Lapetina E G, Watson S P, Cuatrecasas P
Proc Natl Acad Sci U S A. 1984 Dec;81(23):7431-5. doi: 10.1073/pnas.81.23.7431.
In an attempt to establish a system with physiological substrates and phospholipid surfaces to investigate Ca2+- and 1,2-diacylglycerol-dependent protein kinase C activation, saponized platelets were used. Saponin, through interaction with plasma membrane cholesterol, makes cells permeable without major disruption of organelles. Washed platelets, prelabeled with 32P, were treated with 1-50 micrograms of saponin per ml. Permeabilization was evident at a concentration of 10 micrograms of saponin per ml, as indicated by the action of extracellular Ca2+ on the phosphorylation of the 20,000- and 40,000-Da proteins. These proteins are, respectively, the substrates for myosin light chain kinase and protein kinase C. Activation of these enzymes occurred when the estimated free [Ca2+] was changed from approximately equal to 80 nM to 300 nM. The effect of Ca2+ on kinase C-induced phosphorylation was potentiated by 1,2-didecanoylglycerol (1 microM). myo-Inositol 1,4,5-trisphosphate (5-20 microM) increased phosphorylation of the 20,000- and 40,000-Da proteins. This action was time and concentration dependent. The effect of myo-inositol 1,4,5-trisphosphate on the activation of kinase C was additive with 1,2-didecanoylglycerol. The action of myo-inositol 1,4,5-trisphosphate could be due to mobilization of Ca2+ from platelet organelles and/or to a direct effect on protein kinases.
为了建立一个具有生理底物和磷脂表面的系统来研究钙离子和1,2 - 二酰甘油依赖性蛋白激酶C的激活，使用了皂化血小板。皂角苷通过与质膜胆固醇相互作用，使细胞具有通透性，而不会对细胞器造成重大破坏。用32P预标记的洗涤血小板，每毫升用1 - 50微克皂角苷处理。每毫升10微克皂角苷的浓度下通透性明显，这由细胞外钙离子对20,000和40,000道尔顿蛋白质磷酸化的作用表明。这些蛋白质分别是肌球蛋白轻链激酶和蛋白激酶C的底物。当估计的游离[Ca2+]从约80 nM变为300 nM时，这些酶被激活。1,2 - 二癸酰甘油（1 microM）增强了钙离子对激酶C诱导的磷酸化的作用。肌醇1,4,5 - 三磷酸（5 - 20 microM）增加了20,000和40,000道尔顿蛋白质的磷酸化。这种作用具有时间和浓度依赖性。肌醇1,4,5 - 三磷酸对激酶C激活的作用与1,2 - 二癸酰甘油具有加和性。肌醇1,4,5 - 三磷酸的作用可能是由于从血小板细胞器中动员钙离子和/或对蛋白激酶的直接作用。
