Lobo L I, Wilton D C
Department of Biochemistry, University of Southampton, U.K.
Biochem J. 1997 Feb 1;321 ( Pt 3)(Pt 3):829-35. doi: 10.1042/bj3210829.
The breakdown of normal substrates by lipases requires an interfacial binding step prior to hydrolysis. Interfacial binding and subsequent hydrolysis will be affected by the lipid components and hence physical properties of the substrate surface. In order to investigate in detail the effect of lipid structure on the activity of lipoprotein lipase (LPL), triolein-containing emulsion particles of defined composition have been used as substrates. In addition, lipase activity has been measured using a continuous fluorescence displacement assay that monitors the release of long-chain fatty acids as an alternative to normal radiochemical assays. Using this fluorescence assay, rates of hydrolysis of triolein were the same as when using a standard radiochemical assay under identical conditions. Activation by apolipoprotein CII was very similar by both methods; however, the extent of activation (2-3-fold) was less than has been reported previously using different assay conditions. In order to investigate the effect of cholesterol on LPL activity, emulsion particles were prepared in which the cholesterol/egg-phosphatidylcholine ratio was increased up to a 1:1 molar ratio. A pronounced stimulatory effect of cholesterol was observed under these assay conditions, with up to a 5-fold increase in rate compared with emulsion particles without cholesterol. Since high molar ratios of cholesterol are reported to exclude triacylglycerol from the phospholipid surface [Spooner and Small (1987) Biochemistry 26, 5820-5825], these results are not consistent with a mechanism involving LPL hydrolysis of surface triacylglycerol. Instead, they support an interfacial penetration model, allowing the enzyme's active site direct access to triacylglycerol in the lipoprotein core. Perturbation of the surface phospholipid monolayer of the emulsion particle as a result of hydrolysis by Naja naja phospholipase A2 resulted in a 10-fold activation of LPL, providing further support for an interfacial penetration model. The stimulatory effect of apolipoprotein CII was not modulated by modification of the interface with cholesterol.
脂肪酶对正常底物的分解在水解之前需要一个界面结合步骤。界面结合及随后的水解会受到脂质成分以及底物表面物理性质的影响。为了详细研究脂质结构对脂蛋白脂肪酶（LPL）活性的影响，已使用特定组成的含三油酸甘油酯乳液颗粒作为底物。此外，已使用连续荧光置换分析法来测量脂肪酶活性，该方法监测长链脂肪酸的释放，以此替代常规的放射化学分析法。使用这种荧光分析法，在相同条件下，三油酸甘油酯的水解速率与使用标准放射化学分析法时相同。两种方法对载脂蛋白CII的激活作用非常相似；然而，激活程度（2至3倍）低于先前使用不同分析条件时所报道的程度。为了研究胆固醇对LPL活性的影响，制备了胆固醇/蛋黄卵磷脂摩尔比增至1:1的乳液颗粒。在这些分析条件下观察到胆固醇有显著的刺激作用，与不含胆固醇的乳液颗粒相比，速率提高了多达5倍。由于据报道高摩尔比的胆固醇会将三酰甘油从磷脂表面排除[Spooner和Small（1987年），《生物化学》26，5820 - 5825]，这些结果与涉及LPL水解表面三酰甘油的机制不一致。相反，它们支持一种界面渗透模型，使酶的活性位点能够直接接触脂蛋白核心中的三酰甘油。眼镜蛇磷脂酶A2水解导致乳液颗粒表面磷脂单分子层受到扰动，从而使LPL激活了10倍，这为界面渗透模型提供了进一步支持。载脂蛋白CII的刺激作用不会因用胆固醇修饰界面而受到调节。
