Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F₁), in the absence of Mg²⁺, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure.2. Since during the binding experiments the ‘tightly’ bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters.3. The binding data show that only one tight binding site (K_d about 0.5 μM) for ADP is present.4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 μM.     

Specific photolabelling of beef-heart mitochondrial ATPase by 8-azido-ATP

1. 8-Azido-ATP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F₁).2. 8-Azido-ATP is hydrolysed slowly by F₁ in the dark. Photolysis at 350 nm in the presence of F₁ leads to inhibition of the ATPase activity. The presence of ATP during illumination prevents the inhibition. Illumination of F₁ in the absence of 8-azido-ATP causes no inhibition.3. Added Mg²⁺ is not necessary for the binding of the 8-azido-ATP to F₁.4. 8-Azido-ATP binds specifically to the β subunits of F₁.5. The ATPase activity is completely inhibited when 2 mol of 8-azido-ATP are bound per mol F₁.     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.     

Specific photolabelling of beef-heart mitochondrial ATPase by 8-azido-ATP.

1. 8-Azido-ATP is a suitable photoaffinity label for beef-heart mitochondrial ATPase (F1) 2. 8-Azido-ATP is hydrolysed slowly by F1 in the dark. Photolysis at 350 nm in the presence of F1 leads to inhibition of the ATPase activity. The presence of ATP during illumination prevents the inhibition. Illumination of F1 in the absence of 8-azido-ATP causes no inhibition. 3. Added Mg2+ is not necessary for the binding of the 8-azido-ATP to F1. 4. 8-Azido-ATP binds specifically to the beta subunits of F1. 5. The ATPase activity is completely inhibited when 2 mol of 8-azido-ATP are bound per mol F1.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.     

Conformational change in beef-heart mitochondrial F1 ATPase to ATP synthesis mode induced by dimethylsulfoxide and ATP revealed by sulfhydryl group labeling

Treatment of beef-heart mitochondrial F1 ATPase with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) results in the incorporation of 1 mol DTNB/mol F1 without loss of ATPase activity. Incorporation is not prevented by ATP. Labeling occurs predominantly on an alpha-subunit, but also with a significant degree of modification of gamma- and epsilon-subunits. It is suggested that the modified sulfhydryl groups of the alpha-, gamma- and epsilon-subunits are in proximity so that only one can be modified by the reagent. Guanidine hydrochloride (0.3 M) dissociates F1 into its subunits. Eight sulfhydryl groups/mol F1 can be modified under these conditions. Guanidine hydrochloride does not cause dissociation of F1 in the presence of 30% (v/v) dimethylsulfoxide (Me2SO) and 2 mM ATP. Under these conditions a second molecule of DTNB is incorporated into F1 with nearly equal modification of the epsilon-subunit and an alpha-subunit. It is proposed that Me2SO and ATP induce a more stable conformation of F1, which is resistant to dissociation by guanidine hydrochloride, but in which the site of reaction with DTNB is made more accessible by the guanidine hydrochloride to permit the simultaneous modification of an alpha-subunit and the epsilon-subunit. This conformation is probably that which occurs during ATP synthesis by F1 in the presence of Me2SO.     

The binding of aurovertin to isolated F1 (mitochondrial ATPase).

1. Isolated F1 contains 14.9% N, indicating the presence of at least 8% non-protein material. The Lowry method, standardized with bovine serum albumin, correctly measures the protein content. 2. An extinction coefficient of 28.5 mM-1.cm-1 at 367.5 nm was found for aurovertin D in ethanol. 3. The fluorescence enhancement of aurovertin bound to F1 at pH 7.5 was found to be more than 100-fold. 4. Binding parameters calculated from the fluorescence enhancement with fixed F1 and variable aurovertin concentrations, and vice versa, indicate two binding sites per F1 molecule. 5. The fluorescence data are not readily interpreted on the basis of successive binding of aurovertin by 3-component binding reactions of the form E + A in equilibrium EA, but fit closely a model of two non-interacting sites binding aurovertin in a 4-component reaction, EF + A in equilibrium EA + F, with an equilibrium constant of about 2.     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying.

