Zubrzycka E, MacLennan D H
J Biol Chem. 1976 Dec 25;251(24):7733-8.
Temporal patterns of biosynthesis of the sarcoplasmic reticulum protein, calsequestrin, were analyzed and compared with rates of ATPase synthesis in primary cultures of rat skeletal muscle cells. Rates of synthesis were measured by the incorporation of radioactive leucine into the isolated proteins. Cells at various stages of differentiation were incubated for 2 h with tritium-labeled leucine and extracted with detergent. The extracts were incubated with antibodies specific against calsequestrin or the ATPase and immunoprecipitates were separated by disc gel electrophoresis. Incorporation of radioactivity into bands identified as calsequestrin or the ATPase was analyzed by counting of gel slices. In Dulbecco's modified Eagles medium (DME medium) containing 0.1 volume of horse serum and 0.005 volume of chick embryo extract, the cells began to fuse after about 50 h in culture, forming multinucleated myotubes. Calsequestrin synthesis was barely detectable after 24 h in culture. After 44 h, before fusion of myoblasts began, the rate of calsequestrin synthesis increased severalfold. The rate of synthesis continued to increase until about 72 h and then diminished. If cells were transferred at 44 h to DME medium containing 0.2 volume of fetal calf serum and 0.08 volume of chick embryo extract, fusion was delayed by about 20 h. In this medium the rate of calsequestrin synthesis diminished after a peak at 44 h but, by contrast, the rate of synthesis of the ATPase increased dramatically following fusion at about 80 h. If cells were transferred at about 40 h to DME medium containing 0.1 volume of horse serum and only 60 muM Ca2+ the cells did not fuse and, again, the rate of calsequestrin synthesis was diminished after a peak at about 40 h. By contrast the rate of ATPase synthesis increased sharply in spite of the lack of fusion. Both proteins were degraded with a half-life of about 20 h. These studies show that the synthesis of calsequestrin, an extrinsic membrane protein, and the ATPase, an intrinsic protein of the same membrane, are synthesized under separate control.
分析了肌浆网蛋白（钙结合蛋白）生物合成的时间模式，并将其与大鼠骨骼肌细胞原代培养物中ATP酶的合成速率进行了比较。通过将放射性亮氨酸掺入分离的蛋白质中来测量合成速率。将处于不同分化阶段的细胞与氚标记的亮氨酸孵育2小时，并用去污剂提取。提取物与针对钙结合蛋白或ATP酶的特异性抗体孵育，免疫沉淀物通过圆盘凝胶电泳分离。通过对凝胶切片计数来分析放射性掺入被鉴定为钙结合蛋白或ATP酶的条带中的情况。在含有0.1体积马血清和0.005体积鸡胚提取物的杜尔贝科改良伊格尔培养基（DME培养基）中，细胞在培养约50小时后开始融合，形成多核肌管。培养24小时后几乎检测不到钙结合蛋白的合成。44小时后，在成肌细胞开始融合之前，钙结合蛋白的合成速率增加了几倍。合成速率持续增加直到约72小时，然后下降。如果在44小时将细胞转移到含有0.2体积胎牛血清和0.08体积鸡胚提取物的DME培养基中，融合会延迟约20小时。在这种培养基中，钙结合蛋白的合成速率在44小时达到峰值后下降，但相比之下，ATP酶的合成速率在约80小时融合后急剧增加。如果在约40小时将细胞转移到含有0.1体积马血清且仅含60μM Ca2+的DME培养基中，细胞不会融合，并且同样，钙结合蛋白的合成速率在约40小时达到峰值后下降。相比之下，尽管没有融合，ATP酶的合成速率仍急剧增加。两种蛋白质的降解半衰期约为20小时。这些研究表明，外在膜蛋白钙结合蛋白和同一膜的内在蛋白ATP酶的合成是在分开的控制下进行的。
