Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.
Activation of protein kinase C by oleic acid. Determination and analysis of inhibition by detergent micelles and physiologic membranes: requirement for free oleate.
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DOI：
10.1016/s0021-9258(19)50567-1
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
W. Khan;G. Blobe;Y. Hannun
中科院分区：
文献类型：
--
作者：
W. Khan;G. Blobe;Y. Hannun
油酸钠能够激活可溶性蛋白激酶C（Murakami，K.，Chan，S.是的，和Liattenberg，A.（1986）J.Biol.Chem.261，15424-15429），但不能激活膜结合酶（El Touny，S.，Khan，W.，和Hannun，Y.（1990）J.Biol.Chem.265，16437-16443）。由于脂肪酸与蛋白激酶C的生理相互作用发生在膜的存在下，进行了以下研究，以评估表面（去污剂胶束或血小板膜）对油酸激活蛋白激酶C的影响。在浓度等于或高于Triton X-100的临界胶束浓度（CMC）时，油酸酯主要存在于Triton X-100/油酸酯混合胶束中，通过凝胶渗透色谱法和平衡透析结合研究确定。在略低于Triton X-100的CMC的浓度下，油酸酯的存在导致形成有限数量的混合胶束。在不同浓度的Triton X-100存在下，油酸钠对纯化的血小板蛋白激酶C的剂量依赖性激活研究表明，只有未结合的油酸酯能够激活蛋白激酶C。血小板蛋白激酶C被分解为两种主要同工酶（II型（β）和III型（α）），它们与油酸的相互作用几乎相同。在生理环境中，采用血小板底物和内源性血小板蛋白激酶C，研究油酸对蛋白激酶C的激活作用。油酸在胞浆区室中有效激活蛋白激酶C。在血小板匀浆以及在重建的血小板胞质溶胶和膜系统中，蛋白激酶C对油酸的剂量依赖性显示出显着的向右移位。约30%的油酸与血小板胞浆相关，70%与血小板膜相关。油酸分配到两个血小板室显示pH值，温度或孵育时间的变化不大。当校正游离油酸浓度时，油酸对蛋白激酶C的激活在胞质溶胶和匀浆中表现出相同的剂量依赖性。花生四烯酸，蛋白激酶C的一种潜在的生理激活剂，表现出类似的行为，油酸，虽然只有30%的花生四烯酸分配到血小板膜与花生四烯酸的大部分（70%）留在胞浆级分。（400字处截断摘要）
Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)