INHIBITION OF HUMAN SECRETORY CLASS-II PHOSPHOLIPASE A(2) BY HEPARIN
INHIBITION OF HUMAN SECRETORY CLASS-II PHOSPHOLIPASE A(2) BY HEPARIN
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DOI：
10.1111/j.1432-1033.1994.tb18761.x
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发表时间：
1994-04-01
期刊：
影响因子：
--
通讯作者：
CHO, W
中科院分区：
文献类型：
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作者：
DUA, R;CHO, W
用Triton X-100/脱氧胆酸/二月桂酰甘油磷乙醇胺(摩尔比为4：2：1)混合胶束进行动力学分析，考察了肝素对几种磷脂酶A(2)(PLA(2))活性的影响。肝素能与阳离子聚乳酸(2)、S(含人分泌物II类聚乳酸(2))强烈结合，从而抑制其在混合胶束中对磷脂的水解。初始速度测量表明，肝素对人类分泌II类解放军(2)和密切相关的A.H.具有竞争性抑制作用。Blomhoffii pla和A.P.Pisciorus pla(2)。特别是，肝素对人类分泌II类解放军的特异性最高(2)。在混合胶束中没有脱氧胆酸的情况下，A.H.肝素对Blomhoffii pla(2)也有很强的抑制作用。这种抑制作用不是由于肝素与聚乳酸(2)活性中心的相互作用所致，因为肝素不抑制聚乳酸(2)对单体底物的水解作用。在不同肝素存在下，8-苯胺基-1-萘磺酸盐与聚乳酸(2)结合的动力学和荧光测定结果表明，一个肝素分子与聚乳酸(2)的7个分子结合。当A.H.氨基末端的四个赖氨酸带正电荷时。Blomhoffii pla(2)通过有限的氨甲酰化反应被中和，肝素既不能与氨甲酰化的A.H.blomhoffii pla(2)结合，也不能抑制氨甲酰化的A.H.Blomhoffii pla(2)，保持了原生A.H.Blomhoffii pla(2)。此外，肝素不能抑制7，10-二(辛酰基)AP对混合胶束的水解性。鱼属(2)，其中氨基末端的α-螺旋中的两个赖氨酸是酰化的。这些结果表明，肝素对人分泌II类聚乳酸(2)及其相关阳离子聚乳酸(2)S的抑制作用来源于肝素与形成界面结合部位一部分的氨基末端的阳离子残基的相互作用。此外，人类分泌II类聚乳酸(2)的独特结构特征，以及它与肝素相互作用的独特模式，推测该聚乳酸(2)可能具有额外的肝素结合位点。尽管肝素与聚乳酸(2)的结合随着反应介质离子强度的增加而减弱，但在生理离子强度下，肝素对人分泌II类聚乳酸(2)的抑制作用仍然显著。生理条件下抑制常数(K-I)的估计值为0.1uM，表明正常药物剂量的肝素可能抑制人分泌II类磷脂酶A(2)并调节其生物学效应。
By means of kinetic analyses using Triton X-100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4:2:1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A(2) (PLA(2)). Heparin avidly bound cationic PLA(2)s including human secretory class II PLA(2) and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class II PLA(2) and closely related A.h. blomhoffii PLA, and A.p. piscivorus PLA(2). In particular, heparin showed the highest specificity for human secretory class II PLA(2). In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA(2) was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA(2) because heparin did not inhibit the hydrolysis of monomeric substrates by PLA(2)s. Both kinetic measurements and fluorescence measurements of PLA(2)-bound 8-anilino-1-naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA(2). When positive charges of four lysines in the amino-terminal region of A.h. blomhoffii PLA(2) were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A. h. blomhoffii PLA(2) nor inhibited the hydrolysis of Triton X-100/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blomhoffii PLA(2) that retained 50% activity of native A.h. blomhoffii PLA(2). Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,10-bis(octanoyl)ated A.p. piscivorus PLA(2) in which two lysines in the amino-terminal alpha-helix are acylated. These results indicate that the inhibition of human secretory class II PLA(2) and related cationic PLA(2)s by heparin Originates from the interaction of heparin with cationic residues in the aminoterminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class II PLA(2), together with its unique mode of interaction with heparin, su that this PLA(2) might have an additional heparin-binding site. Although the heparin-PLA(2) binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class II PLA(2) by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (K-i) was 0.1 mu M under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class II PLA(2) and regulate its biological effects.