CORE--ENZYMES AND ANTIBODIES FACILITY

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The overall purpose of the Enzymes and Antibodies Facility Core is to provide expertise and facilities for the characterization of enzymes that are important in the biotransformation of environmental chemicals. The application provides a detailed list of specific services to be provided by the Enzymes and Antibodies Facility Core. The Enzyme and Antibody Facility Core was formed in October of 1996 by combining two pre-existing Cores, namely the Monoclonal Antibody Production and Assay Laboratory (which was directed by P.E. Thomas), and the Laboratory for Protein Purification and Characterization (which was directed by C.S. Yang). The new Core Facility is directed by Dr. Thomas, which provides continuity. Dr. Thomas will be assisted by Dr. Hong, who will oversee the Core Facility?s new role in providing expertise and resources for expressing recombinant enzymes, for analyzing genetic polymorphisms in xenobiotic-metabolizing enzymes (XME?s) and for preparing DNA probes. The Enzyme and Antibody Facility Core is located in laboratories in three different buildings: the EOSHI building, the Laboratory for Cancer Research (located next to the EOSHI building) and Dr. Chada?s laboratory in the RWJMS (also located next to the EOSHI building). This Core Facility is readily accessible to members of the Center. The Enzyme and Antibody Facility Core has produced one of best, if not the best, inventory of monoclonal and polyclonal antibodies against various P450 enzymes and other xenobiotic-metabolizing enzymes (XME). The inventory is impressive not only in terms of the number of antibodies that have been prepared, but also for the degree to which these antibodies have been characterized with respect to their specificity and properties (such as whether they recognize the denatured P450 enzyme on western immunoblots, and whether they inhibit P450-dependent metabolism, a highly desirable property of such antibodies). The Enzyme and Antibody Facility Core has supported an impressive number and variety of research projects carried out by Center members. The facility was used to prepare recombinant P450 enzymes for studies of methyl-tert-butyl ether (MTBE) metabolism (for C.S. Yang and J.Y. Hong); to purify P450 enzymes, the inducible form of nitric oxide synthase and muconaldehyde cross-linked proteins (for P.E. Thomas, J. Laskin and G. Witz); to prepare monoclonal antibodies against heme oxygenase (for M. Iba); to provide antibodies for immunoquantitation and immunocytochemistry (for M. Iba, D. Morse, H. Lowndres, C.S. Yang, J. Landau, C. Gardner, D. Laskin and numerous outside investigators); to conduct antibody-inhibition experiments to identify the contribution of individual P450 enzymes to the metabolism of MTBE (for C.S. Yang and J.Y. Hong) and estradiol (for B.T. Zhu and A.H. Conney); and to screen for polymorphisms in the human genes encoding CYP2A6, CYP2E1, the pi and mu forms glutathione S-transferases and quinone oxidoreductase (for J.Y. Hong and S. Mohr). The Enzyme and Antibody Facility Core also provides training in a variety of immunochemical and molecular biology techniques. The Enzyme and Antibody Facility Core is used by members in each of the research cores (albeit to varying extent), and by numerous outside researchers. The application identifies a large number of future research projects that will be supported by the Enzyme and Antibody Facility Core.