The ultimate goal of this research proposal is the biochemical characterization of human forms of the microsomal flavin-containing monooxygenase (FCM). Studies performed exclusively with FCM derived from animal sources show that the FCM exists in multiple forms and functions in a variety of metabolic transformations, some of which are bioactivation reactions resulting in mutagenic and carcinogenic events. The major hypothesis to be tested in this proposal is that multiple forms of FCM exist in human pulmonary and hepatic tissue. Our preliminary studies indicate that purified hepatic and pulmonary forms of FCM from animal sources may be differentiated on the basis of their stereoselective metabolism of prochiral thioethers. This research proposal will address specifically; a) if these prochiral substrates have a general utility as probes of the active site topology of FCM isozymes, b) whether these probe substrates can identify multiple forms of FCM in human tissue microsomes, c) the purification of FCM from human tissues, and d) the catalytic competency of purified human forms, their immunochemical relatedness and extent of homology of their N-terminal sequences. Therefore, we propose to probe the active site of human pulmonary and hepatic FCM by investigating the stereoselective sulfoxidation of a series of prochiral thioethers. That the degree of stereoselective sulfoxidation of members of such a series is indeed a function of the FCM isozyme involved, will be assessed following analysis by chiral phase GC or HPLC using several purified isozymes from animal sources. A sensitive GC/Ms assay combined with specific antibodies to human liver P-450 reductase will provide the tools necessary to dissect the microsomal FCM component of sulfoxidation in human tissues. Conclusions drawn from these studies regarding inter- and intra-tissue heterogeneity of FCM will be tested by purification of the component isozymes. Subsequent comparisons of their immunochemical relatedness, substrate specificities and, ultimately, their N-terminal sequences as deduced from FABMS/MS will fully delineate isozymic status.
