Structural preferences for the binding of chromium nucleotides by beef heart mitochondrial ATPase

The mono- and bidentate forms of adenosine 5'-diphosphate, chromium (III) salt (CrADP) were separated using Sephadex G-10 column chromatography. The isomeric purity of the two forms was monitored using high voltage electrophoresis and column chromatography. The same techniques were employed to assess the purity of the mono-, bi-, and tridentate forms of adenosine 5'-triphosphate, chromium (III) salt (CrATP). Distinct differences in the interaction of beef heart mitochondrial ATPase with the various isomers of chromium nucleotides were seen in kinetic studies. Monodentate CrADP was a competitive inhibitor of the ATP hydrolysis activity of both purified ATPase and submitochondrial particles. However, when ITPase activity was examined, noncompetitive inhibition was observed. The bidentate isomer of CrADP did not affect ATPase activity. Enzymatic synthesis of the transition state analog of ATP synthesis and hydrolysis, Pi-CrADP occurred exclusively with the monodentate isomer of CrADP. It was also found that only the mono- and tridentate forms of CrATP were potent inhibitors of ATP hydrolysis by beef heart mitochondrial ATPase. These results are discussed in terms of possible ATP synthesis and hydrolysis mechanisms.     

Effect of inosine 5' -(beta, gamma-imido) triphosphate and other nucleotides on beef heart mitochondrial ATPase.

The effects of various substrates and alternative substrates on the hydrolytic activity of beef heart mitochondrial ATPase was examined. It was found that ATP or ADP, ITP hydrolysis showed positive cooperativity. IDP inhibited ITP hydrolysis and caused positive cooperativity. When ITP was present during an ATP hydrolysis assay, the rate of ATP hydrolysis was stimulated. IDP had no effect on ATP hydrolysis rates. A nonhydrolyzable ITP analog, inosine 5'-(beta, gamma-imido)triphosphate (IMP-P(NH)P), was synthesized and purified. It was found to be a potent competitive inhibitor of ITP and GTP hydrolytic activity. However, this beta-gamma-imido-bridged ITP analog was found to change the ITP and GTP hydrolysis kinetics from linear to positively cooperative. This compound inhibited ATP hydrolysis at substrate concentrations of 100 muM and lower, and stimulated ATP hydrolysis at substrate concentrations between 100 muM and 2 mM. IMP-P(NH)P had no effect on ATP hydrolysis when the substrate concentration was above 2 mM. In the presence of the activating anion, bicarbonate, IMP-P(NH)P inhibited ATP hydrolysis competitively, and induced positive cooperativity. IMP-P(NH)P had no effect on the ATP equilibrium Pi exchange, the ITP equilibrium Pi exchange, or ATP synthesis catalyzed by beef heart submitochondrial particles.     

Occurrence and significance of oxygen exchange reactions catalyzed by mitochondrial adenosine triphosphatase preparations.

The capacity of various ATPase preparations from beef heart mitochondria to catalyze exchange of phosphate oxygens with water has been evaluated. Oligomycin-sensitive ATPase preparations retain a capacity for considerable intermediate Pi equilibrium HOH exchange per Pi formed during ATP hydrolysis at relatively high ATP concentration (5 mM). Submitochondrial particles prepared by an ammonia-Sephadex procedure with 5 mM ATP showed more rapid ATPase, less oligomycin sensitivity, and less capacity for intermediate exchange. With these particles, intermediate Pi equilibrium HOH exchange per Pi formed was increased as ATP concentration was decreased. The purified, soluble ATPase from mitochondria catalyzed little or no intermediate Pi equilibrium HOH exchange at 5 mM ATP but showed pronounced increase in capacity for such exchange as ATP concentration was lowered. The ATPase also showed a weak catalysis of an ADP-stimulated medium Pi equilibrium HOH exchange. The results support the alternating catalytic site model for ATP synthesis or cleavage. They also demonstrate that a transmembrane protonmotive force is not necessary for oxygen exchange reactions. At lower ATP concentrations, ADP and Pi formed at a catalytic site appear to remain bound and continue to allow exchange of Pi oxygens until ATP binds at another site on the enzyme.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of beef heart submitochondrial particle-catalyzed Pi goes to and comes from ATP exchange by nucleotides and the ATPase inhibitor protein

