Sharma R V, Bhalla R C
Am J Physiol. 1986 Jan;250(1 Pt 1):C65-75. doi: 10.1152/ajpcell.1986.250.1.C65.
A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.
通过用含有ATP和Ca2+的低离子强度缓冲液提取粗微粒体部分，已从牛颈动脉中分离出质膜部分。此步骤之后是在0.6 M KCl存在下进行蔗糖密度梯度离心。质膜囊泡中Na+-K+-三磷酸腺苷酶、5'-核苷酸酶和磷酸二酯酶I的活性提高了60至80倍。这些标记酶的最终产量为核后上清液中总活性的12 - 18%，蛋白质产量为每克颈动脉湿重100 - 120微克。分别根据琥珀酸 - 细胞色素c还原酶和NADPH - 细胞色素c还原酶的低活性判断，线粒体和肌浆网对质膜部分的污染较低。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳以及用平滑肌特异性肌动蛋白抗体进行的免疫沉淀表明，质膜部分基本没有肌球蛋白和肌动蛋白污染。质膜囊泡在ATP存在下积累Ca2+，并且钙调蛋白可增加这种积累。在有或没有钙调蛋白存在的情况下积累的Ca2+，几乎可通过添加Ca2+离子载体A23187从囊泡中完全释放，但不能通过乙二醇双（β - 氨基乙基醚） - N，N'-四乙酸释放，这表明在ATP存在下的Ca2+摄取是在囊泡内进行的。磷酸盐和草酸盐对质膜中Ca2+摄取的影响彼此不同。磷酸盐以浓度和时间依赖性方式增加Ca2+摄取，并且早在1分钟就可观察到Ca2+摄取增加。另一方面，在长达5 mM的浓度下，草酸盐在30分钟孵育期间并未显著增加Ca2+摄取。这些质膜可证明对研究离子跨质膜运输、激素结合、钙通道表征以及制备抗质膜蛋白抗体有用。
