Purification and characterization of NADPH-cytochrome P-450 reductase from rat epidermis.
NADPH-cytochrome P-450 oxidoreductase (P-450 red) transfers reducing equivalents from NADPH to cytochrome P-450 (P-450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2',5'-ADP-agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH-cytochrome c reductase activity increased by 95-fold in the purified enzyme. Epidermal P-450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P-450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P-450 red immunologically cross reacts to liver P-450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with beta-naphthoflavone, which results in a substantial increase in P-450 1A1 activity. Quantitative assessment of P-450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P-450 red, fully capable of supporting P-450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.
Takahara
H
H
Department of Dermatology, University of California, San Francisco 94143.
Zaidi
S I
SI
Mukhtar
H
H
Handa
M
M
Epstein
W L
WL
Fukuyama
K
K
D000818:Animals; D000831:Animals, Newborn; D001571:Benzoflavones / Q000494:pharmacology; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis / Q000201:enzymology*; D005260:Female; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D009249:NADP; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase