Initial Rates Of ATP Hydrolysis Research Articles

Interactions of Human Rad54 Protein with Branched DNA Molecules

The Rad54 protein plays an important role during homologous recombination in eukaryotes. The protein belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that yeast and human Rad54 (hRad54) specifically bind to Holliday junctions and promote branch migration. Here we examined the minimal DNA structural requirements for optimal hRad54 ATPase and branch migration activity. Although a 12-bp double-stranded DNA region of branched DNA is sufficient to induce ATPase activity, the minimal substrate that gave rise to optimal stimulation of the ATP hydrolysis rate consisted of two short double-stranded DNA arms, 15 bp each, combined with a 45-nucleotide single-stranded DNA branch. We showed that hRad54 binds preferentially to the open and not to the stacked conformation of branched DNA. Stoichiometric titration of hRad54 revealed formation of two types of hRad54 complexes with branched DNA substrates. The first of them, a dimer, is responsible for the ATPase activity of the protein. However, branch migration activity requires a significantly higher stoichiometry of hRad54, approximately 10 +/- 2 protein monomers/DNA molecule. This pleomorphism of hRad54 in formation of oligomeric complexes with DNA may correspond to multiple functions of the protein in homologous recombination.

The effect of inorganic pyrophosphate on the activity and Pi-binding properties of mitochondrial F1-ATPase

Interaction of F1-ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1-ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady-state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP-dependent reactivation of an inactive complex formed by F1-ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1-ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non-preincubated enzyme. PPi stimulates the binding of Pi to F1-ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high-affinity Pi binding. It follows from the results obtained that PPi interacts with the non-catalytic site(s) of F1-ATPase.

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES.

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.

Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1.

Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 microM) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.

Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation.

The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated. Superfusate collected during EFS rapidly metabolised exogenous ATP (100 microM) and 50% was broken down in 5.67+/-0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 40 microM and V(max) of 20.3 nmol ATP metabolized min(-1) ml(-1) superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80 degrees C for 10 min. ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC(50)=25 microM (95% confidence limits=22-27 microM), Hill slope=-1.06+/-0.04). When EFS was applied three times at 30 min intervals, ATP metabolism was 20-30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz. In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent.

The Activity of the ATP Synthase from Escherichia coli Is Regulated by the Transmembrane Proton Motive Force

The ATP synthase from Escherichia coli was reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential, and one second after energization, the electrochemical proton gradient was dissipated by uncouplers, and the ATP hydrolysis measurement was started. In the presence of ADP and P(i), the initial rate of ATP hydrolysis was up to 9-fold higher with pre-energized proteoliposomes than with proteoliposomes that had not seen an electrochemical proton gradient. After dissipating the electrochemical proton gradient, the high rate of ATP hydrolysis decayed to the rate without pre-energization within about 15 s. During this decay the enzyme carried out approximately 100 turnovers. In the absence of ADP and P(i), the rate of ATP hydrolysis was already high and could not be significantly increased by pre-energization. It is concluded that ATP hydrolysis is inhibited when ADP and P(i) are bound to the enzyme and that a high Delta mu(H(+)) is required to release ADP and P(i) and to convert the enzyme into a high activity state. This high activity state is metastable and decays slowly when Delta mu(H(+)) is abolished. Thus, the proton motive force does not only supply energy for ATP synthesis but also regulates the fraction of active enzymes.

Mutational analysis of arginine 177 in the nucleotide binding site of β‐actin

Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R177 side chain is in a position similar to that of the catalytically essential lysine 71 in heat shock cognate protein Hsc70, another member of the actin-fold family of proteins. Therefore, actin residue R177 has been implicated in the mechanism of ATP hydrolysis. This paper compares wild-type beta-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant beta-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced thermal stability and an increased nucleotide exchange rate, suggesting a weakened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D replacement, showing that this residue is not involved in catalysis. In the presence of polymerizing salts, ATP hydrolysis by both wild-type Mg-beta-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the polymers formed exhibited much lower viscosity. The critical concentration of polymerization (Acc) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant beta-actin bound phalloidin.

Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin

Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R177 side chain is in a position similar to that of the catalytically essential lysine 71 in heat shock cognate protein Hsc70, another member of the actin-fold family of proteins. Therefore, actin residue R177 has been implicated in the mechanism of ATP hydrolysis. This paper compares wild-type β-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant β-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced thermal stability and an increased nucleotide exchange rate, suggesting a weakened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D replacement, showing that this residue is not involved in catalysis. In the presence of polymerizing salts, ATP hydrolysis by both wild-type Mg-β-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the polymers formed exhibited much lower viscosity. The critical concentration of polymerization (Acc) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant β-actin bound phalloidin.

Isolation and characterization of a processive DNA helicase from the fission yeast Schizosaccharomyces pombe that translocates in a 5'-to-3' direction.

We report here the isolation and characterization of a novel DNA helicase from extracts of the fission yeast Schizosaccharomyces pombe. The enzyme, called DNA helicase II, also contains an intrinsic DNA-dependent ATPase activity. Both the helicase and ATPase activities co-purified with a 63 kDa polypeptide on an SDS/polyacrylamide gel. The protein has a sedimentation coefficient of 4.8 S and a Stokes radius of 36 A (3.6 nm); from these data the native molecular mass was calculated to be 65 kDa. The enzyme translocates in a 5'-to-3' direction with respect to the substrate strand to which it is bound. Unwinding reactions carried out in the presence of increasing enzyme showed a sigmoidal curve, suggesting either co-operative interactions between monomers or multimerization of DNA helicase II in the presence of single-stranded DNA and/or ATP. This enzyme favoured adenosine nucleotides (ATP and dATP) as its energy source, but utilized to limited extents GTP, CTP, dGTP and dCTP. Non-hydrolysable ATP analogues did not support helicase activity. Kinetic analyses showed that the unwinding reaction was rapid, being complete after 50-100 s of incubation. Addition of unlabelled substrates to the helicase reaction after preincubation of the enzyme with substrate did not significantly diminish unwinding. The ATPase activity of DNA helicase II increased proportionally with increasing lengths of single-stranded DNA cofactor. In the presence of circular DNA, ATP hydrolysis continued to increase up to the longest time tested (3 h), whereas it ceased to increase after 5-10 min in the presence of shorter oligonucleotides. The initial rate of ATP hydrolysis during the first 5 min of incubation time was not affected by DNA species used. These data indicate that the enzyme does not dissociate from the single-stranded DNA once it is bound and is therefore highly processive.

GroES promotes the T to R transition of the GroEL ring distal to GroES in the GroEL-GroES complex.

Curves of initial rates of ATP hydrolysis by GroEL as a function of ATP concentration, in the presence of fixed concentrations of GroES, were found to deviate from sigmoidal kinetics. Instead of the lag phase typical of sigmoidal curves, a linear phase is observed at low ATP concentrations. Consequently, a good fit of the data to the Hill equation could not be achieved. Such curves could be simulated using a linear combination of Hill equations, thus indicating that more than one allosteric transition is taking place in the ATP concentration range studied. The data were fitted to a fractional saturation equation for ATP binding to GroEL based on a partition function that includes both GroES and ATP-liganded states of GroEL. Using this equation, it was possible to estimate in a reliable manner the value of the allosteric constant, L2', for the transition of the ring distal to GroES in the GroEL-GroES complex from the low (T)- to the high (R)-affinity state for ATP. The value of L2' is found to be 4 x 10(-5) whereas the value of the allosteric constant, L2, for the transition of the second ring of GroEL from the T to R state is 2 x 10(-9) [Yifrach, O., & Horovitz, A. (1995) Biochemistry 34, 5303-5308]. Comparison of these values shows that GroES promotes the T to R transition of the ring distal to GroES in the GroEL-GroES complex. Owing to the relatively low affinity of the R conformation for nonfolded proteins, this transition will lead to release of protein substrates from trans ternary complexes of GroEL, GroES, and protein substrate. The role of this release mechanism may be to assist the folding of relatively large proteins that cannot form cis ternary complexes and/or to facilitate degradation of damaged proteins which cannot fold.

ATP Synthase: CONDITIONS UNDER WHICH ALL CATALYTIC SITES OF THE F1 MOIETY ARE KINETICALLY EQUIVALENT IN HYDROLYZING ATP

Conditions have been reported under which the F1 moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10(6)-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F1 by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F1 to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with Pi as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single Vmax exceeding 100 micromol of ATP hydrolyzed per min/mg of F1 (turnover number = 635 s-1) and a single Km for ATP of about 57 microM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F1 moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.

ATP synthesis catalyzed by the mitochondrial F 1-F 0 ATP synthase is not a reversal of its ATPase activity

The ADP(Mg 2+)-deactivated oligomycin-sensitive F 1-F 0 ATPase of coupled submitochondrial particles treated with the substoichiometric amount of oligomycin was studied to test whether ATP synthesis and hydrolysis proceed in either direction through the same intermediates. The initial rates of ATP hydrolysis, oxidative phosphorylation, ATP-dependent, succinate-supported NAD + reduction, and ATP-induced Δ μ H+ generation were measured using deactivated ATPase trapped by azide [Biochem. J. (1982) 202, 15–23]. Three ATP consuming reactions were strongly inhibited when azide was present in the assay mixtures, whereas ATP synthesis was not altered by azide. The unidirectional effect of azide is not consistent with three alternating binding sites mechanism operating in ATP synthesis and support our hypothesis on the existence of nucleotide(Mg 2+)-controlled ‘synthase’ and ‘hydrolase’ states of the mitochondrial F 1-F 0 ATPase.

Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL

Initial rates of ATP hydrolysis by wild-type GroEL were measured as a function of ATP concentration from 0 to 0.8 mM. Two allosteric transitions are observed: one at relatively low ATP concentrations (< or = 100 microM) and the second at higher concentrations of ATP with respective midpoints of about 16 and 160 microM. Two allosteric transitions were previously observed also in the case of the Arg-196-->Ala GroEL mutant [Yifrach, O., & Horovitz, A. (1994) J. Mol. Biol. 243, 397-401]. On the basis of these observations a mathematical model for nested cooperativity in ATP hydrolysis by GroEL is developed in which there are two levels of allostery: one within each ring and the second between rings. In the first level, each hepatameric ring is in equilibrium between the T and R states, in accordance with the Monod-Wyman-Changeux (MWC) model of cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. A second level of allostery is between the rings of the GroEL particle which undergoes sequential Koshland-Némethy-Filmer (KNF)-type transitions from the TT state via the TR state to the RR state [Koshland et al. (1966) Biochemistry 5, 365-385]. Using our model, we estimate the values of the Hill coefficient for the negative cooperativity between rings in wild-type GroEL and the Arg-196-->Ala mutant to be 0.003 (+/- 0.001) and 0.07 (+/- 0.02), respectively. The inter-ring coupling free energies in wild-type GroEL and the Arg-196-->Ala mutant are -7.5 (+/- 0.4) and -3.9 (+/- 0.3) kcal mol-1, respectively.

Deactivation of F0F1 ATPase in intact plant mitochondria

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (delta mu H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl tin and aurovertin), used at non-saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase deactivation.

Beef heart mitochondrial F 1-ATPase: Inhibition by azidoadenyl-5′-yl imidodiphosphates and cooperative binding of substrate

Two ATP analogs, 2- and 8-azidoadenyl-5′-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F 1-ATPase under non-photolytical conditions. In the range of 5 μM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of K m and V max values (3 μM, 8.5 μmol ATP/min per mg; 255 μM, 42.0 μmol ATP/min per mg). With increasing inhibitor concentrations, the apparent K m,2 increases as in competitive inhibition, while V max,1 decreases as in non-competitive inhibition. The K i values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 μM and 460 μM, respectively). The decrease of the high-affinity V max is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.

On the coupling between ATP usage and DNA transport by yeast DNA topoisomerase II.

The initial rates of ATP hydrolysis and relaxation of negatively supercoiled DNA by highly purified wild-type and mutant yeast DNA topoisomerase II were measured under identical conditions to study the coupling between the ATPase activity of a type II DNA topoisomerase and its catalysis of the transport of one DNA segment through another. The results indicate that the binding of the enzyme to DNA stimulates its intrinsic ATPase activity by about 20-fold, and ATP binding to the pair of ATPase sites in a DNA-bound dimeric enzyme appears to be cooperative. The cooperativity in ATP binding may be significant in the coordination of the two halves of a DNA-bound enzyme dimer. At low ATP concentrations, the rate-limiting step in ATP usage appears to be slower than that in DNA transport, and DNA transport is relatively efficient in terms of ATP consumption: 1.9 +/- 0.5 ATP molecules are hydrolyzed/DNA transport event. At a saturating ATP concentration, however, there appears to be a reversal of these rate-limiting steps, and DNA transport is less efficient: 7.4 +/- 1.0 ATP molecules are hydrolyzed/DNA transport event. These data are interpreted in terms of a model in which a DNA-bound enzyme acts as an ATP-operated clamp for the capture and transport of a second DNA segment.

Dependence of the proton translocation stoichiometry of cyanobacterial and chloroplast H +-ATP synthase on the membrane composition

The high proton translocation stoichiometry (approx. 9 H + / ATP) of ATPase proteoliposomes reconstituted from a thermophilic cyanobacterium (Van Walraven et al. (1986) FEBS Lett. 208, 138–142) has also been observed with chloroplast ATP synthase when reconstituted with cyanobacterial lipids. Both enzyme complexes in isolated and reconstituted form show highest stable trypsin-activated ATP hydrolysis activity at the same temperature (55°C). Also, both isolated ATP synthases require the same reconstitution procedure for maximal coupling quality. The proton translocation stoichiometry has been deduced from the relation between the initial rates of ATP hydrolysis at varying sizes of the electrochemical potential gradient ( Δ \\ ̄ gm H + ). A Δ \\ ̄ gm H + was imposed by valinomycin-induced K + diffusion potentials or by base-pulses which were equally efficient in inhibiting ATP hydrolysis. Kinetic experiments with the use of the pH indicator Cresol red confirm the high proton translocation stoichiometry of both types of ATPase proteoliposome. Functional co-reconstitution of both types of ATPase proteoliposome with cyanobacterial cytochrome b 6 f complex leads to a decrease in proton translocation stoichiometry to about 7 H + / ATP. Cyanobacterial membrane vesicles take up 4.4 protons per ATP hydrolyzed. A value of 4.5 H + / ATP is observed with chloroplasts in equilibrium (Gräber, P., Junesch, U. and Thulke, G. (1986) in Progress in Photosynthesis Research (Biggins, J., ed.), pp. 177–184, Martinus Nijhoff, Dordrecht). These results indicate that the proton translocation stoichiometry of the ATP synthase depends on the membrane composition. The consequence of this finding for the mechanism of proton translocation and the possible physiological relevance are discussed.

The effect of inorganic pyrophosphate on the activity and Pi-binding properties of mitochondrial F1-ATPase.

Interaction of F1-ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1-ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady-state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP-dependent reactivation of an inactive complex formed by F1-ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1-ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non-preincubated enzyme. PPi stimulates the binding of Pi to F1-ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high-affinity Pi binding. It follows from the results obtained that PPi interacts with the non-catalytic site(s) of F1-ATPase.

Temperature dependence and mercury inhibition of tonoplast-type H +-ATPase

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H +-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 °C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was ~14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 °C. Lineweaver-Burke analysis of the activities showed that the K m of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg 2+. The proton pumping process was more sensitive to the presence of Hg 2+. Double-reciprocal analysis of kinetic data indicated that Hg 2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg 2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H + pumping and ATP hydrolysis.