Inhibition Of DT-diaphorase Research Articles

Lapachol inhibition of DT-Diaphorase (NAD(P)H:Quinone Dehydrogenase)

Lapachol has been found to be a potent inhibitor of the enzyme DT-Diaphorase. Inhibition is competitive versus NADH, K i = 0.15 μM. Lapachol was not a good substrate for cytochrome P450 reductase, thus inhibition of DT-Diaphorase should not promote its metabolism via radical generating pathways. DT-Diaphorase has been used to test a lapachol affinity chromatography column designed for purification of another coumarin anticoagulant and lapachol sensitive enzyme, vitamin K epoxide reductase.

Indirect inhibition of vitamin K epoxide reduction by salicylate.

Salicylate antagonizes the vitamin K-dependent biosynthesis of clotting factors in the rat and produces an elevation of the ratio of vitamin K epoxide to vitamin K in the liver. Vitamin K epoxide is reduced to vitamin K by a vitamin K epoxide reductase, and 1 mM salicylate was required to cause a 50% inhibition of the dithiothreitol-dependent in-vitro reduction of vitamin K epoxide by this enzyme. This enzyme was, however, inhibited 50% by as little as 70-80 microM salicylate when reducing equivalents for the reaction were furnished by endogenous cytosolic reductants. This effect on the cytosolic reductant supply was shown to be unrelated to a previously demonstrated inhibition of DT-diaphorase by salicylate. The concentrations of salicylate at which significant inhibitory effects are exerted in-vitro (50-100 microM) are below the 200 microM levels observed in the livers of rats given an anticoagulating dose of salicylate.

Studies on the reaction mechanism of DT diaphorase: Action of dead-end inhibitors and effects of phospholipids

The relationship between the inhibition of DT diaphorase [NAD(P)H dehydrogenase (quinone): EC.1.6.99.2] by 4-hydroxycoumarin and the 1,3-indandione derivates was investigated. Evidence is presented that these two classes of anticoagulants, although both acting as competitive inhibitors with respect to NAD(P)H, bind to different sites of the enzyme in a synergistic fashion. These findings are interpreted as indicative of a cooperativity between different substrate-binding sites of the enzyme. Neutral phospholipids exert effects on partially purified rat-liver DT diaphorase similar to those earlier obtained with nonionic detergents. The effects concern several kinetic parameters of the enzyme, including V, K m for electron donor and acceptor, and K i for various inhibitors. The changes in the kinetic parameters vary in extent and direction according to the individual phospholipids.