Simard G, Perret B, Durand S, Collet X, Chap H, Douste-Blazy L
Laboratoire de Biochemie Médicale, CHU d'Angers, France.
Biochim Biophys Acta. 1989 Feb 6;1001(2):225-33. doi: 10.1016/0005-2760(89)90152-5.
(1) Human HDL2 (d 1.063-1.125) and HDL3 (d 1.125-1.210), labelled with 2-[14C]oleoylphosphatidylcholine (PC), and with/without tri[3H]oleoylglycerol, were incubated with a partially purified human hepatic triacylglycerol lipase, at pH 8.5. PC hydrolysis was linear up to 90-120 min incubation and within a range of lipase activities, from 50 to 500 mIU/ml. At low degrees of lipolysis, the hydrolysis of triacylglycerol was linearly related to that of PC, but the relative degradation rate was 10-fold higher for the former, which was thus very rapidly consumed. HDL subfractions were then differentiated in terms of PC hydrolysis. Km values were 0.32 and 0.43 mM for HDL2 PC and HDL3 PC, respectively. The corresponding Vmax values expressed for 200 mIU/ml hepatic lipase activity were 41.0 nmol PC hydrolysed/ml per h (HDL2) and 28.6 nmol PC/ml per h (HDL3). (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis in VLDL with a semi-purified human plasma or bovine milk lipoprotein lipase (LPL). Lipolysis-modified HDL3 (LIP-HDL3) were mostly enriched in free cholesterol (+80%, P less than 0.05) and to a lesser extent in triacylglycerol (+33%). As a consequence, 45% of the LIP-HDL3 was reisolated in the HDL2-density interval, and is referred to as light LIP-HDL3. LIP-HDL3 displayed a 65% increase in its reactivity towards hepatic lipase compared to control HDL3. The light LIP-HDL3 showed the lowest Km (0.19 mM PC) and the highest Vmax (69 nmol/ml per h) of all HDL tested. Coincubation of HDL3 with VLDL and albumin did not alter the further reactivity of HDL3 towards hepatic lipase. Cholesterol loading of HDL3 by celite-cholesterol dispersions also led to an enhanced reactivity, though less important than with the lipolysis modification. (3) HDL3 were also modified by coincubation with VLDL and the lecithin-cholesterol acyltransferase-inhibited plasma fraction of d greater than 1.21 g/ml, thus allowing the cholesteryl ester transfer reaction to occur. The modified HDL3 (CET-HDL3) were depleted in esterified cholesterol (-25%, P less than 0.05) and enriched in triacylglycerol (+70%, P less than 0.05). However, these particles behaved like control HDL3 in their reactivity towards hepatic triacylglycerol lipase. Thus, the hydrolysis of HDL PC mediated by hepatic triacylglycerol lipase appears to be influenced by changes occurring in the particle's surface rather than in the lipid core.
(1) 用2-[¹⁴C]油酰磷脂酰胆碱（PC）标记，并添加/不添加三[³H]油酰甘油的人HDL2（密度1.063 - 1.125）和HDL3（密度1.125 - 1.210），在pH 8.5条件下与部分纯化的人肝脏三酰甘油脂肪酶一起孵育。PC水解在长达90 - 120分钟的孵育时间内以及50至500 mIU/ml的脂肪酶活性范围内呈线性。在低脂肪分解程度下，三酰甘油的水解与PC的水解呈线性相关，但前者的相对降解速率高10倍，因此消耗非常迅速。然后根据PC水解对HDL亚组分进行区分。HDL2 PC和HDL3 PC的Km值分别为0.32和0.43 mM。以200 mIU/ml肝脏脂肪酶活性表示的相应Vmax值分别为每小时每毫升水解41.0 nmol PC（HDL2）和每小时每毫升28.6 nmol PC（HDL3）。(2) 通过用半纯化的人血浆或牛乳脂蛋白脂肪酶（LPL）诱导VLDL中的三酰甘油脂肪分解，在VLDL存在下对HDL3进行修饰。脂肪分解修饰的HDL3（LIP - HDL3）主要富含游离胆固醇（增加80%，P < 0.05），三酰甘油含量增加程度较小（增加33%）。结果，45%的LIP - HDL3在HDL2密度区间内重新分离出来，称为轻LIP - HDL3。与对照HDL3相比，LIP - HDL3对肝脏脂肪酶的反应性增加了65%。在所有测试的HDL中，轻LIP - HDL3显示出最低的Km（0.19 mM PC）和最高的Vmax（每小时每毫升69 nmol）。HDL3与VLDL和白蛋白共同孵育不会改变HDL3对肝脏脂肪酶的进一步反应性。通过硅藻土 - 胆固醇分散体对HDL3进行胆固醇负载也导致反应性增强，尽管不如脂肪分解修饰那么显著。(3) 通过将HDL3与VLDL以及密度大于1.21 g/ml的卵磷脂 - 胆固醇酰基转移酶抑制血浆部分共同孵育进行修饰，从而使胆固醇酯转移反应发生。修饰后的HDL3（CET - HDL3）的酯化胆固醇减少（-25%，P < 0.05），三酰甘油增加（+70%，P < 0.05）。然而，这些颗粒对肝脏三酰甘油脂肪酶的反应性与对照HDL3相似。因此，肝脏三酰甘油脂肪酶介导的HDL PC水解似乎受颗粒表面而非脂质核心发生的变化影响。
