Three of the most highly expressed drug-metabolizing enzymes in lung are cytochrome P450 IIB and IVB and the flavin-containing monooxygenase (FMO). Previously, we have purified and characterized these enzymes and have derived their sequences from the nucleotide sequences of cloned cDNAs. During the past year our efforts have concentrated on further studies of the FMO. The FMO, which was thought to consist of two enzymes at the most, has been discovered to encompass a family of genes with at least five members, each of which belongs to a separate gene subfamily. Expression of these FMOs is both tissue and species dependent. The major liver enzyme in rabbits, for example, is a minor hepatic enzyme in mouse, but a major enzyme in mouse kidney. In lung, only one species, rabbit, appears to express only the """"""""lung"""""""" enzyme. In all other species examined -- mouse, rat, hamster, and guinea pig -- both """"""""liver"""""""" and """"""""lung"""""""" FMOs are expressed in lung. Multiple protein products for fMO in guinea pig and rabbit lung appear to be allelic variants whose primary structures differ at only two positions. Differences among the FMO enzymes are now being studied by expression in cDNAs in various systems, including COS-1, yeast and E. coli. Expression in COS-1 cells has allowed us to demonstrate that the kinetic properties of FMOs follow simple Michaelis-Menton equations and that the reported substrate activation of purified FMOs is likely due to more than one enzyme present in the preparations used. Work with chimeric proteins is being carried out in order to define the active sites of the FMOs. A second major area of this work involves the localization of enzymes by immunochemical and in situ hybridization techniques. With both methods we have defined areas of the vasculature endothelium that contain cytochrome P450, but not FMO. In addition, these, areas do not appear to express cytochrome P450 reductase, which suggests that the function of the P450 has nothing to do with monooxygenation.
