Abstract
We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.
Highlights
From the Laboratories of Biochemistry,Department ofAnimal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
We showed that the antibody to microsomal P-45Oc detected a 54-kDa protein, a putative mitochondriaspecific P-450 in mitochondria from 3-MC-treated rat liver which could not be detected in sufficient quantity in control liver mitochondria [25]
A proteinof similar size can be purified by affinity binding to theanti-P-450 IgG column from PEG fractions of mitochondria induced with both of these compoundsA. lthough not shown, the PEG fraction from P-NF-induced liver mitoplasts was able to metabolize benzo(a)pyrene in an in vitro system reconstituted with Adx + Adr as well as NADPH-cytochrome
Summary
From the Laboratories of Biochemistry,Department ofAnimal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Ne 450 have been purified and characterized with respect to their of these isoforms showing an apparentmolecular mass molecular and catalytic properties from the microsomes of of 52 kDa is termed P-450mtl and thseecond isoform hepatic and extrahepatictissues [2, 10, 11].two with 54-kDamolecular mass is termed P-450mt.Cy- isoforms of P-450 involved in cholesterol side chain cleavage tochrome P-450mt comigrates with similarliynduced and 1la-hydroxylation of cholesterol have been purified from microsomal P-45Oc (the major B-NF-inducible form) mitochondria of steroidogenic tissues, like adrenal cortex, and on sodium dodecyl sulfate-polyacrylamide gels and fully characterized [12,13,14]. A distinctive property of mitochondrial P-450mtl and P-450mt was that their catalytic activitcioeusld be reconstituted with both NADPH-cytochromeP-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, It is well established that hepatic mitochondria contain cytochrome P-450 which is able to catalyze 25-hydroxylation of vitamin Ds and 26-hydroxylation of C-27 sterols [17,18,19,20]. It is seen that there is a minor difference between the elution profile of control and p-
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