Hornberger W, Patscheke H
Institute for Clinical Chemistry, Klinikum Mannheim, University of Heidelberg, Federal Republic of Germany.
Eur J Biochem. 1990 Jan 12;187(1):175-81. doi: 10.1111/j.1432-1033.1990.tb15292.x.
Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.
血小板中类二十烷酸的形成取决于游离花生四烯酸的浓度，而游离花生四烯酸主要由磷脂酶A2从膜磷脂中释放出来。游离花生四烯酸的浓度还受再酰化酶花生四烯酰辅酶A合成酶和溶血磷脂酰转移酶活性的控制。在人血小板微粒体中，我们测定了花生四烯酰辅酶A合成酶的高酶活性为5.9 nmol·min-1·(10^9个血小板)-1，溶血磷脂酰转移酶的活性为37 nmol·min-1·(10^9个血小板)-1。这些再酰化酶的活性被过氧化氢（H2O2）和甲基汞强烈降低，而过氧化氢和甲基汞是完整血小板中花生四烯酸释放的主要刺激物。H2O2抑制花生四烯酰辅酶A合成酶，IC50为3.3 mmol/L，而不影响溶血磷脂酰转移酶。3-巯基-1,2-丙二醇对巯基的保护不能克服这种抑制作用，但谷胱甘肽可防止H2O2对花生四烯酰辅酶A合成酶的抑制。这表明谷胱甘肽借助谷胱甘肽过氧化物酶还原H?O?，而不是保护花生四烯酰辅酶A合成酶的游离巯基。甲基汞不影响花生四烯酰辅酶A合成酶的活性，但抑制溶血磷脂酰转移酶的活性，IC50为3.4 μmol/L。这种抑制可能是由于溶血磷脂酰转移酶的巯基被阻断所致，因为当加入浓度高于甲基汞的3-巯基-1,2-丙二醇时，抑制作用消失。凝血酶作为生理性完全激动剂、Ca2+≤1 mmol/L、钙离子载体A23187以及佛波醇12-肉豆蔻酸酯13-乙酸酯（TPA）和1-油酰-2-乙酰甘油作为蛋白激酶C的模型刺激物，均不影响花生四烯酰辅酶A合成酶和溶血磷脂酰转移酶。结论是，H2O2和甲基汞分别对花生四烯酸再酰化酶花生四烯酰辅酶A合成酶或溶血磷脂酰转移酶的抑制作用，是它们在完整细胞中刺激类二十烷酸释放能力的原因。凝血酶及其细胞内信使Ca2+和二酰甘油不直接影响花生四烯酰辅酶A合成酶和溶血磷脂酰转移酶。
