Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities - Selective action of human paraoxonase allozymes Q and R
Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities - Selective action of human paraoxonase allozymes Q and R
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DOI：
10.1161/01.atv.18.10.1617
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发表时间：
1998-10-01
影响因子：
8.7
通讯作者：
La Du, B
中科院分区：
文献类型：
--
作者：
Aviram, M;Billecke, S;La Du, B
人血清对氧磷酶（PON 1）存在2种主要的多态性形式（Q和R），它们在位置191处的氨基酸（分别为谷氨酰胺和精氨酸）不同。这些PON等位酶水解有机磷酸酯和芳香酯，并且两者也保护LDL免受铜离子诱导的氧化。我们比较了纯化的血清脑桥的两种形式，并评估其对LDL氧化的影响，在他们的芳基酯酶/对氧磷酶的活动。通过孵育4小时后过氧化物和醛的产生来测量铜离子诱导的LDL氧化，PON Q分别使其降低高达61%和58%，但等效浓度的PON R仅分别使其降低高达46%和38%。这些现象是PON浓度依赖性。重组PON Q和PON R表现出与纯化血清同种酶相似的模式，在谷胱甘肽过氧化物酶（GPx）存在下进一步评价PON Q和PON R在保护LDL免受氧化方面的差异。孵育4小时后，单独使用GPx（0.1 U/mL）可使铜离子诱导的LDL氧化降低20%。在上述体系中加入PON R对LDL氧化有相加的抑制作用，而PON Q则无此相加作用。2种PON等位酶抑制LDL氧化起始和增殖的能力也不同，如果在氧化起始时加入PON Q，则PON Q在阻断LDL氧化方面更有效，而PON R在LDL氧化起始后1小时加入时更有效。这些数据表明，这两种等位酶作用于不同的底物。两种PON等位酶也能够减少磷脂和胆固醇酯的氧化，PON Q芳基酯酶活性在LDL氧化4小时后仅降低28%，而PON R的芳基酯酶活性降低高达55%。通过使用金属螯合剂EDTA或通过在Chelex柱上去除钙离子来灭活钙依赖性PON芳基酯酶活性，并没有改变PON抑制LDL氧化的能力。然而，在位置283与p-羟基汞苯甲酸酯的PON自由巯基的阻断抑制其芳基酯酶活性和其保护LDL氧化。重组PON突变体中的PON自由巯基被替换为丙氨酸或丝氨酸不再能够防止LDL氧化，即使他们保留对氧磷酶和芳基酯酶的活动。总之，这些研究表明PON的芳基酯酶/对氧磷酶活性和对LDL氧化的保护作用并不以完全相同的方式涉及酶上的活性位点，PON保护LDL免受氧化的能力需要283位的半胱氨酸残基。
Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion-induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion-induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes, PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion-induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation, PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester, PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON's ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON's arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON's ability to protect LDL from oxidation requires the cysteine residue at position 283.