F1I, the specific ATPase inhibitor protein, and the chromium(III) analogs of ATP and ADP, CrATP and CrADP, were used to study the inhibition of Pi goes to and comes from ATP exchange reaction catalyzed by beef heart submitochondrial particles. F1I was found to be an uncompetitive inhibitor of the exchange reaction. CrATP and CrADP, both competitive inhibitors of ATP hydrolysis in isolated F1 (Schuster, S. M., Ebel, R. E., and Lardy, H. A. (1975) ARch. Biochem. Biophys. 171, 656-661) were shown to be competitive and noncompetitive inhibitors of Pi goes to and comes from ATP exchange, respectively. Dual inhibitor studies were done using combinations of F1I and the chromium nucleotides, or the nucleotide analogs in combination. All cases show sets of intersecting Dixon plots indicative of interacting inhibitors. Upward curvature is also evident on some of the plots. This phenomenon was explained using the concept of multiple synergistic binding of the inhibitors. Binding mechanisms and their relevant kinetic equations were postulated to explain the results of the dual inhibitor studies. They support the notion that in addition to the catalytic site, there are two types of regulatory binding sites on F1, one specific for nucleotides and one specific for F1I. When one of these sites is occupied, other sites are either opened or other inhibitors become more potent.     

Inhibition of steady-state mitochondrial ATP synthesis by bicarbonate, an activating anion of ATP hydrolysis.

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.     

Synthesis and hydrolysis of ATP and the phosphate-ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide.

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al-Shawi, M.K. & Senior, A.E. (1992), Biochemistry 31, 886-891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1 [Gómez-Puyou, A., Tuena de Gómez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni-site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni-site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1.ADP + Pi <--> F1.ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1.ATP. The DeltaG degrees of the reaction with 0.54 microM F1, 2 mM Pi and 10 mM Mg2+ at pH 6.8 was -2.7 kcal.mol-1 in favor of F1.ATP. The DeltaG degrees of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, DeltaG degrees became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate-ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.     

Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.

Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state. The covalent bond forming and breaking step at the catalytic site (ADP + Pi in equilibrium ATP + HOH) appears relatively insensitive to uncouplers. Thus to the extent that uncouplers dissipate transmembrane proton-motive force, it is unlikely that such a force is used to drive ATP formation by direct protonations of Pi oxygens. When only Pi and ADP are added and formation of ATP from added ADP by adenylate kinase and subsequent ATP hydrolysis are adequately blocked, no Pi in equilibrium HOH exchange can be observed, demonstrating a requirement of energization by ATP binding and cleavage for such an exchange. This uncoupler-insensitive energization is suggested to represent a conformationally energized state that can be used reversibly to develop a transmembrane protonmotive force accompanying ADP and Pi release. Rates of various exchanges as estimated by improved procedures are compatible with all oxygen exchanges occurring by dynamic reversal of ATP hydrolysis at the catalytic site.     

The effect of Co(III)(NH3)4ATP on the kinetics of beef heart mitochondrial ATPase

Bidentate cobalt(III)tetraamine adenosine triphosphate [Co(NH3)4ATP] was investigated as an inhibitor of the beef heart mitochondrial F1-ATPase. The compound was found to have a mixed noncompetitive mechanism with a Ki of 0.4 mM and an alpha of 1.4 during ATP hydrolysis. Co(NH3)4ATP also noncompetitively inhibited ATP hydrolysis in the presence of bicarbonate. ITP hydrolysis was similarly affected. Co(NH3)4ATP was also used in dual inhibitor studies with adenylylimidodiphosphate (AMP-PNP) and azide; it was found to be mutually exclusive with AMP-PNP and azide. The compound also protected the F1 from modification by 4-chloro-7-nitrobenzofurazan. These results are discussed in terms of the regulation of the ATP hydrolysis reaction.     

Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges

We have examined intermediate Pi-water oxygen exchange during [gamma-18O]ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12. Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources. Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules. The epsilon-replete enzyme showed a much higher probability for oxygen exchange. These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release. Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis. Heterogeneity was apparent in the 18O distribution of the product Pi, however. That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit. In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP. This is the only enzyme so far reported with this unusual property. Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP.

Spinach chloroplasts were able to photophosphorylate the ADP analog alpha,beta-methylene adenosine 5'-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and 32Pi, the 32P label was incorporated into alpha,beta-methylene adenosine 5'-triphosphate (AOPCPOP). In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-Pi exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9. Photophosphorylation of AOPCP was inhibited by the alpha,beta- and beta,gamma-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-Pi exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-Pi exchange was inhibited by the beta,gamma-methylene analog of ATP. Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Characterization of phosphate oxygen exchange reactions catalyzed by myosin through measurement of the distribution of 18-O-labeled species

