title: NADPH-cytochrome P-450 reductase (pig liver). Studies on the sequence of the cyanogen bromide peptides from the catalytic domain and on the reactivity of the thiol groups.
abstract: The reactivity of the cysteine residues in the non-denatured catalytic domain of the NADPH-cytochrome P-450 reductase (pig liver) was studied using the -SH reagent monobromobimane. Prerequisite was the characterization of the cysteine residues by their surrounding amino-acid sequences. In pursuit of these aims the CNBr fragments obtained from the catalytic domain were sequenced. The cysteine residues are distributed on six CNBr fragments of the catalytic domain [Vogel and Lumper (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1074]. Only the 11-kDa CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr- and Ala-Ser-Ser-Ser-, respectively, contain two cysteine residues each. The cysteine residues of the catalytic domain accessible to monobromobimane were localized on three CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser- and Ala-Arg-Asp-Val-, respectively. Inactivation of the trypsin-solubilized enzyme by -SH-directed reagents is caused by the modification of the accessible cysteine residue (which can be protected by NADPH) in the 11-kDa CNBr fragment (N-terminal sequence: Val-Gly-Pro-Thr-). The cosubstrate NADPH protected a second cysteine residue localized in the 11-kDa CNBr peptide with the N-terminal sequence Ala-Ser-Ser-Ser-, which is however modified at a distinctly slower rate than the critical cysteine residue characterized by the sequence -Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg-Arg. Five non-reacting thiol groups were localized on CNBr fragments with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser-, Ser-Leu-Asn-Asn-, Gly-Lys-Tyr-Val-Asp- and Ala-Ala-Asp-Pro-.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D010446:Peptide Fragments; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase; D003545:Cysteine; D003488:Cyanogen Bromide