Activation of hepatocyte protein kinase C by redox-cycling quinones.
Activation of hepatocyte protein kinase C by redox-cycling quinones.
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氧化还原循环醌激活肝细胞蛋白激酶 C。
DOI：
10.1042/bj2600499
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
Orrenius,S
中科院分区：
文献类型：
--
作者：
Kass,GE;Duddy,SK;Orrenius,S
研究了醌类化合物产生的活性氧对大鼠肝细胞蛋白激酶C的影响。在与氧化还原循环醌、甲萘醌、杜醌或2，3-二甲氧基-1，4-萘醌孵育的肝细胞中，细胞溶质蛋白激酶C的比活性增加2-3倍，而颗粒蛋白激酶C的比活性或Ca 2 +-和脂质非依赖性激酶活性没有改变。氧化还原循环醌没有刺激蛋白激酶C的易位，但是，激活的蛋白激酶C被重新分配从细胞质到颗粒部分时，醌处理的肝细胞暴露于12-O-十四酰佛波醇13-乙酸酯（TPA）。醌处理没有改变胞质佛波醇12，13-二丁酸酯（PDBu）的结合能力，和对照组和醌处理的肝细胞的胞质溶胶表现出2 nM的PDBu结合的Kd。醌介导的细胞溶质蛋白激酶C的活化通过与10 mM-β-巯基乙醇、二硫苏糖醇或GSH在4 ℃下孵育24 h来逆转。此外，在空气中孵育的控制胞质溶胶中的蛋白激酶C的比活性增加了超过100%，在3小时内，这种增加被逆转的巯基还原剂。类似地，在空气中孵育部分纯化的大鼠脑蛋白激酶C，或在GSH存在下与低浓度的GSSG孵育，导致Ca 2+和脂质依赖性激酶活性增加2-2.5倍。与氧化还原循环醌的作用相反，当用硫醇剂N-乙基马来酰亚胺（NEM）、对苯醌（pBQ）或对氯汞苯甲酸（pCMB）处理肝细胞时，细胞溶质Ca 2+和脂质依赖性激酶活性被显著抑制，但颗粒相关蛋白激酶C活性不受影响。的Ca 2+和脂质的非依赖性激酶活性的胞质和颗粒馏分显着刺激NEM，但不受pBQ和pCMB。这些结果表明，肝细胞胞浆蛋白激酶C被醌产生的活性氧物质激活为高Vmax形式，这种效应是由于蛋白激酶C的巯基/二硫键状态的还原敏感性修饰。
The effects of quinone-generated active oxygen species on rat hepatocyte protein kinase C were investigated. The specific activity of cytosolic protein kinase C was increased 2-3-fold in hepatocytes incubated with the redox-cycling quinones, menadione, duroquinone or 2,3-dimethoxy-1,4-naphthoquinone, without alterations in particulate protein kinase C specific activity or Ca2+- and lipid-independent kinase activities. Redox-cycling quinones did not stimulate translocation of protein kinase C; however, activated protein kinase C was redistributed from cytosol to the particulate fraction when quinone-treated hepatocytes were exposed to 12-O-tetradecanoylphorbol 13-acetate (TPA). Quinone treatment did not alter cytosolic phorbol 12,13-dibutyrate (PDBu) binding capacity, and the cytosol of both control and quinone-treated hepatocytes exhibited a Kd for PDBu binding of 2 nM. Quinone-mediated activation of cytosolic protein kinase C was reversed by incubation with 10 mM-beta-mercaptoethanol, dithiothreitol or GSH, at 4 degrees C for 24 h. Furthermore, protein kinase C specific activity in control cytosol incubated in air increased by over 100% within 3 h; this increase was reversed by thiol-reducing agents. Similarly, incubation of partially-purified rat brain protein kinase C in air, or with low concentrations of GSSG in the presence of GSH, resulted in a 2-2.5-fold increase in Ca2+- and lipid-dependent kinase activity. In contrast with the effects of the redox-cycling quinones, when hepatocytes were treated with the thiol agents N-ethylmaleimide (NEM), p-benzoquinone (pBQ) or p-chloromercuribenzoic acid (pCMB), the cytosolic Ca2+- and lipid-dependent kinase activity was significantly inhibited, but the particulate-associated protein kinase C activity was unaffected. The Ca2+- and lipid-independent kinase activity of both the cytosolic and particulate fractions was significantly stimulated by NEM, but was unaffected by pBQ and pCMB. These results show that hepatocyte cytosolic protein kinase C is activated to a high-Vmax form by quinone-generated active oxygen species, and this effect is due to a reduction-sensitive modification of the thiol/disulphide status of protein kinase C.