Factors that control the reactivity of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi.

The amino acid sequences and X-ray structures of homodimeric triosephosphate isomerase from the pathogenic parasites Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM) are markedly similar. In the two TIMs, the side chain of the only interface cysteine (Cys14) of one subunit docks into loop 3 of the other subunit. This portion of the interface is also markedly similar in the two enzymes. Nonetheless, Cys14 of TcTIM is nearly 2 orders of magnitude more susceptible to the thiol reagent methylmethane thiosulfonate (MMTS) than Cys14 of TbTIM. The causes of this difference were explored by measuring the second-order rate constant of inactivation by MMTS (k(2)) under various conditions. At pH 7.4, k(2) in TcTIM is 70 times higher than in TbTIM. The difference decreases to 30 when the amino acid sequence of loop 3 and adjoining residues of TbTIM are conferred to TcTIM (triple mutant). The pK(a) values of the thiol group of the interface cysteine of TcTIM and the triple mutant were 0.7 pH unit lower than in TbTIM. Because this difference could account for the different sensitivity of the enzymes to thiol reagents, we determined the k(2) of inactivation at equal levels of ionization of their interface cysteines. Under these conditions, the difference in k(2) between TcTIM and TbTIM became 8-fold, whereas that of the triple mutant to TbTIM was 1.5 times. The substrate analogue phosphoglycolate did not modify the pK(a) of the thiol group of the interface, albeit it diminished the rate of its derivatization by MMTS. In the presence of phosphoglycolate, under conditions in which the interface cysteines of the enzymes had equal levels of protonation, the difference in k(2) of TcTIM and TbTIM became smaller, whereas k(2) of the triple mutant was almost equal to that of TbTIM. Thus, from measurements of the reactivity of the interface cysteine in various conditions, it was possible to obtain information on the factors that control the dynamics of a portion of the dimer interface.

Nucleotide binding drives conformational changes in the isolated alpha and beta subunits of the F(1)-ATPase from Escherichia coli.

The modeling of the rotatory mechanism performed by the F(1)-ATPase complex during ATP synthesis shows that the beta, but not the alpha subunit, undergoes large conformational changes that depend on the occupancy of the catalytic site. Here we determined by fluorescence spectroscopy the changes in tertiary structure and hydrophobic exposed area of the isolated alpha and beta subunits of the F(1)-ATPase complex from Escherichia coli upon adenine nucleotide binding. The results show that in the absence of intersubunit contacts, the two subunits exhibit markedly similar conformational movements.

Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane.

To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-A resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 A from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

Derivatization of the interface cysteine of triosephosphate isomerase from Trypanosoma brucei and Trypanosoma cruzi as probe of the interrelationship between the catalytic sites and the dimer interface.

In the interface of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), one cysteine of each monomer forms part of the intersubunit contacts. The relatively slow derivatization of these cysteines by sulfhydryl reagents induces progressive structural alterations and abolition of catalysis [Garza-Ramos et al. (1998) Eur. J. Biochem. 253, 684-691]. Derivatization of the interface cysteine by 5, 5-dithiobis(2-nitrobenzoate) (DTNB) and methylmethane thiosulfonate (MMTS) was used to probe if events at the catalytic site are transmitted to the dimer interface. It was found that enzymes in the active catalytic state are significantly less sensitive to the thiol reagents than in the resting state. Maximal protection against derivatization of the interface cysteine by thiol reagents was obtained at near-saturating substrate concentrations. Continuous recording of derivatization by DTNB showed that catalysis hinders the reaction of sulfhydryl reagents with the interface cysteine. Therefore, in addition to intrinsic structural barriers, catalysis imposes additional impediments to the action of thiol reagents on the interface cysteine. In TcTIM, the substrate analogue phosphoglycolate protected strongly against DTNB action, and to a lesser extent against MMTS action; in TbTIM, phosphoglycolate protected against the effect of DTNB, but not against the action of MMTS. This indicates that barriers of different magnitude to the reaction of thiol reagents with the interface cysteine are induced by the events at the catalytic site. Studies with a Cys14Ser mutant of TbTIM confirmed that all the described effects of sulfhydryl reagents on the trypanosomal enzymes are a consequence of derivatization of the interface cysteine.

Comparison of plasma membrane H+-ATPase activity in vesicles obtained from dry and hydrated maize embryos.

