Wientzek M, Katz S
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
J Mol Cell Cardiol. 1991 Oct;23(10):1149-63. doi: 10.1016/0022-2828(91)90204-y.
We have demonstrated for the first time the isolation of sarcoplasmic reticulum (SR) membranes from adult rat ventricular myocytes obtained from a single rat heart. The myocyte SR preparation exhibits similar Ca(2+)-transport and Ca2+/K(+)-ATPase activity as well as a similar protein profile to SR membranes isolated from intact rat heart tissue. This SR preparation exhibited a Ca2+/K(+)-ATPase activity of 371 +/- 55 nmol/min/mg protein (mean +/- S.E.M.; n = 5) and an oxalate-stimulated Ca(2+)-uptake activity of 103 +/- 4 nmol/min/mg protein (mean +/- S.E.M.; n = 6). Pretreatment of the SR vesicles with 5 microM ruthenium red increased the oxalate-stimulated Ca(2+)-uptake to 204 +/- 12 nmol/min/mg protein demonstrating the presence of junctional SR membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that the isolated SR membranes contained protein bands at 430 (Ca(2+)-release channel), 100 (Ca2+/K(+)-ATPase), 55 (calsequestrin and/or calreticulin) and 53 kDa (glycoprotein). Western blots of myocyte SR membranes stained with ruthenium red detected 2 major Ca(2+)-binding protein bands in this preparation at 53-55 kDa (calsequestrin and/or calreticulin) and 97-100 kDa (Ca2+/K(+)-ATPase). The presence of phospholamban, a regulatory protein of the Ca2+/K(+)-ATPase of cardiac SR, was confirmed in the myocyte SR membranes by western blots probed with a monoclonal antibody to phospholamban. Isoproterenol stimulation of intact [32P]orthophosphate equilibriated myocytes was associated with an increase in the phosphorylation of 3 distinct proteins (27, 31 and 152 kDa) in myocyte homogenates. The 27 kDa phosphorylated protein was identified in purified SR membranes as phospholamban my migration on electrophoretic gels and by immunoblotting. The ability to prepare SR membranes from intact isolated adult rat ventricular myocytes makes this system a potentially useful model for the study of SR regulation by protein phosphorylation.
我们首次展示了从取自单只大鼠心脏的成年大鼠心室肌细胞中分离肌浆网（SR）膜。该肌细胞SR制剂表现出与从完整大鼠心脏组织分离的SR膜相似的Ca(2+)转运和Ca2+/K(+) - ATP酶活性以及相似的蛋白质谱。这种SR制剂表现出Ca2+/K(+) - ATP酶活性为371±55 nmol/分钟/毫克蛋白质（平均值±标准误；n = 5），草酸盐刺激的Ca(2+)摄取活性为103±4 nmol/分钟/毫克蛋白质（平均值±标准误；n = 6）。用5 microM钌红预处理SR囊泡可将草酸盐刺激的Ca(2+)摄取增加到204±12 nmol/分钟/毫克蛋白质，证明存在连接性SR膜。十二烷基硫酸钠聚丙烯酰胺凝胶电泳表明，分离的SR膜在430（Ca(2+)释放通道）、100（Ca2+/K(+) - ATP酶）、55（肌集钙蛋白和/或钙网蛋白）和53 kDa（糖蛋白）处含有蛋白条带。用钌红染色的肌细胞SR膜的蛋白质免疫印迹在该制剂中检测到两个主要的Ca(2+)结合蛋白条带，分别在53 - 55 kDa（肌集钙蛋白和/或钙网蛋白）和97 - 100 kDa（Ca2+/K(+) - ATP酶）。通过用抗磷酸受纳蛋白单克隆抗体探测的蛋白质免疫印迹在肌细胞SR膜中证实了磷酸受纳蛋白（心脏SR的Ca2+/K(+) - ATP酶的调节蛋白）的存在。异丙肾上腺素刺激完整的[32P]正磷酸盐平衡的肌细胞与肌细胞匀浆中3种不同蛋白质（27、31和152 kDa）的磷酸化增加有关。通过其在电泳凝胶上的迁移和免疫印迹，在纯化的SR膜中鉴定出27 kDa的磷酸化蛋白为磷酸受纳蛋白。从完整分离的成年大鼠心室肌细胞制备SR膜的能力使该系统成为研究蛋白质磷酸化对SR调节的潜在有用模型。
