Comparison of the Effects of 2-Phenylindolone and N-Ethylmaleimide on Energy-Linked Reactions in Submitochondrial Particles

Abstract

In a recent paper Sweetman et al. (1971) described the effects of a novel energy-transfer inhibitor, 2-phenylisatogen, on rat liver mitochondria. The effects of 2-phenylindolone, one of the reduction products of the isatogen, on energy-linked reactions in rat liver submitochondrial particles have now been investigated by A. P. G., A. J. S. and M. H. at the Sunderland Polytechnic. Since the results obtained showed a marked similarity to those obtained by K. J. C., C. R. L. and R. B. B. at Shell Research Ltd., who have been using [‘4C]N-ethylmaleimide to correlate inhibition of the partial reactions of oxidative phosphorylation with the binding of the thiol-blocking reagent to mitochondria1 membranes, we present them as a joint communication. Previous work (Sanadi et al., 1968; Foucher et al., 1969; Foucher & Gaudemer, 1971) suggested that thiol groups were involved in the reactions leading to ATP synthesis, but no attempt had been made to label isolated membrane components containing these thiol groups. Ox heart submitochondrial particles were used in the work on N-ethylmaleimide to avoid problems with the mercurial-sensitive phosphate-porter (Tyler, 1968 ; Fonyo & Bessman, 1968). The reactions studied with 2-phenylindolone and N-ethylmaleimide included NADH and succinate oxidation, energy-linked and non-energy-linked transhydrogenases, Mg’+-stirnulated adenosine-triphosphatase and the ATP-driven reduction of NAD+ by succinate. Without preincubation, lOnmol of 2-phenylindolone/mg of submitochondrialparticle protein inhibited the non-energy-linked transhydrogenase by 37.7 %, the ATPdriven transhydrogenase by 55.7 % and the aerobically driven reaction by 65.1 %. The same concentration inhibited the ATP-driven reduction of NAD+ by succinate by 72.5 %, but had no effect on the oxidation of succinate or NADH or on the Mg’+-stirnulated adenosine triphosphatase. The degree of inhibition of the two transhydrogenases was very similar to that seen with the thiol-blocking reagent N-ethylmaleimide. It was found that lOnmol of Nethylmaleimide/mg of submitochondrial-particle protein inhibited the non-energylinked reaction by 60.2 % and the ATP-driven reaction by 89.7 %. The N-ethylmaleimide was incubated with the particles for 14h at 2°C before measurement of the reaction. It was also found that lOnmol of inhibitor/mg of submitochondrial-particle protein failed to inhibit the Mg*+-stimulated adenosine triphosphatase during 2.5 h at 2°C; on the contrary, a variable stimulation of between 10 and 50% was observed. The ATP-driven reduction of NAD+ by succinate was inhibited by 35 % with lOnmol of N-ethylmaleimide/mg of submitochondrial-particle protein during 2.5 h at 2°C. However, the same concentration of N-ethylmaleimide inhibited NADH oxidation by 33% after lOmin at 2°C. It was therefore concluded that the inhibition of the ATPdriven reduction of NAD+ by succinate by N-ethylmaleimide was probably due to an interaction with the NADH dehydrogenase enzyme system present in the particles. The binding of [14C]N-ethylmaleimide was correlated with its inhibitory activity against the non-energy-linked and energy-linked transhydrogenases and with its stimulation of the Mg’+-stimulated adenosine triphosphatase (Fig. 1). The transhydrogenase reaction was chosen for this work since it does not involve the electrontransport-chain enzymes, some of which are sensitive to thiol-blocking reagents. After measurement of the reactions the submitochondrial particles were solubilized in a sodium dodecyl sulphate-dithiothreitol-EDTA solution and subjected to electrophoresis in polyacrylamide gels (Cattell et al., 1971). The distribution of radioactivity was then determined in each gel. The results obtained were surprising, since the single major