Antiarteriosclerosis activity due to disposal of oxidized cholesterol by plasma lecithin ; cholesterol acyltransferase reaction.

低密度脂蛋白(LDL)的氧化修饰在动脉粥样硬化形成中起着重要作用。我们前期的研究表明，氧化型低密度脂蛋白(OX-LDL)对胆固醇逆向转运系统中的重要酶卵磷脂：胆固醇酰基转移酶(LCAT；EC 2.3.1.41)具有抑制作用。然而，氧化低密度脂蛋白抑制LCAT活性的确切机制尚不清楚。本研究探讨了氧化低密度脂蛋白对LCAT分子及其在高密度脂蛋白中的激活性脂蛋白载脂蛋白A-I(apo A-I)的影响。将纯化的LCAT和d&GT；1.063 g/ml人血浆作为LCAT的来源，分别与低密度脂蛋白和氧化低密度脂蛋白在4℃和37℃孵育1h。采用Superose-12凝胶过滤柱，通过FPLC系统对LCAT进行凝胶渗透色谱分离。经37℃孵育后，LCAT活性仅在从氧化低密度脂蛋白中分离出来时才被显著抑制(纯化组和血浆组的抑制率分别为对照的20%和85%)。用溴化钠调节密度的序贯超速离心法从重组血浆(d&gt；1.063 g/ml血浆&低密度脂蛋白/氧化低密度脂蛋白)中分离高密度脂蛋白。将分离的高密度脂蛋白用[^&lt；14&gt；C]-胆固醇-BSA乳剂标记，作为LCAT的底物，发现底物组与对照组相比活性降低21.18%。在高密度脂蛋白中，LCAT比载脂蛋白A-I更容易受到氧化低密度脂蛋白的不利影响。LCAT活性被DTNB保护，而谷胱甘肽对氧化低密度脂蛋白没有任何保护作用。因此，在37℃时，低密度脂蛋白的氧化产物(S)转移到高密度脂蛋白中的低密度脂蛋白和载脂蛋白A-I，并与之紧密结合，对这些分子造成不可逆的修饰。多种氧化产物参与了对LCAT活性的抑制，可能遵循不同的机制。

Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Our previous studies demonstrated that Lecithin : cholesterol acyltransferase (LCAT ; EC 2.3.1.41) an important enzyme in the reverse cholesterol transport system was inhibited by oxidized LDL (ox-LDL). However the precise mechanism of inhibition of LCAT activity by ox-LDL is not clear still. In this study, we investigated the effect of ox-LDL on LCAT molecule and it's activator lipoprotein in HDL, apolipoprotein A-I (apo A-I). Purified LCAT and d>1.063 g/ml fraction of humanplasma as source of LCAT were incubated with LDL and ox-LDL for 1h at 4℃ and 37℃. LCAT was separated by gel permeation chromatography using Superose-12 gel filtration column through FPLC system. LCAT activity was found inhibited significantly only when it was separated from ox-LDL after incubation at 37℃ (inhibition was 20% and 85% of control in purified and plasma LCAT respectively). HDL was isolated form recombined plasma (d>1.063 g/ml fraction of plasma ＆ LDL/ox-LDL) by sequential ultrcentrifugation using NaBr to adjust density. This isolated HDL was then labeled by [^<14>C]-Cholesterol-BSA emulsion and used as substrate for LCAT.Activity was found 21.18% lower in substrate group, which was previously exposed to ox-LDL, compared to control. LCAT was more susceptible to adverse effect of ox-LDL compared to apo A-I in HDL.LCAT activity was found protected by DTNB but GSH was found not afforded any protection to LCAT activity from ox-LDL.We therefore concluded that oxidation product (s) of LDL was transferred to LCAT and apo A-I in HDL at 37℃ and tightly bound to it, which caused irreversible modification to these molecules. More than one type of oxidation product was involved in the inhibition of LCAT activity, which may be follow different mechanism.