ATP hydrolysis from H+-ATPase of plasma membrane was measured in vesicles from maize embryos imbibed at times between 0 and 5 h. The activity had a maximum at 2 h of imbibition. In order to detect whether the enzyme had the same characteristics through the first 5 h of imbibition, vanadate and lysophophatydilcholine sensitivities, as well as trypsin, pH and temperature effects on the activity of the H+-ATPase from plasma membrane vesicles isolated from embryos imbibed at 0 or 5 h were studied. The results indicate that the activity expressed at 0 h is very different from the activity at 5 h. The activity from embryos imbibed for 5 h was less sensitive to vanadate, trypsin and lysophosphatidylcholine, more sensitive to denaturing temperatures and with a broader pH dependence, as compared to the activity from embryos that were not imbibed. When vanadate-sensitive ATPase activity was purified by anion exchange chromatography, the peaks obtained from the 0 and 5 h imbibed embryos were different and non-overlapping. These data could be interpreted in terms of different enzyme structures from dry and imbibed embryos due to either different primary structures or covalent modifications, or differences in membrane vicinities.

Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of suramin.

The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2-32 microg of T. brucei TIM per ml and 0.2-5 microg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 microg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM.

Unisite hydrolysis of [gamma 32 P]ATP by soluble mitochondrial F1-ATPase and its release by excess ADP and ATP. Effect of trifluoperazine.

Some of the characteristics of unisite hydrolysis of [gamma 32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [gamma 32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [gamma 32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [gamma 32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [gamma 32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30-40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [gamma 32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [gamma 32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.

Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues. The cysteine residues of triosephosphate isomerase.

The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S-phenyl-p-toluenethiosulfonate (MePhSO2-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhSO2-SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhSO2-SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pombe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSO2-SPh. Methyl metanethiosulfonate (MeSO2-SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeSO2-SMe inhibited by 40-50% the activity of that from S. pombe, 20-25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeSO2-SMe protected against the strong inhibiting action of MePhSO2-SPh. The latter observations suggest that MeSO2-SMe and MePhSO2-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K(m) for glyceraldehyde 3-phosphate and a 65% decrease in Vmax. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cys14 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.

A column centrifugation method for the reconstitution in liposomes of the mitochondrial F0F1 ATP synthase/ATPase.

A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F0F1 ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-beta-D maltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F0F1 complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H+ gradients. They also catalyzed the ATP-Pi exchange reactions. Thus the F0F1 complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.

Water-induced transitions in the K+ requirements for the activity of pyruvate kinase entrapped in reverse micelles.

The activity of pyruvate kinase was studied in reverse micelles formed with cetyltrimethylammonium bromide, n-octane, hexanol, and various amounts of water. In systems with 100% water, K+ is an essential activator of pyruvate kinase [Kachmar, J. F., & Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683]; i.e., without and with K+, the activities observed were 0.07 and 300 mumol/(min.mg), respectively. In the micellar system with 3.6% water (v/v), pyruvate kinase exhibited an activity of about 45 mumol/(min.mg), in the absence of K+. The kcat was about 450 times larger than that in 100% water without K+. Km values for ADP and phosphoenolpyruvate differed, but not markedly from those in 100% water with or without K+. The kinetics of pyruvate kinase in reverse micelles were not affected by K+. The activity curve of pyruvate kinase in reverse micelles without K+ in a pH range of 6.0-8.5 was almost superimposable to that of the enzyme in 100% water with K+, and it differed drastically from that in 100% water without K+. The fluorescence emission spectra of pyruvate kinase in 100% water exhibited a blue shift of 3 nm upon the addition of ligands (Mg2+, phosphoenolpyruvate, and K+) that cause a transition of the enzyme to its active state. Without ligands, the entrapment of pyruvate kinase in reverse micelles with 3.0% water produced a blue shift of nearly 2 nm with respect to that of the enzyme in 100% water without ligands. As water was raised to 7.0% (v/v), the maximal emission shifted to longer wavelengths; these changes paralleled the appearance of the K(+)-dependent activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Are there different water requirements in different steps of a catalytic cycle? Hydration effects at the E1 and E2 conformers of sarcoplasmic reticulum Ca(2+)-ATPase studied in organic solvents with low amounts of water.

The Ca(2+)-ATPase from sarcoplasmic reticulum was transferred in an active form to a low-water system composed of toluene, phospholipids, and Triton X-100 (TPT). The Ca(2+)-ATPase activity in the TPT system with 4.0% water (by vol. was about 50% of the activity observed in all-aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme-catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.

Binding of adenine nucleotides to the F1-inhibitor protein complex of bovine heart submitochondrial particles.

The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of photophosphorylation and electron transport by N,N-dimethylformamide.

