, 2030-2036, Apr, 1976

Assembly of the sarcoplasmic reticulum. Biosynthesis of the adenosine triphosphatase in rat skeletal muscle cell culture

P. C. Holland and D. H. MacLennan

Temporal patterns of biosynthesis of the Ca2+ + Mg2+-dependent adenosine triphosphatase of sarcomplasmic reticulum were obtained from studies with primary cultures of rat skeletal muscle cells. Rates of synthesis at various stages of differentiation were estimated from the incorporation of tritium-labeled leucine into the ATPase. Cells were solubilized with detergent, and newly synthesized ATPase was isolated from cells by antibody precipitation in the presence of carrier ATPase. Radioactivity incorporated into the ATPase was determined after gel electrophoresis of the precipitates and counting of gel slices containing the ATPase band. In Dulbecco's modified Eagle's medium containing 10% horse serum and 0.5% chick embryo extract, mononucleated myoblast cells began to form multinucleated myotubes after about 50 hours in culture. Prior to fusion little ATPase synthesis was detectable; during fusion the ATPase was synthesized at an accelerating rate for a period of about 30 hours. The rate of synthesis levelled off after about 90 hours coincident with termination of fusion. In Dulbecco's modified Eagle's medium containing 20% fetal calf serum and 8% embryo extract, the onset of fusion was delayed for 30 to 40 hours. In this medium biosynthesis of the ATPase was also delayed so that biosynthesis of the ATPase appeared to be correlated with fusion of muscle cells. Cells cultured in Culbecco's modified Eagle's medium containgin 10% horse serum, but only 60 muM Ca2+, proliferated but did not fuse. Under these conditions, synthesis of the ATPase was measurable at 50 to 60 hours, and the rate of synthesis accelerated until 120 hours when it declined. Under all conditions degradation of the ATPase occurred with a half-life of 20 hours whereas the half-life of total protein degradation was 40 hours. Synthesis of the sarcoplasmic reticulum ATPase, like that of a number of other muscle-specific proteins, is greatly accelerated as myoblasts fuse and differentiate into myotubes. Fusion is not essential for this phenomenon, however, although it is normally concomitant with it.
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