Yokoyama N, Furuyama S, Wang J H
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
J Biol Chem. 1990 May 15;265(14):8170-5.
A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.
采用针对脑磷酸酶VJ6和VA1亚基特异性单克隆抗体的免疫沉淀和免疫印迹相结合的方法，对心脏、肌肉、肺、脾、胰腺、子宫和肝脏等组织进行检测。各种组织提取物用β亚基特异性VA1免疫吸附剂进行免疫沉淀，免疫沉淀物分别用α和β亚基特异性抗体VJ6和VA1进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹分析。在所有样品中均检测到对应于α和β亚基的蛋白条带以及β亚基的免疫染色，而α亚基仅在脑提取物中强烈染色，在心脏和肌肉提取物中染色较弱，在所有其他样品中基本呈阴性染色。相比之下，牛脑钙调蛋白刺激磷酸酶的多克隆抗血清可对所有组织中的α和β亚基进行免疫染色。来自多种牛组织的钙调蛋白结合蛋白组分均显示含有可免疫沉淀的α亚基以及钙调蛋白刺激的对硝基苯磷酸酶活性。微肽图谱分析表明，牛脑和牛肺钙调蛋白刺激磷酸酶同工酶的α亚基是不同的分子种类。这些结果为哺乳动物组织中存在钙调蛋白刺激磷酸酶同工酶提供了直接证据。
