Isolation and characterization of the protein components of the liver microsomal O2-insensitive NADH-benzamidoxime reductase.
Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochrome b5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83-92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b5 and NADH cytochrome b5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxic N-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason why N-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.
Clement
B
B
Pharmazeutisches Institut, Christian-Albrechts-Universität zu Kiel, Gutenbergstr. 76, 24118 Kiel, Germany.
Lomb
R
R
Möller
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology*; D013552:Swine
D001550:Benzamidines; D009249:NADP; C053561:benzamidoxime; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; C418330:NADH-benzamidoxime reductase; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D010100:Oxygen