Use of an adenosine triphosphate analog, adenylyl imidodiphosphate, to evaluate adenosine triphosphate-dependent reactions in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), an analog of adenosine triphosphate (ATP), was found to be an effective inhibitor of adenine nucleotide translocation in rat liver mitochondria. Inhibition by AMP-PNP was shown to be competitive with ATP. Therefore, studies designed to evaluate the interaction of ATP with mitochondrial adenosine triphosphatase (ATPase) in the presence of AMP-PNP were carried out on submitochondrial particles which lack a membrane barrier between the enzyme and the test medium. The effect of AMP-PNP on the ATP-driven reversed electron transfer reaction in sonically prepared submitochondrial particles was further examined by using oligomycin to induce coupling. The ATPase of oligomycin treated particles did not show significantly different sensitivity to AMP-PNP. Submitochondrial particles which were sensitive to AMP-PNP were less efficient in driving energy-coupled reactions. Results from these studies indicate that uncoupling in mitochondria is not only due to a leaky membrane but may also result from an altered membrane-ATPase association.     

Action of the adenosine triphosphate analog, adenylyl imidodiphosphate in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), and analog of adenosine triphosphate (ATP), is a potent competitive inhibitor of mitochondrial ATPase activity. It inhibits both the soluble oligomycin-insensitive ATPase (K_i = 9.2 × 10^?7 M) and the bound oligomycin-sensitive APTase (K_i = 1.3 × 10^?6 M). ATPase activity of inside-out submitochondrial preparations are more sensitive to AMP-PNP in the presence of an uncoupler (K_i = 2.0 × 10^?7 M). Mitochondrial ATP-dependent reactions (reversed electron transfer and potassium uptake) do not proceed if ATP is replaced with AMP-PNP; however, the analog does affect these systems. Oxidative phosphorylation of whole mitochondria and submitochondrial preparations were unaffected by AMP-PNP.     

Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum.

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca2+-ATPase and Mg2+-ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave Km and V values of 17.3 muM and 1.25 mumol Pi/min per mg, and 2.4 muM and 0.12 mumol Pi/min per mg, respectively. Adenylyl imidodiphosphate, a beta-gamma-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with Ki values equal to 350 and 12 muM in the presence of Ca2+ and Mg2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate (t1/2 = 24 h) by the ATP analog, 6,6'-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate (t1/2 approximately 10 min) skeletal and cardiac myosins rapidly by reacting with key cysteines.     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca²⁺ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca²⁺-ATP on the association of the ATPase inhibitor peptide to F₁-ATPase. This action of Ca²⁺ may explain why mitochondria utilize respiratory energy for the transport of Ca²⁺ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

Characterization of nucleotide binding sites on chloroplast coupling factor 1.

A study of the equilibrium binding of ADP, 1,N6-ethenoadenosine diphosphate, adenylyl imidodiphosphate, and 1,N6-ethenoadenylyl imidodiphosphate to solubilized spinach chloroplast coupling factor 1 (CF1) has been carried out. All four nucleotides were found to bind to two apparently identical "tight" sites, with characteristic dissociation contants generally less than 10 muM. The binding to these "tight" sites is similar in the presence of Mg2+ and Ca2+, is stronger in 0.1 M NaC1 than in 20 mM Tris-C1, and is only slightly altered by heat activation. The slow rate of association of ADP and 1,N6-ethenoadenosine diphosphate at these sites rules out the possibility that they are catalytic sites for ATPase activity on the solubilized enzyme. A third tight site for adenylyl imidodiphosphate was found on the heat-activated enzyme. The dissociation constant for this interaction (7.6 muM) is similar to the adenylyl imidodiphosphate competitive inhibition constant for ATPase activity (4 muM). ADP, which inhibits ATPase activity but is not a strong competitive inhibitor, binds only weakly at a third site (dissociation constant greater than 70 muM). One mole of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted per mole of CF1 prevents ADP and adenylyl imidodiphosphate binding at the "catalytic" site and abolishes the ATPase activity. A model is proposed in which the "tight" nucleotide binding sites act as allosteric conformational switches for the ATPase activity of solubilizedCF1.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Inhibitors of the slow stage of proton transfer in the link connecting respiration with mitochondrial phosphorylation]

