Structural and functional analysis of NADPH-cytochrome P-450 reductase from human liver: complete sequence of human enzyme and NADPH-binding sites.
The complete amino acid sequence of human liver NADPH-cytochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linked glycosylation. The human enzyme exhibits sequence homology in the range of 92-95% with other mammalian enzymes. Sequence differences were mainly confined to several hydrophilic regions in the NH2-terminal and COOH-terminal domains. Since the human enzyme is immunochemically distinct from the rabbit enzyme despite similar enzymatic properties, it is likely that these variable hydrophilic regions are potential antigenic determinants. The NADPH-depleted enzyme is inactivated by either fluorescein isothiocyanate, a lysine-specific reagent, or 5-(iodoacetamido)fluorescein, a cysteine-specific reagent. In both cases, protection by NADP(H) prevents enzyme inactivation by the reagents. Isolation of fluorescent peptide from 5-(iodoacetamido)fluorescein-inactivated enzyme identified Cys 565 as the specifically NADPH-protected residue.
Haniu
M
M
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
McManus
M E
ME
Birkett
D J
DJ
Lee
T D
TD
Shively
J E
JE
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D013861:Thiocyanates / Q000494:pharmacology
D005452:Fluoresceins; D013861:Thiocyanates; C015296:5-iodoacetamidofluorescein; D009251:NADPH-Ferrihemoprotein Reductase; D016650:Fluorescein-5-isothiocyanate