Studies on the Respiratory Chain-linked Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase: XVII. REACTION SITES OF PIERICIDIN A AND ROTENONE

Abstract

Abstract An improved estimate from Scatchard plots of the number of specific binding sites of the electron transport particle for 14C-piericidin A gives a value of 2 moles of inhibitor bound per mole of calculated NADH dehydrogenase content. The affinity of these two specific binding sites for piericidin A is very similar, since the binding curve is a smooth hyperbola and the Scatchard plot is linear. The two sites may be distinguished, however, by their unequal contribution to the inhibition of NADH oxidation. This is shown by the sigmoidicity of the curves relating inhibition of NADH oxidase, NADH-coenzyme Q reductase, and energy-linked NAD reductase activities to piericidin concentration. The inhibition of NADH oxidase and energy-linked NAD reductase activities but not of NADH-coenzyme Q reductase activity are partially reversed by washing of inhibited particles with bovine serum albumin. Hence both specifically and unspecifically bound piericidin contribute to the inhibition of the first two activities while only specifically bound inhibitor is involved in the block of external coenzyme Q reduction. The titration curve for inhibition of NADH oxidase by rotenone is more complex, apparently because of the contribution of inhibition at unspecific sites. The number of specific binding sites for piericidin may be reduced to near 1 per mole of NADH dehydrogenase by treatment of the electron transport particle with mercurials and by dissociation into the component complexes with bile salts. The results are discussed in terms of the location of the binding and inhibition sites of rotenone and piericidin in the respiratory chain and of the possible participation of NADH dehydrogenase itself and other membrane components in the binding of these inhibitors.