Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N terminus and requires mitochondrial specific electron transfer proteins for activity.
Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.
Robin
M A
MA
Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6047, USA.
Anandatheerthavarada
H K
HK
Fang
J K
JK
Cudic
M
M
Otvos
L
L
Avadhani
N G
NG
D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D019392:Cytochrome P-450 CYP2E1 / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D010446:Peptide Fragments; D011720:Pyrazoles; D000325:Adrenodoxin; C031280:pyrazole; D019392:Cytochrome P-450 CYP2E1; D005287:Ferredoxin-NADP Reductase