Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations 3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.