HUMAN SERUM PARAOXONASE: ROLE IN PESTICIDE METABOLISM

人血清对氧磷酶/芳基酯酶水解芳香酯， 包括一些有机磷的有毒代谢物 农药 这种酶在人类中是多态的，至少有两种 描述的等位基因形式。 一个是低周转率， 对氧磷（对氧磷的有毒代谢物），另一种具有较高的 这种有毒农药代谢物的周转数。 我们有 最近显示对氧磷酶也水解毒死蜱-氧磷， 杀虫剂毒死蜱的有毒代谢物。 的 毒死蜱-氧磷的水解是单峰分布的。 因此，在本发明中， 对氧磷表现出底物选择性多态性。 自然 这种酶的底物尚未鉴定。 该项目的具体目标分为五个方面： 1)人和兔的纯化和进一步表征 血清对氧磷酶/芳基酯酶; 2）对底物的进一步研究 酶的特异性，特别强调 有机磷农药底物和可能的生理 底物; 3）大鼠毒理学研究，探索 农药代谢中的酶和遗传多态性 以及纯化酶在某些情况下的治疗用途的可能性， 有机磷中毒的研究; 4）DNA的克隆和鉴定 编码高活性和低活性等位基因形式的 酶;和5）对黄原胶酶的表达和分泌， 大肠埃希菌 酶将通过柱色谱法纯化， 制备凝胶电泳。 的底物特异性 对氧磷酶将用常规酶测定法测定。 潜在的农药底物以及生理底物 将被审查。 将对大鼠进行毒理学研究 注射纯化的兔或人对氧磷酶。 兔子 酶是迄今为止所描述的最具活性的对氧磷酶之一。 给大鼠注射纯化兔酶可提高其血清 水平高达200倍或可能更多，允许 酶在农药中作用的直接评价 新陈代谢. 标准克隆技术将用于克隆一个 编码人血清对氧磷酶的cDNA。 cDNA克隆将是 双脱氧法测序。 分离与 基因组DNA克隆的特征应该提供信息 多态性的分子差异 该信息将用于合成等位基因特异性证据。 血清中存在低浓度的对氧磷酶。 到 获得更大量的蛋白质，用于结构和活性 研究中，克隆的cDNA将被量身定制地表达和分泌 在我们开发的大肠杆菌菌株中。 这些 菌株分泌高纯度的蛋白质到培养基中， 非生长条件。

Human serum paraoxonase/arylesterase hydrolyzes aromatic ester, including the toxic metabolites of some organophosphorus pesticides. This enzyme is polymorphic in humans with at least two allelic forms described. One has a low turnover number for paraoxon (toxic metabolite of parathion) and the other has a higher turnover number for this toxic pesticide metabolite. We have recently shown that paraoxonase also hydrolyzes chlorpyrifos-oxon, the toxic metabolite of the pesticide chlorpyrifos (Dursban). The hydrolysis of chlorpyrifos-oxon is unimodally distributed. Thus, paraoxon exhibits a substrate selective polymorphism. The natural substrates for this enzyme have not yet been identified. The specific aims for this project are divided into five areas: 1) purification and further characterization of human and rabbit serum paraoxonase/arylesterase; 2) further studies on the substrate specificity of the enzyme with particular emphasis on organophosphate pesticide substrates and possible physiological substrates; 3) toxicology studies with rats that explore the role of the enzyme and the genetic polymorphism in pesticide metabolism and the possibility of therapeutic uses of purified enzyme in cases of poisoning by organophosphates; 4) cloning and characterizing DNA clones that encode the high and low activity allelic forms of the enzyme; and 5) the expression and secretion of paraoxanase by Escherichia coli. The enzyme will be purified by column chromatography and preparative gel electrophoresis. The substrate specificity of paraoxonase will be determined with conventional enzyme assays. Potential pesticide substrates as well as physiological substrates will be examined. Toxicology studies will be pursued with rats injected with purified rabbit or human paraoxonase. The rabbit enzyme is one of the most active paraoxonases so far described. Injection of purified rabbit enzyme into rats raises their serum levels by as much as 200-fold or possibly much more, allowing for the direct assessment of the role of the enzyme in pesticide metabolism. Standard cloning techniques will be used to clone a cDNA that encodes human serum paraoxonase. The cDNA clone will be sequenced by the dideoxy procedures. Isolation and characterization of genomic DNA clones should provide information about the molecular differences responsible for the polymorphism. This information will be used to synthesize allele specific proves. Paraoxonase is present in the serum in low concentrations. To obtain larger quantities or protein for structural and activity studies, the cloned cDNAs will be tailored expression and secretion in strains of Escherichia coli that we have developed. These strains secrete highly pure protein into the medium under nongrowing conditions.