AuthorFraser, David John, 1968-
KeywordsHealth Sciences, Pharmacology.
AdvisorHalpert, 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 these studies was to isolate and characterize novel members of the 3A subfamily of canine cytochromes P450. This subfamily is of interest due to the wide range of endogenous and exogenous compounds metabolized. Previous studies led to the identification, isolation, and heterologous expression of 3A12, the major P450 form in canine liver. This enzyme demonstrated the ability to metabolize steroidal compounds and macrolide antibiotics in reconstituted systems. However, several lines of evidence suggested the presence of additional 3A enzymes with different substrate specificities. Initial experiments employed the same library used to clone the 3A12 cDNA and led to the isolation of a cDNA encoding P450 3A26. This new cDNA encoded a protein of 503 amino acids with 33 nucleotide differences encoding 22 amino acid variations compared with 3A12. Immunoblots indicated that 3A26 comigrates with a previously unidentified 3A protein in PB-induced canine microsomes. The 3A26 cDNA was modified for heterologous expression and cloned into the pSE380 expression vector. Expression of 3A26 and 3A12 in E. coli was accomplished using slightly modified protocols developed in this laboratory. Steroid hydroxylase assays indicated that these two enzymes have distinct catalytic profiles, with 3A12 exhibiting a rate of 6β-hydroxysteroid product formation ranging from 4 to 50-fold higher than 3A26. The vast differences in the activities of 3A12 and 3A26 in contrast to their similarity in structure made these enzymes an excellent model system for the identification of structure-function relationships. Studies were done to identify which of the 22 amino acid residue differences between 3A12 and 3A26 confer differences in rates of hydroxylation of progesterone, testosterone and androstenedione. Ten different 3A12/3A26 chimeras were generated using restriction endonuclease sites. Hydroxylase assays indicated that the first four residue changes and the six differences found within an internal PstI fragment were at least partially responsible for differences in the hydroxylation rates of all three steroids tested. Site-directed mutagenesis revealed the importance of 3A26 residues Ile-187, Ser-368, and Val-369. Conversion of these positions to 3A12 residues increased the rates of 6β-hydroxysteroid formation by 10-20 fold for progesterone, testosterone, and androstenedione. These studies identified a new member of the P450 3A subfamily and are the first to use catalytically distinct cytochromes P450 3A from the same species in the elucidation of 3A structure-function relationships.
Degree ProgramGraduate College
Pharmacology and Toxicology