Studies on the Respiratory Chain-linked Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase: XV. Interactions of piericidin with the mitochondrial respiratory chain

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Abstract Titration of submitochondrial particles with 14C-piericidin A, labeled by biosynthesis, results in linear binding which continues well beyond the point at which NADH oxidase activity is completely inhibited. However, when the titrated particles are treated with a bovine serum albumin solution, excellent proportionality is found between the binding and inhibition curves, indicating that binding is only at the specific site associated with NADH oxidase activity, and not at unspecific sites. The titer for 14C-piericidin A at the specific site in membrane fragments of varying complexity in cytochrome composition is 1 to 1.5 times the estimated concentration of NADH dehydrogenase. These findings confirm and extend previous studies with 14C-rotenone which binds at the same site but with a lower affinity, requiring indirect rather than direct calculation of the titer. The binding of 14C-piericidin A to the specific site is noncovalent, since the radiocarbon bound to this site is released by protein denaturants, phospholipase A, and proteolytic enzymes. Rotenone and Amytal, which bind at the same specific site as piericidin A, do not effectively compete with piericidin A nor remove it from this site, while piericidin A readily displaces bound rotenone from the specific site. Thus, piericidin A is a more specific and more tightly bound inhibitor of the NADH oxidase segment of the respiratory chain than either rotenone or barbiturates. Several labeled 14C-compounds related to piericidin A, although qualitatively acting in the same manner, are less effective inhibitors and bind less tightly. The inhibition of succinoxidase activity by piericidin A reported in the literature requires high inhibitor concentration and is probably associated with unspecific binding. Only cytochrome b reduction accompanies NADH oxidation in piericidin A-inhibited submitochondrial particles, whereas all the cytochromes are slowly reduced in piericidin A-inhibited mitochondria.