Natsukari N, Uezato T, Ohta H, Fujita M
National Institute for Physiological Sciences, Division of Active Transport, Okazaki, Japan.
Biochim Biophys Acta. 1992 Jan 13;1133(2):193-205. doi: 10.1016/0167-4889(92)90069-n.
Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.
通过半定量免疫印迹分析（Natsukari, N., Ohta, H. 和 Fujita, M. (1989) J. Immunol. Methods 125, 159 - 166）估计，在经乙二醇双四乙酸（EGTA）洗涤的大脑皮质突触体膜（SM）制剂中，内源性钙调蛋白（CaM）含量低于3微克/毫升蛋白质。在经EGTA洗涤、未处理（对照）和Ca(2+)处理的大脑皮质突触体膜（SM）以及添加了CaM的富集SM中，通过免疫电子显微镜证实了膜结合CaM的存在。CaM的密度按上述顺序增加。在所有测试的Ca2+浓度下，CaM依赖性腺苷酸环化酶和CaM依赖性蛋白激酶II（CaM - 激酶II）的活性得以恢复，而磷酸二酯酶（PDE）的活性不受外源性CaM的影响。在pCa 6.2时，腺苷酸环化酶要么被GTP和CaM协同激活，要么被CaM和β - 肾上腺素能激动剂（±） - 异丙肾上腺素协同激活，这反映了SM中信号转导途径的完整性。同时还证实了SM中存在蛋白激酶A、CaM - 激酶II及其内源性底物。基于32P放射自显影和125I - CaM覆盖数据，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）上鉴定出了某些CaM结合蛋白，如CaM - 激酶II和突触素I。通过有或无Ca2+的125I - CaM凝胶覆盖区分了Ca(2+)依赖性和非依赖性CaM结合蛋白（CaMBPs）。前者的分子大小（大于或等于49 kDa）比后者（小于或等于34 kDa）大。在制备SM的过程中，Ca(2+)依赖性CaMBPs的产量不受Ca2+浓度的影响，而Ca(2+)非依赖性CaMBPs的产量因暴露于100微摩尔Ca2+而降低。与脑SM的CaMBPs相比，肠上皮细胞和红细胞质膜的CaMBPs，尤其是肠上皮细胞的微绒毛膜，显示出截然不同的CaMBP谱。目前的研究结果表明，经EGTA洗涤的SM制剂为研究CaM在脑SM中的作用提供了一个有用的系统。
