Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae.
The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Brian
W R
WR
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Srivastava
P K
PK
Umbenhauer
D R
DR
Lloyd
R S
RS
Guengerich
F P
FP
D002384:Catalysis; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006591:Hexobarbital / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008617:Mephenytoin / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013426:Sulfaphenazole / Q000494:pharmacology; D014044:Tolbutamide / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D013426:Sulfaphenazole; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D006591:Hexobarbital; D006899:Mixed Function Oxygenases; C053553:tolbutamide 4-hydroxylase; D008617:Mephenytoin