HUMAN SERUM PARAOXONASE: ROLE IN PESTICIDE METABOLISM

人血清对氧磷酶/芳香酯酶水解芳香酯， 包括某些有机磷的有毒代谢物 杀虫剂。这种酶在人类中是多态的，至少有两个 所描述的等位基因形式。其中一个人的营业额较低， 对氧磷(对硫磷的有毒代谢物)和另一种具有较高的 这种有毒农药代谢物的成交量。我们有 最近发现对氧磷酶也能水解毒死磷， 杀虫剂毒死蜱(敌百虫)的有毒代谢产物。这个 毒死蜱-氧磷的水解物呈单峰分布。因此， 对氧磷表现出底物选择性多态。自然的 这种酶的底物还没有确定。 这项计划的具体目标分为五个方面： 1)人和兔的纯化及进一步鉴定 血清对氧磷酶/芳香酯酶；2)底物的进一步研究 该酶的专一性，特别强调 有机磷农药底物及其可能的生理作用 底物；3)大鼠的毒理学研究，探索其作用 酶与农药代谢中的遗传多态 以及纯化酶在病例中治疗应用的可能性 4)DNA的克隆和鉴定 编码高活性和低活性等位基因形式的克隆 5)对氧聚糖酶的表达和分泌。 大肠埃希菌。 该酶将通过柱层析和 制备凝胶电泳法。底物专一性 对氧磷酶的测定将采用常规的酶分析方法。 潜在的农药底物和生理底物 将会被检查。毒理学研究将在老鼠身上进行 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活跃的对氧磷酶之一。 大鼠注射兔酶提纯后血清含量升高 水平提高多达200倍或更多，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 编码人血清对氧磷酶的基因。克隆的cdna将是 通过双脱氧程序进行测序。隔离和 基因组DNA克隆的特征应该提供信息 关于导致这种多态的分子差异。 这些信息将被用来合成等位基因特异性证据。 对氧磷酶存在于低浓度的血清中。至 获得更大数量或蛋白质的结构和活性 研究表明，克隆的cDNA将被量身定做表达和分泌 在我们培育的大肠杆菌菌株中。这些 在这种情况下，菌株能将高纯度的蛋白质分泌到培养基中 不生长的条件。

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.