Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6.
High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.
Born
S L
SL
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, USA.
Fraser
D J
DJ
Harlow
G R
GR
Halpert
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001571:Benzoflavones / Q000494:pharmacology*; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000378:metabolism*; D011817:Rabbits
D001571:Benzoflavones; D011374:Progesterone; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System