Electrophilic agents--derivatives of carbonic acids--are found to inhibit respiration, ATP synthesis and reverse electrone transport in intact mitochondria. The inhibition of respiration and ATPase was observed in intact mitochondria at 3 and 3u states (by Chance). Inhibitors concentrations, which caused 50% inhibition, were approximately the same. Sharp decrease of the effect of electrophilic inhibitors on respiration and ATPase activity in mitochondria and submitochondrial particles with substantially impaired coupling system was observed. The following conclusions are drawn on the basis of the data obtained: 1) electrophilic inhibitor attack the coupling site of respiration and ATP synthesis in mitochondria; 2) the reaction of the proton transport from the respiration proton pump to ATP synthetase is one of the slowest steps of the process of ATP-synthesis in mitochondria. A scheme of working the coupling system is suggested which includes the step of proton lateral diffusion.     

Regulation of the mitochondrial adenosine 5'-triphosphatase in situ during ischemia and in vitro in intact and sonicated mitochondria from slow and fast heart-rate hearts

In the present study we examined the regulation of the cardiac muscle mitochondrial ATPase both in situ and in vitro in intact and sonicated mitochondria from rabbit, pigeon, and rat. We chose to study these three species because each is representative of one of the three classes into which all species thus far studied may be placed with respect to the in situ activity of their cardiac muscle mitochondrial ATPase inhibitor and with respect to the amount of ATPase inhibitor present in their cardiac muscle mitochondria (1). Class A species (rabbit) contain a full complement of ATPase inhibitor and show a marked ATPase inhibition during ischemia. Class B species (pigeon) also contain a full complement of inhibitor but exhibit only a low level of ATPase inhibition in situ. Class C species (rat) contain only low levels of inhibitor and, like class B species, don't appear to utilize the inhibitor they possess during ischemia in situ. We found that, while hearts from all three species developed a marked cytosolic acidosis during ischemia, only rabbit exhibited a marked ATPase inhibition in situ. In in vitro experiments in which matrix pH values close to 6.2 and delta psi values close to zero were measured in intact mitochondria from all three species, matrix pH appeared to be an important factor regulating ATPase inhibition in rabbit, but it had little effect upon ATPase--inhibitor interaction in pigeon and rat despite the lack of membrane potential. However, a pH-dependent further release of ATPase inhibitor was observed in sonicated pigeon heart mitochondria only. This latter observation suggests that, while slow heart-rate heart mitochondria appear to be designed for ATPase down regulation during ischemia by inhibitor binding to the ATPase, fast heart-rate heart mitochondria appear to be designed primarily for ATPase up regulation by a further release of inhibitor from the enzyme.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

The Membrane-Bound Forms of the Mitochondrial ATPase of Turnip (Brassica napus L.) and Mung Bean (Phaseolus aureus): Characterization and Investigation of Factors Controlling Activity

Isolated intact plant mitochondria, including those from turnipand mung bean, show low endogenous Mg²⁺-ATPase activity and,unlike mammalian mitochondria, lack significant uncoupler-stimulatedATPase activity. In contrast, the rates of respiration-drivenATP synthesis are comparable to those in mammalian mitochondria,suggesting the presence of an ATPase inhibitor. Disruption ofintact turnip mitochondria only results in limited increasesin ATPase activity, indicating that a permeability barrier toATP transport is not primarily responsible for the low endogenousactivity. The ATPase activity of turnip mitochondria and membraneparticles can be increased up to 50-fold when assayed underoptimum conditions. Time-dependent increases in activity inducedby ageing, exposure to salts and trypsin treatment, are allconsistent with an inhibitor protein being responsible for thelow endogenous activity and lack of uncoupler-stimulation. TheATPase activity of particles under optimum conditions and afterageing is sufficient to account for the rates of ATP synthesis.After activation, turnip mitochondrial ATPase activity is similarto the mammalian enzyme in inhibitor sensitivity, pH optimum,bivalent cation requirement, and sensitivity to ‘activatinganions’. In mung bean mitochondria, a permeability barrierto ATP is only partly responsible for the low endogenous ATPaseactivity, together with the inhibitory factor. On the basisof variation in the relative Ca²⁺ and Mg²⁺ -ATPase activitiesafter various treatments, a Ca²⁺-regulatory site which affectsATPase activity is proposed to exist in the F₁ATPase complex. Key words: Plant mitochondrial ATPase, calcium/magnesium -ATPase, inhibitor+ nucleotide specificity, cation/anion effects     

Increased ATP-hydrolase activity of the brain mitochondria under the effect of aminazine (electron cytochemical study)]

The localization of Mg-stimulated ATPase activity was determined in the sensomotor cortex mitochondria of Wag rats after aminazin administration (15 mg/kg). Apart from normal mitochondria containing no reaction product, a number of altered mitochondria with different localization in them of the ATPase reaction product were recorded. The intact animals showed no sediment in the mitochondria. It is suggested that aminazin-induced increase in ATP-hydrolase activity is caused by two factors: neuroleptic-induced decrease in glycolytic and oxidative cell activity and increased permeability of mitochondrial membranes.     

