Levitsky D O, Aliev M K, Kuzmin A V, Levchenko T S, Smirnov V N, Chazov E I
Biochim Biophys Acta. 1976 Sep 7;443(3):468-84. doi: 10.1016/0005-2736(76)90466-1.
The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca2+-dependent ATPase and are also able to accumulate up to 10 mumol Ca2+ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a3 and an increase in the content of [32P]phosphoenzyme. The basic components in "calcium-oxalate preparation" from hearts are proteins with molecular weights of about 100000 (Ca2+-dependent ATPase) and 55000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca2+-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12-16 mumol Pi/min per mg protein. Enzyme activity of purified Ca2+-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca2+-dependent ATPases isolated from heart and skeletal muscles.
本文描述了从鸽子和狗的心脏中分离高活性肌浆网组分的方法。该方法基于用钙离子和草酸根离子部分加载心脏微粒体，并在蔗糖和氯化钾浓度梯度中沉淀加载的囊泡。所获得的制剂具有高活性的Ca2+依赖性ATP酶，并且每毫克蛋白质还能够积累高达10微摩尔的Ca2+。肌浆网膜的纯化伴随着细胞色素a+a3浓度的降低和[32P]磷酸酶含量的增加。心脏“草酸钙制剂”中的基本成分是分子量约为100000（Ca2+依赖性ATP酶）和55000的蛋白质。鸽子心脏的草酸钙制剂用于随后纯化Ca2+依赖性ATP酶。来自鸽子心脏的纯化酶的比活性为每毫克蛋白质12-16微摩尔无机磷/分钟。纯化的Ca2+依赖性ATP酶的酶活性受到EGTA的抑制，并且对叠氮化物、2,4-二硝基苯酚和哇巴因不敏感。所获得的数据证明了从心脏和骨骼肌中分离的钙泵系统和Ca2+依赖性ATP酶的相似性。
