CHARACTERIZATION OF A PHENOBARBITAL-INDUCIBLE CYTOCHROME-P-450, NADPH-CYTOCHROME P-450 REDUCTASE AND RECONSTITUTED CYTOCHROME-P-450 MONOOXYGENASE SYSTEM FROM RAT-BRAIN - EVIDENCE FOR CONSTITUTIVE PRESENCE IN RAT AND HUMAN BRAIN

被引:25
作者
ANANDATHEERTHAVARADA, HK
BOYD, MR
RAVINDRANATH, V
机构
[1] NATL INST MENTAL HLTH & NEUROSCI,DEPT NEUROCHEM,HOSUR RD,BANGALORE 560029,KARNATAKA,INDIA
[2] NCI,DRUG DISCOVERY RES & DEV LAB,FREDERICK,MD 21702
关键词
D O I
10.1042/bj2880483
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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).
引用
收藏
页码:483 / 488
页数:6
相关论文
共 34 条
[1]   RAT-BRAIN CYTOCHROMES P-450 - CATALYTIC, IMMUNOCHEMICAL PROPERTIES AND INDUCIBILITY OF MULTIPLE FORMS [J].
ANANDATHEERTHAVARADA, HK ;
SHANKAR, SK ;
RAVINDRANATH, V .
BRAIN RESEARCH, 1990, 536 (1-2) :339-343
[2]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[3]   CLONED AND EXPRESSED NITRIC-OXIDE SYNTHASE STRUCTURALLY RESEMBLES CYTOCHROME-P-450 REDUCTASE [J].
BREDT, DS ;
HWANG, PM ;
GLATT, CE ;
LOWENSTEIN, C ;
REED, RR ;
SNYDER, SH .
NATURE, 1991, 351 (6329) :714-718
[4]   ALZHEIMERS-DISEASE, PARKINSONS-DISEASE, AND MOTONEURON DISEASE - ABIOTROPIC INTERACTION BETWEEN AGING AND ENVIRONMENT [J].
CALNE, DB ;
MCGEER, E ;
EISEN, A ;
SPENCER, P .
LANCET, 1986, 2 (8515) :1067-1070
[5]  
DEMONTELLANO O, 1986, CYTOCHROME P 450 STR
[6]  
GREENLEE WF, 1978, J PHARMACOL EXP THER, V205, P596
[7]  
GUENGERICH FP, 1977, MOL PHARMACOL, V13, P911
[8]  
GUENGERICH FP, 1987, MAMMALIAN CYTOCHROME, V1, P1
[9]  
GUENGERICH FP, 1984, PRINCIPLES METHODS T, P609
[10]   CHARACTERIZATION OF 3 CYTOCHROME-P450S PURIFIED FROM RENAL MICROSOMES OF UNTREATED MALE-RATS AND COMPARISON WITH HUMAN RENAL CYTOCHROME-P450 [J].
IMAOKA, S ;
NAGASHIMA, K ;
FUNAE, Y .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1990, 276 (02) :473-480