Studies on the Respiratory Chain-linked Nicotinamide Adenine Dinucleotide Dehydrogenase: XXII. RHEIN, A COMPETITIVE INHIBITOR OF THE DEHYDROGENASE

Abstract

Abstract Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) inhibits the mitochondrial oxidation of reduced nicotamide adenine dinucleotide (NADH) but not of succinate. The inhibition is competitive with respect to substrate and involves a block between NADH and the flavin of NADH dehydrogenase. The Ki is about 2 µm at 30°. Evidence for this localization has come from kinetic analysis of the effect of rhein on the transhydrogenation and oxidation of NADH by purified NADH dehydrogenase. Rhein inhibits at similar concentrations the NADH-ferricyanide reaction and energy-linked NAD reduction in submitochondrial particles, but in these instances a secondary, noncompetitive inhibition is also apparent. Rhein has a pronounced tendency to combine with proteins, resulting in shifts of its absorption spectrum. These spectral changes may be used to monitor the combination of rhein with submitochondrial particles and the release of rhein from NADH dehydrogenase by NAD. The displacement of rhein by NAD suggests that this inhibitor may be used as a selective probe of the active site of NADH dehydrogenase. Combination of rhein with bovine serum albumin may be used to prevent or reverse the inhibition of NADH dehydrogenase. Combination with other proteins may mask the inhibition of respiratory chain-linked NADH dehydrogenase in tissues or mitochondria having a relatively low content of NADH dehydrogenase, e.g. liver. Rhein is not entirely specific for NADH dehydrogenase; at similar concentrations it also inhibits mitochondrial transhydrogenation and DT-diaphorase and at somewhat higher concentrations lactate and malate dehydrogenases from heart muscle but is without effect on a number of other pyridine nucleotide-linked enzymes, flavoproteins, and membrane-bound enzymes tested.