大鼠心肌肌浆网的纯化及其性质研究

Purification and Properties of Rat Cardiac Sarcoplasmic Reticulum

用超声波破碎心肌细胞,差速离心法纯化大鼠心肌肌浆网(CSR)。SDS-聚丙烯酰胺凝胶电泳测得Ca^(2+)-ATPase分子量为98kD;电镜观察膜制备为完整的CSR微囊;标志酶哇巴因敏感型Na^(+),K^(+)-ATPase和叠氮化钠敏感型Mg^(2+)-ATPase活性表明膜制备中肌膜含量很低,但仍有线粒体污染。 用^(45)Ca^(2+)示踪微孔滤膜法研究Ca^(2+)跨膜转运,CSRCa^(2+)蓄集最大值为57nmol/mg蛋白。CSR Ca^(2+)-ATPase在4℃—21℃和21℃—49℃两区间反应活化能不同,前者大于后者。酶的最适pH为7.4。以ATP为底物,该酶有两个表观Km值:Km_1为3.7μmol/LKm_2为713μmol/L。

Cardiac sarcoplasmic reticulum membrane vesicles (CSR) were purified from rat by means of supersonic disruption of ventricular cells and differential cen-trifugation.The protein composition and molecular weights of CSR were determined by SDS-PAGE, the molecular weight of Ca2+-ATPase is 98000 dalton. The micros-tructure observed by electron microscope showed that the CRS vesicles were intact.The activity measurements of ouabain-sensitive Na+,K+-ATPase and NaN3-sensitive Mg2+-ATPase demonstrated that there was almost no contamination of sarcolemma,but some of mitochondria.Ca2+ transport was determined by Millipore filter technique with 45Ca2+. The maximum Ca2+ content of Ca2+ sequestration was 57 nmol/mg protein;in the presence of 5 mmol/L oxalate,Ca2+ uptake was 3.0 μmol/mg protein (at 30 min),which was only about one third of SSR Ca2+ uptake (7.8 μmol/mg protein) . Ca2+-ATPase activity was determined by a coupled enzyme reaction.The activity of Ca2+-ATPase was found to be dependent on temperature, the reaction energy between 4℃ and 21℃ was found to be larger than that between 21℃ and 49℃. The optimum pH was 7.4.The enzyme had two apparent Km constants. Km1 = 3.7 μmol/L and Km2 = 713 μmol/L (withATP as substrate) .