Mitochondrial adenosine triphosphatase of wild-type and poky Neurospora crassa.

We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Km for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 +/- 0.03 mumol/min-mg of protein compared with 1.33 +/- 0.07 mumol/min-mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 +/- 0.2 mumol/min-mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky.     

Inhibition of mitochondrial ATPase by 2,4,3',5'-tetrahydroxystilbene.

2,4,3',5'-tetrahydroxystilbene (THS) wa s found to inhibit rat liver mitochondrial adenosine triphosphatase (ATPase) activity induced by various concentrations of 2,4-dinitrophenol (DNP). The I50 was found to be 17 nmoles/mg mitochondrial protein. The maximum inhibitory effects of oligomycin and atractyloside on the DNP-activated mitochondrial ATPase activity can be enhanced by adding THS. The atractyloside-insensitive ATPase activity of Lubrol-treated rat liver mitochondria was also inhibited by low concentration of THS. The tetramethoxyderivative of THS was much less effective than the parent compound in depressing the ATPase activity of both intact and Lubrol-treated mitochondria. These observations suggest that the phenolic groups are essential for the mitochondrial actions of THS, and this compound most probably acts by a mechanism different from oligomycin on the mitochondrial ATPase complex.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Investigations on the mitochondria of the house fly,Musca domestica L. I. Adenosinetriphosphatases

1. The ATPase activity of insect mitochondria has been investigated. A comparison was made to determine the distribution and nature of such activity in other isolated fractions of the house fly, Musca domestica L. 2. The ATPase in insect mitochondria is specific in that orthophosphate can be cleaved only from ATP. The Michaelis-Menten constant K(8) = 2.78 x 10(-3)M and V(max.) = 76 micrograms P min.(-1) mg.(-1) dry weight. 3. Mg(++) and Mn(++) activate this enzymatic reaction in mitochondria, but Ca(++) does not. The extent of activation is 60 per cent with the optimal concentration 6 x 10(-4)M. Experiments with combinations of Mg(++) and Mn(++) show that either ion can replace the other and that the effects are additive, depending solely on the final concentration of the combination. Concentrations of Mg, Mn, or Ca ions higher than 6 x 10(-3)M inhibit the enzyme. 4. Fluoride does not inhibit the ATPase of insect mitochondria, whereas azide and chloromercuribenzoate do. The per cent inhibition depends on the concentration of inhibitor. 5. Finely dispersed mitochondrial particles have much greater ATPase activity than intact mitochondria. The possible relationship of this observation to latent ATPase is considered. 6. A magnesium-activated adenylate kinase is present in these mitochondria. The liberated orthophosphate, derived from ADP, is the result of the activity of adenylate kinase followed by the specific ATPase. 7. ATP can be dephosphorylated by enzymes found in the muscle fibrils, and in a "soluble" fraction, as well as in mitochondria. The fibrillar ATPase is Ca(++)-activated. The "soluble" fraction, however, like the mitochondria, is Mg(++)-activated. The "soluble" ATP dephosphorylation mechanism is distinguished from the mitochondrial ATPase in that it is inhibited by fluoride. 8. The "soluble" fraction also contains a magnesium-activated inorganic pyrophosphatase. Fluoride completely inhibits this enzymatic reaction. 9. The possible mechanism of ATP dephosphorylation in the "soluble" fraction is discussed.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.     

Requirement for lysine-19 of the yeast mitochondrial ATPase inhibitor for the stability of the inactivated inhibitor-F1Fo complex at higher pH

The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase. In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis. Two amino acids (Gln and Glu) were substituted for the Lys19. The purified mutant inhibitor (Lys19-->Gln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4. ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH. These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity, but increases the stability of the inhibitor-F1Fo complex at higher pH.