NAD(P)H dehydrogenase and its role in the vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone)-dependent carboxylation reaction.
A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
D000818:Animals; D001360:Avidin / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D014812:Vitamin K / Q000378:metabolism*; D014859:Warfarin / Q000494:pharmacology
D011725:Pyridines; D014812:Vitamin K; D001360:Avidin; D014859:Warfarin; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D004229:Dithiothreitol