Simard G, Loiseau D, Girault A, Perret B
Laboratoire de Biochimie Médicale, C.H.U. d'Angers, France.
Biochim Biophys Acta. 1989 Oct 17;1005(3):245-52. doi: 10.1016/0005-2760(89)90044-1.
(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.
(1) 用未酯化的[14C]胆固醇标记的人HDL2（密度1.070 - 1.125）和HDL3（密度1.125 - 1.21）与卵磷脂胆固醇酰基转移酶来源一起孵育。为获得最佳活性，反应需要加入过量白蛋白，其浓度至少比HDL游离胆固醇浓度高3倍。在这种条件下，反应呈现饱和状态。发现HDL3是最有效的底物，对于1.5 IU LCAT/ml且白蛋白/游离胆固醇比率为3时，HDL3和HDL2的Vmax值分别为每小时每毫升8.3 nmol游离胆固醇酯化和4.1 nmol/ml。(2) 在VLDL存在下，通过用来自牛乳的半纯化脂蛋白脂肪酶诱导三酰甘油脂解来修饰HDL3。新形成的HDL获得了游离胆固醇和磷脂，因此约50%的这些修饰后的HDL（称为轻LIP - HDL3）在HDL2密度范围内被重新分离出来。轻LIP - HDL3主要富含游离胆固醇（增加160%）和磷脂（增加40%）。与亲本HDL3相比，它们对LCAT的反应性降低了一半，这与它们的磷脂/游离胆固醇摩尔比降低密切相关。此外，人工富含游离胆固醇且具有可比PL/FC的HDL3在对LCAT的反应性方面表现得像脂解修饰的HDL。(3) HDL3也通过与VLDL共同孵育（VLDL后HDL3）或与VLDL和脂质转运蛋白来源共同孵育（CET - HDL3）进行修饰。后一种处理极大地影响了核心颗粒的脂质组成（酯化胆固醇减少25%，甘油三酯增加190%）。在这两种情况下，观察到的LCAT反应性适度降低可能与磷脂/游离胆固醇比率有关。因此，就像在人工底物中一样，HDL表面的脂质组成可能控制LCAT介导的胆固醇酯化速率。
