Font J, Marino A, Ravelingien N, Dehaye J P, Trueba M, Macarulla J M
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad del País Vasco, Spain.
Rev Esp Fisiol. 1990 Dec;46(4):325-30.
Protein kinase C (PKC) has been widely studied from different tissues of mammals. Human platelets display higher levels of PKC activity, if compared with other sources. The PKC activity from calf platelets crude extract was determined in the presence of various protease inhibitors such as PMSF, Leupeptin or Trypsin inhibitro, and the Ca(2+)-chelators EGTA and EDTA. The free calcium requirement was 0.25 mM, calculated with the help of the Solgas-water computer program, which represents 1 mM CaCl2, in these assay conditions. Optimum PKC activity was obtained at 4 min in the presence of PS plus DAG or TPA, using H1 type III-S histone as substrate. Phospholipid-interacting drugs, such as trifluoperazine, chlorpromazine and tetracaine, inhibited the PKC activity in a dose-dependent manner. Triton X-100, a non-ionic detergent, which is usually employed to solubilize the membrane fraction, in different translocation assays, inhibited PKC activity at concentrations higher than 0.01%. In these conditions, non-proteolytic PKC activity from calf platelets was easily determined, and shares similar activity levels with those described in human platelets.
蛋白激酶C（PKC）已在哺乳动物的不同组织中得到广泛研究。与其他来源相比，人血小板显示出更高水平的PKC活性。在存在各种蛋白酶抑制剂（如苯甲基磺酰氟、亮抑酶肽或胰蛋白酶抑制剂）以及钙螯合剂乙二醇双四乙酸（EGTA）和乙二胺四乙酸（EDTA）的情况下，测定了小牛血小板粗提物中的PKC活性。在这些测定条件下，借助Solgas - water计算机程序计算得出，游离钙的需求量为0.25 mM，相当于1 mM氯化钙。以H1 III - S型组蛋白为底物，在磷脂酰丝氨酸（PS）加二酰甘油（DAG）或佛波酯（TPA）存在的情况下，4分钟时可获得最佳PKC活性。磷脂相互作用药物，如三氟拉嗪、氯丙嗪和丁卡因，以剂量依赖性方式抑制PKC活性。在不同的转位测定中，通常用于溶解膜部分的非离子去污剂Triton X - 100在浓度高于0.01%时抑制PKC活性。在这些条件下，小牛血小板的非蛋白水解性PKC活性易于测定，且与报道的人血小板的活性水平相似。
