Salgo M G, Corongiu F P, Sevanian A
Department of Pathology, University of Southern California, Los Angeles 90033.
Arch Biochem Biophys. 1993 Jul;304(1):123-32. doi: 10.1006/abbi.1993.1330.
The hydrolytic action of phospholipase A2 was examined with unilamellar vesicles composed of soybean phosphatidylcholine in terms of the calcium dependency of the enzyme and substrate specificity following lipid peroxidation. Experiments were performed under conditions where enzyme:substrate ratios were low, specifically in the range of one to five enzyme molecules for every 10 vesicle particles. Accordingly, low hydrolytic activities were found where less than 15% of the phospholipids were hydrolyzed under the various conditions of time, enzyme:substrate ratios, calcium concentrations, and extent of peroxidation utilized. Vesicle peroxidation increased the Ca2+ binding potential to a degree comparable to addition of the anionic phospholipid, dioleoylphosphatidic acid (DOPA). A remarkable similarity was found between the binding profiles for Ca2+ and phospholipase A2 activity; however, enzyme activity toward oxidized vesicles was beyond the increases observed for Ca2+ binding. Under conditions where approximately 5% of the phospholipids were peroxidized the effective Ca2+ concentration required for half-maximal activity was less than one-half that required for unoxidized vesicles. Peroxidation of vesicle phospholipids markedly increased the rate and extent of hydrolysis, even in the presence of DOPA or deoxycholate. Deoxycholate is known to induce vesicle fusion such that a larger proportion of enzyme is associated with a fewer number of enlarged vesicles. Using a dual isotope technique to measure hydrolysis of oxidized vs unoxidized phospholipids and covesicle preparations to study enzyme binding and activity, a significantly greater apparent intervesicle exchange of enzyme was found after peroxidation of vesicles with more than a twofold hydrolytic specificity toward the oxidized phospholipids. We postulate that a combination of structural and Ca2+ binding affinity changes are produced in membranes following lipid peroxidation which evoke an additive effect on PLA2 activity. Although oxidized phospholipids may serve as activators of phospholipase A2 by presenting the interface in a form where Ca2+ and enzyme binding and/or specific activity are increased, an additional and important factor appears to involve membrane fusion or vesicle-vesicle interactions. This process facilitates enzyme activity through the replenishment of substrates wherein the otherwise limited interaction of enzyme and substrate is overcome by more rapid or extensive vesicle fusion which increases access to the phospholipids available in the preparation.
以大豆磷脂酰胆碱构成的单层囊泡为材料，从酶的钙依赖性和脂质过氧化后的底物特异性方面，研究了磷脂酶A2的水解作用。实验在酶与底物比例较低的条件下进行，具体为每10个囊泡颗粒对应1至5个酶分子的范围。因此，在不同的时间、酶与底物比例、钙浓度以及过氧化程度条件下，发现水解活性较低，其中不到15%的磷脂被水解。囊泡过氧化使Ca2+结合电位增加到与添加阴离子磷脂二油酰磷脂酸（DOPA）相当的程度。在Ca2+结合曲线和磷脂酶A2活性之间发现了显著的相似性；然而，酶对氧化囊泡的活性超出了Ca2+结合增加所观察到的范围。在约5%的磷脂被过氧化的条件下，达到最大活性一半所需的有效Ca2+浓度不到未氧化囊泡所需浓度的一半。囊泡磷脂的过氧化显著增加了水解的速率和程度，即使在存在DOPA或脱氧胆酸盐的情况下也是如此。已知脱氧胆酸盐会诱导囊泡融合，使得更大比例的酶与数量较少的增大囊泡相关联。使用双同位素技术测量氧化磷脂与未氧化磷脂的水解，并通过共囊泡制剂研究酶的结合和活性，发现囊泡过氧化后，对氧化磷脂具有两倍以上水解特异性的酶的囊泡间表观交换显著增加。我们推测，脂质过氧化后膜中会产生结构和Ca2+结合亲和力变化的组合，这对PLA2活性产生累加效应。虽然氧化磷脂可能通过以增加Ca2+和酶结合及/或比活性的形式呈现界面来充当磷脂酶A2的激活剂，但另一个重要因素似乎涉及膜融合或囊泡 - 囊泡相互作用。这个过程通过补充底物促进酶活性，其中酶与底物原本有限的相互作用通过更快或更广泛的囊泡融合得以克服，这增加了对制剂中可用磷脂的接触。