The change in the distribution of the phosphate species containing 0 to 4 18O oxygens per Pi was investigated during medium Pi equilibrium HOH exchange catalyzed by myosin subfragment 1. At 25 degrees C, a Pi molecule once bound loses an average of 3.9 of its original 4 oxygens prior to release which means that at least 100 reversals of the exchange reaction must have occurred. At 0 degrees C, only 3.4 of the 4 oxygens are lost prior to release indicating an average of 17 reversals. Distribution patterns are consistent with equivalent participation in the exchange reactions of all 4 oxygens of bound Pi. The intermediate exchange of Pi oxygens during hydrolysis of 18O-labeled ATP by myosin has also been investigated. The distribution of the product Pi species shows that there is an ATPase component in myosin preparations which hydrolyzes ATP without intermediate exchange. Presence of this component, which is likely a contaminating ATPase, provides a simple explanation of the apparent nonequivalence of phosphate oxygens which has been observed. When correction is made for this contaminant, characteristics of the myosin intermediate Pi equilibrium HOH exchange are similar to those of myosin subfragment 1 medium exchange, and intermediate exchange data are in much closer agreement with other kinetic measurements.     

Demembranated muscle fibers catalyze a more rapid exchange between phosphate and adenosine triphosphate than actomyosin subfragment 1

The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.     

Activation of ppGpp-3'-pyrophosphohydrolase by a supernatant factor and ATP

The breakdown of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) into GDP and PPi is catalyzed by a Mn2+-dependent 3'-pyrophosphohydrolase, the translation product of the spoT gene. The escherichia coli enzyme is normally found to be associated with the "crude" ribosome fraction. It is reported here that the guanosine 5'-diphosphate, 3'-diphosphate 3'-pyrophosphohydrolase activity in this fraction is activated by ATP in the presence of a relatively heat-stable, low molecular weight, supernatant factor (BS100). This stimulation is not due to a removal of reaction products such as by the phosphorylation of GDP to GTP or by the hydrolysis of PPi. Hydrolysis of ATP is probably required because neither adenosine 5'-(3-thio)triphosphate nor adenosine 5'-(beta, gamma-imido)triphosphate can substitute for ATP. Levallorphan, a morphine analog, which had been shown to inhibit in vivo ppGpp degradation, inhibits specifically the stimulation of ppGpp hydrolysis by ATP and the supernatant factor. The possible relationship of this system and the in vivo energy-dependent control of ppGpp degradation is discussed.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of ATP catalyzed by the (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts. Energy conservation in plasma membranes

The (Ca2+ + Mg2+)-ATPase from erythrocyte ghosts catalyzed the hydrolysis of ATP together with the synthesis of ATP or ATP in equilibrium 'Pi exchange. The modulation of the ATPase reaction cycle was controlled by high- and low-affinity calcium-binding sites asymmetrically located on the enzyme. Calmodulin accelerated the reaction cycle in both directions, stimulating the overall turnover of the enzyme. Calcium transport was achieved utilizing optimal conditions for the expression of the ATP in equilibrium Pi exchange system.     

Kinetic studies on rat liver and beef heart mitochondrial adenosine triphosphatase: the effects of the chromium complexes of adenosine triposphate and adenosine diphosphate on the kinetic properties.

The kinetics of isolated rat liver and beef heart mitochondrial adenosine triphosphatase (ATPase) were studied by using the chromium ATP and ADP complexes as substrate analogs. It was found that both chromium ATP (CrATP) and chromium ADP (CrADP) are competitive inhibitors of ATP hydrolysis. The presence or absence of ATPase-activating anions, e.g., bisulfite, had little effect on the type or potency of the inhibition by these chromium complexes. Both CrADP and CrATP were noncompetitive inhibitors of the hydrolysis of ITP with both the heart and liver-derived enzymes. It was also found that CrADP was a consistently more effective inhibitor than the ATP complex with the beef heart enzyme. These results are consistent with the existence of two types of nucleotide binding sites on mitochondrial ATPases: One site is regulatory and is rather specific for adenosine polyphosphates, while the other site is relatively nonspecific and serves as the hydrolytic site.     

Effect of citreoviridin and isocitreoviridin on beef heart mitochondrial ATPase

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium

The origin of the nuclear magnetic resonance (NMR)-measurable ATP in equilibrium Pi exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP in equilibrium Pi exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO2) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the Pi----ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the Pi----ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. Upon elimination of the glycolytic component, the measured Pi----ATP rates displayed a linear dependence on MVO (micromoles of O consumption rate) with a slope of 2.36 +/- 0.15 (N = 8, standard error of the mean). This linear relationship is expected if the rate determined by CST is the net rate of ATP synthesis by the oxidative phosphorylation process, in which case the slope must equal the P:O ratio. The ATP----Pi rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO2 levels were equal to the corresponding Pi----ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP in equilibrium Pi exchange is inhibited or is specifically shown not to exist, the myocardial Pi in equilibrium ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO2 levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H+-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the "operative" P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value of 3.