The F1 and F1-inhibitor-protein complex synthesized tightly bound ATP from ADP and Pi when the organic solvents dimethylsulfoxide (20 -50% v/v), ethyleneglycol (20 -60% v/v) or poly(ethyleneglyco1) 4000 and 8000 (30 -50% w/v) were included in the assay media. There was no synthesis of tightly bound ATP in the absence of organic solvents.In the presence of 50% dimethylsulfoxide, maximal synthesis of ATP was obtained at pH values between 6.5 and 7.7. In both F1 and F1 -inhibitor-protein there was no synthesis of ATP in the absence of MgC12. The rate of ATP synthesis became faster as the MgC12 concentration in the medium was raised from 0.1 -10 mM. The K , for Pi of F1 was in the range of 0.8-1.5 mM. The K, for Pi of the F1 -inhibitor-protein was much higher than that of F1 and could not be measured.In the presence of 10 mM MgC12 and 2 mM Pi, the rate constants of ATP synthesis by F1 and F1 -inhibitorprotein were 5.2-10.4 h-' and 3.5-5.9 h-' respectively. For both enzymes the rate constant of ATP hydrolysis was 0.69 h-'. The tightly bound ATP of F1 and F1 -inhibitor-protein were hydrolyzed at a much slower rate when either the Pi concentration or the MgClz concentration was suddenly decreased.Both in presence and absence of MgZ+, 40-6OY0 of the radioactive tightly bound ATP synthesized by F1 was hydrolyzed when non-radioactive ATP was added to the assay medium. This was not observed when F -inhibitor-protein was used.Fo-F1 complex of mitochondria, chloroplasts and bacteria catalyzes the synthesis of ATP using the energy of the electrochemicl H + gradient derived from electron transport. The soluble F1 catalyzes an exchange between water oxygen and the oxygen of phosphate [l -41. These studies indicate that the energy of the H f gradient is necessary not for the synthesis of ATP at the catalytic site of the enzyme but instead serves to increase the affinity of the enzyme for Pi and to permit the dissociation from the enzyme of the tightly bound ATP, which forms spontaneously observed the synthesis of about 0.2 mol ATP/mol chloroplast CF, when the enzyme was incubated in a medium containing 100mM Pi. On the other hand several authors [6 -91 have failed to detect the spontaneous synthesis of significant amounts of ATP when the mitochondrial F1 was incubated in media containing ADP and a high Pi concentration (10-500 mM). This has been attributed to the very low affinity of the soluble F1 for Pi [2, 71.Vesicles derived from the sarcoplasmic reticulum retain a membrane-bound Ca2 +-ATPase, which is able to catalyze the synthesis of ATP from ADP and Pi when a CaZ+ gradient is formed across the vesicle membrane (for review, see [lo, 111).In the absence of a Ca2+ gradient the affinity of the Ca2+-ATPase for Pi is increased by several orders of magnitude when part of the water of the incubation medium is replaced by an organic solvent such as MezSO [ll -131. A similar phenomenon is observed with F1 of mitochondria reported that the concentration of Pi required for half-maximal synthesis of tightly bound ATP is higher than...
The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.
When the electrochemical proton gradient is disrupted in the mitochondria, IF1 (Inhibitor Factor-1) inhibits the reverse hydrolytic activity of the F1Fo-ATP synthase, thereby allowing cells to conserve ATP at the expense of losing the mitochondrial membrane potential (Δψm). The function of IF1 has been studied mainly in different cell lines, but these studies have generated contrasting results, which have not been helpful to understand the real role of this protein in a whole organism. In this work, we studied IF1 function in Caenorhabditis elegans to understand IF1´s role in vivo. C. elegans has two inhibitor proteins of the F1Fo-ATPase, MAI-1 and MAI-2. To determine their protein localization in C. elegans, we generated translational reporters and found that MAI-2 is expressed ubiquitously in the mitochondria; conversely, MAI-1 was found in the cytoplasm and nuclei of certain tissues. By CRISPR/Cas9 genome editing, we generated mai-2 mutant alleles. Here, we showed that mai-2 mutant animals have normal progeny, embryonic development and lifespan. Contrasting with the results previously obtained in cell lines, we found no evident defects in the mitochondrial network, dimer/monomer ATP synthase ratio, ATP concentration or respiration. Our results suggest that some of the roles previously attributed to IF1 in cell lines could not reflect the function of this protein in a whole organism and could be attributed to specific cell lines or methods used to silence, knockout or overexpress this protein. However, we did observe that animals lacking IF1 had an enhanced Δψm and lower physiological germ cell apoptosis. Importantly, we found that mai-2 mutant animals must be under stress to observe the role of IF1. Accordingly, we observed that mai-2 mutant animals were more sensitive to heat shock, oxidative stress and electron transport chain blockade. Furthermore, we observed that IF1 is important to induce germ cell apoptosis under certain types of stress. Here, we propose that MAI-2 might play a role in apoptosis by regulating Δψm. Additionally, we suggest that IF1 function is mainly observed under stress and that, under physiological conditions, this protein does not play an essential role.
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...
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