Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.
Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state.
复制标题
短链卵磷脂/长链磷脂单层囊泡的酶水解：磷脂酶对基质相状态的敏感性。
DOI：
10.1021/bi00397a032
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Roberts,MF
中科院分区：
文献类型：
--
作者：
Gabriel,NE;Agman,NV;Roberts,MF
修订稿收到1987年6月30日摘要：短链卵磷脂/长链磷脂单层囊泡(SLUV)，与长链卵磷脂囊泡不同，是水溶性磷脂酶的良好底物。溶血分析表明，99.5%的短链卵磷脂被分配到双层中。在这些二元囊泡中，短链物种是首选底物，而长链磷脂可被视为抑制物(磷脂酶C)或劣质底物(磷脂酶A2)。对于蜡状芽孢杆菌的磷脂酶C，表观Km和Lnax值表明，双分子层增溶的二庚酰磷脂酰胆碱(二庚酰磷脂酰胆碱)几乎和胶束二庚酰磷脂酰胆碱一样好，尽管短链卵磷脂的水解程度取决于长链磷脂的相态。对于磷脂酶A2眼镜蛇来说，Km和Kmax值都表现出更大的范围：在凝胶状态的基质中，二庚酰-PC以胶束状的动力学参数进行水解；在液晶基质中，短链卵磷脂变得与长链卵磷脂相当。在长链磷脂的相变温度附近，这两种酶对二庚酰基-PC的比活性也表现出异常的增加。由于短链卵磷脂没有表现出相变，这一定反映了短链卵磷脂的头基团面积的波动或由周围的长链卵磷脂分子引起的垂直运动。根据SLUV水解物的特殊模型和用水溶性磷脂酶观察到的“界面活化”的一般解释，讨论了这些结果。磷脂酶是一种小的、能催化磷脂酯键水解酶。磷脂酶A2专一性地作用于sn-2脂肪酰基键(Van Deenen&de Haas，1964)，而磷脂酶C专一性地作用于磷脂的磷酸甘油酸键(Little，1981)。这些酶表现出“界面激活”或对聚集形式底物的偏好(Pieterson等人，1974；El-Sayed&Roberts，1985)。此外，磷脂聚集体的类型(如双层、囊泡或胶束)对酶的比活性有显著的影响(Dennis，1983；DeBose&Roberts，1983)。已经提出了一些假设来解释观察到的动力学趋势。其中一些研究集中在聚集引起的底物变化(Brockerhoff，1968；Wells，1974,1978；Apitz-Castro等人，1979；Upreti&Jain，1980)。其他研究人员将界面激活归因于磷脂聚集导致酶的构象变化(Verger&de Haas，1973；Roberts等人，1977；Plunckthun&Dennis，1982)。另一种可能性是，脂质基质聚集状态可能会影响产品的释放(El-Sayed&Roberts，1985)。测试这些不同想法的部分问题是使用了不同的底物链长和物理聚集状态。
Revised Manuscript Received June 30, 1987 abstract: Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlikepure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that> 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, theshort-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Lnax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chainlipid. Forphospholipase A2 Naja naja naja, both Km and Kmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PCaround the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chainlecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysisand a general explanation for the “interfacial activation” observed with water-soluble phospholipases.^^ íospholipases are small, water-soluble enzymes that cata-lyze the hydrolysis of phospholipid ester linkages. Phospholipase A2 acts specifically on the sn-2 fatty acyl bond (Van Deenen & de Haas, 1964) while phospholipase C is specific for thephosphoglycerate bond of phospholipids (Little, 1981). These enzymes exhibit “interfacial activation” or a preference for substrate in aggregated forms (Pieterson et al., 1974; El-Sayed & Roberts, 1985). Furthermore, the type of phospholipid aggregate (eg, bilayer, vesicle, or micelle) has a dramatic effect on the enzyme specific activity (Dennis, 1983; DeBose & Roberts, 1983). A number of hypotheses have been proposed to explain the observed kinetic trends. Several of these focus on aggregation-induced changes in the substrate (Brockerhoff, 1968; Wells, 1974, 1978; Apitz-Castro et al., 1979; Upreti & Jain, 1980). Other workers have attributed interfacial activation to phospholipid aggregationinducing conformational changes in theenzyme (Verger & de Haas, 1973; Roberts et al., 1977; Plunckthun & Dennis, 1982). Yet another possibility is that the lipid matrix aggregation state may affect product release (El-Sayed & Roberts, 1985). Part of the problem in testing these different ideas is that different substrate chain lengths as well as physical aggregation states have been used.