Studies on the Mechanism of Action of Triphenyltin on Proton Conduction by the H+-ATPase of Mitochondria

Abstract

A study is presented of the action of triphenyltin on the kinetics of the anaerobic relaxation of the proton gradient set up by respiration in various type of 'inside-out' inner membrane vesicles obtained by exposure of beef-heart mitochondria to ultrasonic energy.Triphenyltin is shown to act as a powerful inhibitor of the proton conductivity of the H+-ATPase. The inhibition persists after removal of the ATPase protein inhibitor, F 1 and the oligomycin-sensitivity conferral protein (OSCP) from the particles. The inhibitory effect of triphenyltin is exerted, as in the case of oligomycin and N,N'-dicyclohexylcarbodiimide, on the Fo moiety of the ATPase complex.Comparison of the characteristics of the effect of triphenyltin on proton translocation in chloride and nitrate media shows that the inhibition of passive proton conductivity studied here is unrelated to the hydroxide/ anion exchange induced by the organotin.Lack of additivity of the inhibition of H+ conduction by triphenyltin with that exerted by oligomycin and N,N'-dicyclohexylcarbodiimide and the kinetic pattern of the effect of triphenyltin show that the mechanism of action of the organotin is different from that of the other two inhibitors.The relevance of the results obtained with respect to the subunit location and chemical nature of the reaction site of triphenyltin in the H+-ATPase complex is discussed.The membrane sector, Fo, of the H+-ATPase of coupling membranes functions as proton translocator [l -71. In the intact H+-ATPase complex proton translocation by FO is compulsorily coupled to the hydrodehydration reaction catalyzed by the peripheral, F1 moiety of the complex [1,2]. Displacement or removal of F1 from the complex, as occurs in 'insideout' submitochondrial particles, unmasks the proton conductivity of FO [5,6,8 -101. Thus anaerobic relaxation of the electrochemical proton gradient, dpH', set up by respiration in these vesicles, takes place through the proton conducting pathway of FO [5,6,8,9]. FO preparations from different sources consist of a variable number of protein subunits [I 11. The use of DCCD, a specific reagent for glutamic and aspartic residues [I21 which inhibits proton conduction by Fo [1,2,6], has shown that a 7000-8000 proteolipid subunit of Fo, bearing a glutamic [13] or aspartic residue [I41 specifically attacked by DCCD [15], is directly involved in proton translocation [l, 21. Modification with phenylglyoxal of arginine residues and of tyrosine with tetranitromethane results in depression of the proton conductivity of FO of the thermophilic bacteria PS3 [16,17] and mitochondria [S, 91.Organotin compounds, besides their activity in inducing hydroxide/anion exchange-diffusion [18,19], inhibit the ATPase of mitochondria [19-211 and chloroplasts [22,23]. There are reports that this is probably due, as with oligomycin and DCCD [1,2,6], to depression of H+-conduction by FO [23,24]. The inhibition of the H+-ATPase of mitochondria by triAhhrcviurions. DCCD, N,N'-dicyclohexylcarbodiimie; ESMP, submitochondrial particles prepare...

Effect of Thiol Reagents on the Proton Conductivity of the H+-ATPase of Mitochondria

“…1 A show the effect of 3 mM arsenitethis corresponds to the concentration of the reagent effective on thiol-depending dehydrogenases [18,19]-on the cycle of respiration-driven proton uptake and subsequent anaerobic proton releasc in ESMP. The arsenical, in contrast to oligomycin and triphenyltin (see Table 1 and [14]) which inhibited anaerobic proton release, caused a marked acceleration of the process. The extent of aerobic proton uptake did not decrease, evidently due to stimulatory effect of arsenite also on the rate of respiration-driven proton uptake (see also Table 1).…”

“…This substance causes an inhibition of proton conduction which does not change with time and does not perturb the kinetic pattern of the process (cf. [14]). …”

“…Respiration-driven proton translocation was activated by repetitive pulse of 1 -3 H202 (5 pl/ml). The pH of the suspension was monitored potentiometrically [14,24]. For the kinetic analysis of the anaerobic proton release from submitochondria1 particles the potentiometric traces were converted into proton equivalents by double titration with standard HCI and KOH and treated by a double-exponential equation [25].…”

“…The percentage of inversion of submitochondrial particles was found to range from preparation to preparation from 96 to 100 "/d [5,14]. The ATPase protein inhibitor [22] was removed by passing ESMP through a Sephadex column [23].…”

Cotton in Brazil: Importance and Chemical Control of Bolls Rot

“…This fungicide acts as a multi-site inhibitors, prevents spore germination and inhibits the metabolism of fungi, particularly respiration, inhibits oxidative phosphorylation in mitochondria and induces lipid peroxidation. Low concentrations of organostannic compounds inhibit the translocation of H + bound to the membrane, H +ATPase and ions such as Na + and K + (Papa et al, 1982;Powers & Beavis, 1991). Lawrence et al (2007a) while testing the fungicides Quadris ® and 2.08SC Topsin M ® to control bolls rot found that foliar spraying of fungicide 2-4 times during flowering increased the cotton yield.…”