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic approximately 9 nm F1 (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the "etched" surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with freeze-dried TU particles (trypsin-urea treated SMP) that are devoid of F1 (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-"etching."     

Interaction of beef-heart mitochondrial ATPase,coupling factor F1, with aurovertin

The antibiotic aurovertin binds to beef-heart mitochondrial ATPase, coupling Factor F₁, with biphasic fluorescence enhancement. Specific binding effects, polarity and viscosity changes may all contribute to the enhancement. Evidence is presented that it stems from aurovertin binding followed by a slow conformational change in F₁. This occurs more rapidly in dissociated F₁. The effect of substrates of the enzyme on the fluorescence enhancement is examined. Evidence is presented that in the absence of added magnesium, F₁ can hydrolyse low concentrations of added ATP.     

Binding of HQNO to beef-heart sub-mitochondrial particles

1. The fluorescence spectra of HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) in water at pH 7.5 show an emission maximum at 480 nm and an excitation maximum at 355 nm.2. The fluorescence is enhanced by binding to bovine serum albumin, and is completely quenched by binding to sub-mitochondrial particles of beef heart.3. Binding experiments reveal specific binding of HQNO to sub-mitochondrial particles with a dissociation constant of 64 nM and, depending on the protein concentration, a considerable amount of aspecific binding.4. The concentration of specific binding sites for HQNO is identical with that of antimycin-binding sites. Furthermore, the presence of antimycin prevents the binding of HQNO and antimycin releases HQNO from its binding site.5. The binding of HQNO is not sensitive to the redox state of the respiratorychain components.6. Inhibition of electron transfer by HQNO is caused by binding to the specific binding site.7. The relation between inhibition of NADH or succinate oxidation and saturation of the binding site is hyperbolic.8. The increase in the reduction level of cytochrome b on addition of HQNO in the presence of succinate and oxygen, either in the presence or absence of cyanide, does not parallel the inhibition of overall electron transfer.9. All data can be quantitatively described and analysed using the model for electron transfer proposed by Wikström and Berden in 1972 (Wikström, M. K. F. and Berden, J. A. (1972) Biochim. Biophys. Acta 283, 403–420).     

ADP binding to (Na + K)-activated ATPase

[¹⁴C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na⁺, but decreased by K⁺, Mg²⁺ and Ca²⁺. Na⁺ abolished the effect of K⁺ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg²⁺ and Ca²⁺. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na⁺ + K⁺)-activated ATPase.     

Binding of an intrinsic ATPase inhibitor to the F(1)FoATPase in phosphorylating conditions of yeast mitochondria

Yeast mitochondrial ATP synthase has three regulatory proteins; ATPase inhibitor, 9K protein, and 15K protein. A mutant yeast lacking these three regulatory factors was constructed by gene disruption. Rates of ATP synthesis of both wild-type and the mutant yeast mitochondria decreased with decrease of respiration, while their membrane potential was maintained at 170-160 mV under various respiration rates. When mitochondrial respiration was blocked by antimycin A, the membrane potential of both types of mitochondria was maintained at about 160 mV by ATP hydrolysis. ATP hydrolyzing activity of F(1)FoATPase solubilized from normal mitochondria decreased in proportion to the rate of ATP synthesis, while the activity of the mutant F(1)FoATPase was constant regardless of changes in the rate of phosphorylation. These observations strongly suggest that F(1)FoATPase in the phosphorylating mitochondria is a mixture of two types of enzyme, phosphorylating and non-phosphorylating enzymes, whose ratio is determined by the rate of respiration and that the ATPase inhibitor binds preferentially to the non-phosphorylating enzyme.     

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide

Dimethylsulfoxide [Me₂SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F₁-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F₁ with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me₂SO, F₁ bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [³H]ADP to these sites was shown. A change in the conformation of F₁ in 30% (v/v) Me₂SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.