Aviram M, Billecke S, Sorenson R, Bisgaier C, Newton R, Rosenblat M, Erogul J, Hsu C, Dunlop C, La Du B
Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel.
Arterioscler Thromb Vasc Biol. 1998 Oct;18(10):1617-24. doi: 10.1161/01.atv.18.10.1617.
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.
人血清对氧磷酶（PON 1）以两种主要的多态形式（Q型和R型）存在，它们在第191位氨基酸不同（分别为谷氨酰胺和精氨酸）。这些PON同工酶可水解有机磷酸酯和芳香酯，并且两者都能保护低密度脂蛋白（LDL）免受铜离子诱导的氧化。我们比较了两种形式的纯化血清PON，并就其芳基酯酶/对氧磷酶活性评估了它们对LDL氧化的影响。通过孵育4小时后过氧化物和醛的生成来测定，铜离子诱导的LDL氧化分别被PON Q降低了61%和58%，但同等浓度的PON R仅分别降低了46%和38%。这些现象与PON浓度有关。重组PON Q和PON R表现出与纯化血清同工酶相似的模式。在谷胱甘肽过氧化物酶（GPx）存在的情况下，进一步评估了PON Q和PON R在保护LDL免受氧化方面的差异。单独的GPx（0.1 U/mL）在孵育4小时后可使铜离子诱导的LDL氧化降低20%。在上述系统中加入PON R对LDL氧化产生了相加抑制作用，而PON Q没有这种相加作用。这两种PON同工酶在抑制LDL氧化的起始和传播能力上也有所不同。如果在氧化起始时加入，PON Q在阻断LDL氧化方面更有效，而在LDL氧化起始1小时后加入时，PON R更有效。这些数据表明这两种同工酶作用于不同的底物。两种PON同工酶也都能够减少磷脂和胆固醇酯的氧化。在LDL氧化4小时后，PON Q的芳基酯酶活性仅降低了28%，而PON R的芳基酯酶活性降低了高达55%。使用金属螯合剂乙二胺四乙酸（EDTA）或通过在螯合树脂柱上去除钙离子来使依赖钙的PON芳基酯酶活性失活，并没有改变PON抑制LDL氧化的能力。然而，用对羟基汞苯甲酸阻断第283位的PON游离巯基，既抑制了其芳基酯酶活性，也抑制了其对LDL氧化的保护作用。其中PON游离巯基被丙氨酸或丝氨酸取代的重组PON突变体，即使保留了对氧磷酶和芳基酯酶活性，也不再能够防止LDL氧化。总体而言，这些研究表明PON的芳基酯酶/对氧磷酶活性以及对LDL氧化的保护作用并非以完全相同的方式涉及酶上的活性位点，并且PON保护LDL免受氧化的能力需要第283位的半胱氨酸残基。
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