Pontremoli S, Melloni E, Michetti M, Salamino F, Sparatore B, Sacco O, Horecker B L
Biochem Biophys Res Commun. 1986 Apr 14;136(1):228-34. doi: 10.1016/0006-291x(86)90898-3.
Three classes of activators of human neutrophils that induce the intracellular translocation of protein kinase C from the cytosol to the particulate fraction were compared for their effects on the properties of the particulate (membrane-bound) enzyme. In cells stimulated with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) the particulate enzyme is almost fully active in the absence of added Ca2+ or phospholipids and this activity is not released by the Ca2+-chelator EDTA. In contrast, binding of protein kinase C to the particulate fraction in cells treated with the chemotactic factor f-Met-Leu-Phe (fMLF) or with the ionophore A-23187 plus Ca2+ is observed only when the cells are lysed in the presence of 1 mM Ca2+. With these stimuli the particulate enzyme retains a nearly absolute requirement for Ca2+ and phospholipids. Thus only the full intercalation of protein kinase C caused by PMA, which is resistant to removal by chelators stabilizes an active form of protein kinase C in the neutrophil membrane. In confirmation of this conclusion, in isolated plasma membranes loaded with partially purified protein kinase C by incubation with 5 microM Ca2+ further incubation with PMA, but not with fMLF, caused a significant fraction of the bound PKC to become resistant to removal by chelators, and to be nearly fully active in the absence of added activators.
比较了三类可诱导蛋白激酶C从人中性粒细胞胞质溶胶向颗粒部分进行细胞内转运的激活剂对颗粒（膜结合）酶特性的影响。在用10 ng/ml佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）刺激的细胞中，在不添加Ca2+或磷脂的情况下，颗粒酶几乎完全有活性，且这种活性不会被Ca2+螯合剂乙二胺四乙酸（EDTA）释放。相反，只有当细胞在1 mM Ca2+存在下裂解时，才会观察到趋化因子N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLF）或离子载体A-23187加Ca2+处理的细胞中蛋白激酶C与颗粒部分的结合。对于这些刺激，颗粒酶对Ca2+和磷脂仍几乎有绝对需求。因此，只有PMA引起的蛋白激酶C的完全嵌入（对螯合剂去除有抗性）能在中性粒细胞膜中稳定蛋白激酶C的活性形式。为证实这一结论，在通过与5 microM Ca2+孵育加载了部分纯化的蛋白激酶C的分离质膜中，进一步与PMA而非fMLF孵育，导致相当一部分结合的蛋白激酶C对螯合剂去除有抗性，并且在不添加激活剂的情况下几乎完全有活性。
