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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Toxicology Study Section (TOX)
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University of Washington
Schools of Medicine
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