The basal electron transport of pea chloroplasts was inhibited by 78% by 7% (v/v) N,N-dimethylformamide; the inhibition was partially reversed by NH4Cl. N,N-Dimethylformamide also inhibited the Pi-ATP exchange, ATP synthesis and to a smaller extent Mg2+-ATPase activity. Light induced proton uptake was not affected by up to 30% (v/v) N,N-dimethylformamide. Uncoupled electron transport in photosystem II was inhibited to a larger extent by N,N-dimethylformamide than in photosystem I. These results indicate that N,N-dimethylformamide acts as an inhibitor of energy transfer and electron transport.

Catalytic activity of cytochrome oxidase and cytochrome c in apolar solvents containing phospholipids and low amounts of water.

Cytochrome c and cytochrome oxidase, in bovine heart submitochondrial particles and in their purified forms, were transferred to a ternary system that contained phospholipids (10 mg/ml toluene), the apolar solvent toluene, and water at concentrations of 13-15 microliters (high water) and 3 microliters (low water) per milliliter of toluene. When the enzymes were transferred back to an all water system, they exhibited full catalytic capacity. In the low water ternary system, cytochrome c could be reduced by ascorbate introduced via inverted micelles. Also in this system, cytochrome oxidase was reduced by ascorbate and cytochrome c but its oxidation was highly impaired. Data on the kinetics of reduction by ascorbate of cytochrome c and cytochrome oxidase under these conditions are presented. Cytochrome oxidase reduced in the organic solvent by ascorbate failed to form a complex with CO, but formed a complex with cyanide introduced via inverted micelles. The oxidized and the ascorbate-reduced cytochrome oxidase-cyanide complex exhibited a trough at 415 nm and a peak at 433 nm. The extent and rate of formation of the cyanide complex were higher with the reduced form of cytochrome oxidase. To achieve protein-protein interactions (cytochrome c-cytochrome oxidase) in the ternary system, it was necessary to extract the two proteins together. There was no functional interaction when they were extracted separately and mixed. In the high water ternary system reduced cytochrome oxidase was not detected, and it oxidized ascorbate at a higher rate than in the low water system; however, this rate was several orders of magnitude lower than in aqueous media.

Effect of the electrochemical proton gradient and anions on the ATPase activity of soybean submitochondrial particles.

Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38 degrees C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of DeltamuH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by DeltamuH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles.

Thermostability of membrane systems in organic solvents.

Two multisubunit enzymes of the inner mitochondrial membrane, cytochrome oxidase and the H+-ATPase may be transferred into highly apolar solvents as protein-lipid complexes. At 70 degrees C and an initial water concentration of 13 microliters per ml organic solvent (toluene), the half-life of the ATPase was approx. 11 h, whereas that of cytochrome oxidase was about 100 s. Thermostability of cytochrome oxidase could be increased more than 100-times by decreasing the water concentration to 3 microliters per ml toluene. At this latter concentration of water the half-life of the ATPase at 90, 80 and 70 degrees C was 5, 48 and 96 h, respectively.

Release of the inhibitory action of the natural ATPase inhibitor protein on the mitochondrial ATPase.

The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4 mumol min-1 mg-1 to 6-7 mumol min-1 mg-1 upon incubation for 5-6 h at 38 degrees C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762-3769]. At maximal activation approximately 50% of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 125I-labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, delta H not equal to and delta S not equal to of the process were 53.34 kJ/mol, 50.83 kJ/mol and -158.99 J mol-1 K-1, respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.

Regulation of the synthesis and hydrolysis of ATP by mitochondrial ATPase. Role of the natural ATPase inhibitor protein.

The action of the natural ATPase inhibitor protein of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) on the mechanisms of energy conservation of heart mitochondria has been explored. The synthesis and hydrolysis of ATP and the Pi-ATP exchange reaction were studied in submitochondrial particles that possess the ATPase-inhibitor protein complex in two distinguishable states. In addition to their different rates of hydrolysis, the two states of the complex have been identified from their different accessibility to antibodies directed against the inhibitor protein, and from the different action of antibodies and trypsin on the ATPase activity of the two types of particles studied. The steady state rates of hydrolysis and of the Pi-ATP exchange reaction of the particles are determined by the state in which the ATPase-inhibitor complex exists. Apparently by modifying the rate of one of the steps involved in the catalytic reaction of the ATPase, the inhibitor protein determines the extent to which the enzyme is able to catalyze ATP hydrolysis and the Pi-ATP exchange reaction. This action of the inhibitor protein also reflects the rate at which the particles carry out oxidative phosphorylation.

Studies on the mechanism of action of triphenyltin on proton conduction by the H+-ATPase of mitochondria.

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, F1 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 F0 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 interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein.

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.