Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain. Evidence for constitutive presence in rat and human brain.
Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).
Anandatheerthavarada
H K
HK
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore, India.
Boyd
M R
MR
Ravindranath
V
V
D000633:Aminopyrine N-Demethylase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D008297:Male; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013057:Spectrum Analysis; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D000633:Aminopyrine N-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital