Abstract
Coupling factor B activity was measured by the stimulation of the ATP-driven NAD+ reduction by succinate or the 32Pi-ATP exchange activity of Factor B-depleted submitochondrial particles. Half-maximal coupling activity was inhibited by 30 microM cadmium, 5 microM phenylarsine oxide, or 0.3 mM arsenite-2,3-dimercaptopropanol. The inhibition was relieved by slight excess of dithiol but not by a 10-fold molar excess of 2-mercaptoethanol. Inhibition of coupling activity by phenylarsine oxide or cadmium was not due to interference in binding of Factor B to depleted particles. Isolated Factor B binds phenylarsine oxide resulting in loss of ability to stimulate depleted submitochondrial particles. The inhibition was largely overcome by dithiol but not by monothiols. The residual coupling activity of depleted submitochondrial particles was highly resistant to cadmium or arsenical. Moreover, binding of arsenical to the depleted particles per se, did not result in inhibition of Factor B-stimulated activity. Furthermore, the addition of phenylarsine oxide to H+-ATPase resulted in loss of Pi-ATP exchange and stimulation of oligomycin-sensitive ATPase activities. Both effects were further potentiated by 2-mercaptoethanol and reversed by dithiols. These effects parallel uncoupling of oxidative phosphorylation in mitochondria by these inhibitors and point to Factor B as the probable component sensitive to these inhibitors.
Highlights
Coupling factor Bactivity was measured by the stim-pling effects were observed in heart mitochondria and mitoulation of the ATP-drivenNAD' reduction by succinate chondrial fragments, although5 times higher levels of Cd2' or or the 32Pi-ATPexchange activity of Factor B-depleted BAL-arsenite were required ( 6 ) .When used separately, arsensubmitochondrial particles
Dition of phenylarsine oxide to H+-ATPase resulted in As part of theprogramtoelucidatethemechanism of loss of Pi-ATP exchange and stimulationof oligomycin- uncoupling by Cd", we undertook the fractionation of the sensitive ATPase activities
It was shown that 5 p~ cadmium, 50 p~ mol eachof cysteine andcystine/14,600-dalton monomer [13]
Summary
The general procedure for determining the effect of Cd2+, PhAsO, or BAL-arsenite on either Pi-ATexPchange or NAD' reduction by succinate involved incubation of protein (ETPH, AE-P, Fe, or H+-ATPase) in a volume of 50 p1 at 0 "C for 5 min with the reagent being tested.
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