Functional characterization of hepatic microsomal and heterologousely expressed rabbit cytochrome P450 2B enzymes
AuthorGrimm, Scott Wayne.
Committee ChairHalpert, James R.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe objective of the research described in this dissertation was to characterize the function of four closely related cytochrome P450 2B enzymes in the rabbit. Although these enzymes display greater than 97% amino acid sequence identity, their expression is highly variable between different organs and in different individuals. Transient and stable heterologous expression systems were used to study the distinct catalytic properties of each P450 2B enzyme. Cytochrome P450 2B5 was found to have a unique pattern of catalytic activities in comparison to the P450 2B4, 2B-B1, and 2B-Bx forms. The regio- and stereoselectivity of hydroxylation of androstenedione in hepatic microsomes depended upon whether the animal expressed cytochrome P450 2B5. Whereas the catalytic activities of P450 2B5 were characterized by high steroid hydroxylase activities, P450 2B4 had relatively low steroid hydroxylase activity and much higher activity towards the non-steroid substrates than 2B5. Androstenedione 15α-hydroxylation and benzyloxyresorufin O-debenzylation were identified as selective markers of the activity of P450 2B5 and 2B4, respectively. Phencyclidine selectively inactivated P450 2B4 in hepatic microsomes from phenobarbital-induced rabbits as well as the expressed enzyme. The basis for poor inactivation of P450 2B5 by PCP was determined to be the low maximal rate constant for this P450 2B form. N-Aralkylated 1-aminobenzotriazole derivatives were found to be potent inactivators of both P450 2B enzymes and to be much less selective than phencyclidine. These results demonstrate that one or more of the amino acid differences in P450 2B5 are critical to its substrate specificities and selective inactivation. Rabbits which express P450 2B5 have the potential to exhibit different hepatic biotransformation pathways in comparison with animals that lack this enzyme.
Degree ProgramPharmacology & Toxicology