The long-term objective of the proposed studies is to determine the molecular basis for the existence of multiple forms of carboxyesterases in rat liver microsomes, and to determine the function and regulation of these hydrolytic enzymes.
The specific aims of the project are (1) to purify from rat liver microsomes the three major constitutive forms of carboxyesterase (hydrolases A, B and C) and the major phenobarbital-inducible form of carboxyesterase (hydrolase D), and (2) to characterize by biochemical and immunochemical techniques their structure, function and regulation. Monoclonal antibodies and highly specific polyclonal antibodies against hydrolases A-D will be prepared to carry out the proposed immunochemical studies. As part of the studies to determine their function, the purified carboxyesterases will be tested for their ability to hydrolyze selected carboxylic acid esters and amides. In addition, the purified carboxyesterases will be tested for their ability to hydrolyze or covalently bind organophosphates. Studies to determine how the carboxyesterases are regulated will include studies of the tissue and species distribution of hydrolases A-D, and studies of the effects of age, sex and xenobiotic treatment of rats on the levels of hydrolase A- D in rat liver microsomes. Recombinant DNA studies, in conjunction with amino acid sequence and immunochemical analyses, will be performed to establish the molecular basis for the existence of multiple forms of carboxyesterase in rat liver microsomes, and to provide the necessary framework to elucidate the mechanism by which carboxyesterases detoxify organophosphates. cDNA probes will be constructed to study the regulation of hydrolases A-D at the mRNA level. Considerable progress was made toward the goals of this project. Hydrolases A and B have been purified to electrophoretic homogeneity, polyclonal and monoclonal antibodies against hydrolase A and polyclonal antibodies against hydrolase B have been prepared, and the mRNA encoding hydrolase A has been cloned and partially sequenced. Carboxyesterases are known to catalyze the hydrolysis of numerous xenobiotics, including pesticides, environmental contaminants, drugs and mycotoxins. Therefore, the experiments outlined in this proposal will contribute significantly to our basic understanding of carboxyesterases as a family of enzymes involved in the detoxication of foreign compounds.
