Davit-Spraul A, Thérond P, Leroy A, Palmade-Rieunier F, Rousset C, Moatti N, Legrand A
Biochimie, Hôpital Bicêtre, Laboratoire de Biochimie, Le Kremlin Bicêtre, France.
FEBS Lett. 1999 Mar 19;447(1):106-10. doi: 10.1016/s0014-5793(99)00278-1.
To gain insight into the nature of the lecithin-cholesterol acyltransferase inhibitory factor(s), we separated and collected the oxidation products from oxidized lipoproteins after lipoxygenase treatment. Isolated fractions identified by chemiluminescence, as hydroperoxides of phosphatidylcholine, were found to produce a significant reduction of lecithin-cholesterol acyltransferase activity. The reaction kinetics of lecithin-cholesterol acyltransferase with reconstitued high density lipoproteins were studied in the presence of 0.6 and 1.2 microM hydroperoxides of phosphatidylcholine. No significant changes in the apparent Vmax were observed but a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values was observed with increasing hydroperoxide concentrations. These results show that the active site of lecithin-cholesterol acyltransferase is not affected by the presence of phosphatidylcholine hydroperoxides. Nevertheless, hydroperoxides of phosphatidylcholine altered the reactivity of lecithin-cholesterol acyltransferase for reconstitued high density lipoproteins suggesting either an alteration of the binding of lecithin-cholesterol acyltransferase to the reconstitued high density lipoproteins or a competitive inhibition mechanism.
为深入了解卵磷脂胆固醇酰基转移酶抑制因子的本质，我们在脂氧合酶处理后，从氧化脂蛋白中分离并收集了氧化产物。通过化学发光鉴定为磷脂酰胆碱氢过氧化物的分离组分，被发现可使卵磷脂胆固醇酰基转移酶活性显著降低。在存在0.6微摩尔和1.2微摩尔磷脂酰胆碱氢过氧化物的情况下，研究了卵磷脂胆固醇酰基转移酶与重组高密度脂蛋白的反应动力学。未观察到表观Vmax有显著变化，但随着氢过氧化物浓度增加，双倒数图的斜率和表观Km值呈浓度依赖性增加。这些结果表明，磷脂酰胆碱氢过氧化物的存在不会影响卵磷脂胆固醇酰基转移酶的活性位点。然而，磷脂酰胆碱氢过氧化物改变了卵磷脂胆固醇酰基转移酶对重组高密度脂蛋白的反应性，这表明要么是卵磷脂胆固醇酰基转移酶与重组高密度脂蛋白的结合发生了改变，要么是存在竞争性抑制机制。
