Pelech S L, Samiei M, Charest D L, Howard S L, Salari H
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1991 May 15;266(14):8696-705.
The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.
在兔血小板短期暴露于饱和剂量的12 - O - 十四酰佛波醇13 - 乙酸酯（TPA）后，研究了蛋白激酶C（PKC）的亚细胞分布、大小和激活状态。从经TPA处理的血小板制备的胞质和Nonidet P - 40可溶颗粒提取物在Mono Q、羟基磷灰石和Superose 6/12上进行分析柱色谱。根据该酶在激活剂钙、二酰甘油和磷脂酰丝氨酸存在下磷酸化（i）组蛋白H1的能力；（ii）用胰蛋白酶对PKC进行蛋白水解激活后磷酸化组蛋白H1的能力；以及（iii）在无钙和脂质存在下磷酸化鱼精蛋白的能力来测定PKC活性。用所有三种方法评估，在血小板用TPA处理1分钟内，超过95%的PKC活性与颗粒相关。来自1分钟TPA处理细胞的颗粒PKC活性在Mono Q上用约0.35 M NaCl洗脱（峰I），并且其对Ca2 +和脂质高度依赖以实现最佳的组蛋白H1磷酸化。随着血小板与TPA接触时间延长，PKC的Mono Q峰I形式的消失与新的PKC种类的产生相关，这些新种类分别用约0.4 M NaCl（峰II）、约0.5 M NaCl（峰III）和约0.6 M NaCl（峰IV）从Mono Q上洗脱。PKC的这些最后形式仍然被脂质激活，但对Ca2 +的依赖性很小。Mono Q峰III形式在无脂质和Ca2 +时表现出特别高的组蛋白H1磷酸化活性水平。在Superose 6/12上所有这些形式表现为约65 kDa的蛋白质，但在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上，用抗PKC - β抗体进行蛋白质印迹显示约79 kDa（Mono Q峰I、II和IV）和约100 kDa（Mono Q峰III）的免疫反应性多肽。羟基磷灰石柱色谱允许部分分离Mono Q峰I和II形式，它们在典型的PKCβ同工酶的磷酸钾浓度范围（100 - 150 mM）内洗脱。用碱性磷酸酶处理Mono Q峰III和IV的PKC形式导致产生峰I形式，这表明蛋白质磷酸化参与了各种PKC形式的相互转化。
