Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities.
The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIB1 + IIB2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumarin, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.
Bhamre
S
S
Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore, India.
Anandatheerathavarada
H K
HK
Shankar
S K
SK
Boyd
M R
MR
Ravindranath
V
V
D001921:Brain / Q000201:enzymology*; D002474:Cell-Free System; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008875:Middle Aged; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D010105:Oxygenases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase