Characteristics of adenylyl imidodiphosphate- and ADP-binding sites insoluble and particulate mitochondrial ATPase. Studies with methanol

The characteristics of the binding sites for ADP and adenylyl imidodiphosphate have been studied in soluble and particulate F1-ATPase from bovine heart mitochondria. ADP, but not electrochemical gradients, removes the inhibitory effect of adenylyl imidodiphosphate on ATPase activity in coupled submitochondrial particles. In soluble F1-ATPase, methanol at 20% concentration diminishes the ability of ATP and adenylyl imidodiphosphate to inhibit ATP and ITP hydrolysis; these findings suggest that ADP and adenylyl imidodiphosphate inhibit hydrolysis by acting on the same site. Methanol at 20% stimulates the hydrolytic activity of soluble F1-ATPase, but fails to stimulate significantly the activity of the particulate enzyme, even though in particulate F1-ATPase methanol markedly diminishes the inhibiting action of added ADP and adenylyl imidodiphosphate on ATP and ITP hydrolysis. This is consistent with the idea that in the particulate system there are two inhibitory binding sites for ADP, one accessible to methanol, and another which is inaccessible to methanol; the latter is transitorily occupied by ADP arising from ATP hydrolysis. Indeed, experiments on the effect of ADP in ITP hydrolysis by submitochondrial particles show the existence of two ADP inhibitory sites.     

Cold lability of membrane-bound F1-ATPase.

1. Preincubation of MgATP submitochondrial particles with EDTA or Tris.HCl liberated a measurable amount of ATPase inhibitor that could be rapidly purified using only trichloroacetic acid precipitation and heat treatment. 2. In spite of the emergence of high ATPase activity, a considerable amount of ATPase inhibitor was left in the particles. Comparative analysis of other submitochondrial preparations indicated that only AS-particles were effectively depleted. 3. The high ATPase activity of inhibitor-deficient particles, was labile at low temperature provided that the exposure to cold was done in the presence of MgATP. Other nucleotides could not substitute for ATP. Glycerol inhibited and salts enhanced the cold inactivation of membrane-bound F1-ATPase. Isolation of F1-ATPase from cold-inactivated particles yielded a soluble preparation of correspondingly lower activity. 4. It is concluded that together with the increase of ATPase activity, the ATP-dependent cold lability of membrane-bound F1-ATPase and the dislocation of ATPase inhibitor at non operative sites reveal the extent of ATPase complex disorganization.     

Effect of polyamines on mitochondrial F1-ATPase catalyzed reactions

The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons. (a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added. (b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid. (c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.     

Nickel(II) transport in human blood serum. Studies of nickel(II) binding to human albumin and to native-sequence peptide, and ternary-complex formation with l-histidine

1. The initial rapid phase of ATP hydrolysis by bovine heart submitochondrial particles or by soluble F1-ATPase is insensitive to anion activation (sulphite) or inhibition (azide). 2. The second slow phase of ATP hydrolysis is hyperbolically inhibited by azide (Ki approximately 10(-5) M); the inosine triphosphatase activity of submitochondrial particles or F1-ATPase is insensitive to azide or sulphite. 3. The rate of interconversion between rapid azide-insensitive and slow azide-sensitive phases of ATP hydrolysis does not depend on azide concentration, but strongly depends on ATP concentration. 4. Sulphite prevents the interconversion of the rapid initial phase of the reaction into the slower second phase, and also prevents and slowly reverses the inhibition by azide. 5. The presence of sulphite in the mixture when ADP reacts with ATPase of submitochondrial particles changes the pattern of the following activation process. 6. Azide blocks the activation of ATP-inhibited ATPase of submitochondrial particles by phosphoenolpyruvate and pyruvate kinase. 7. The results obtained suggest that the inhibiting effect of azide on mitochondrial ATPase is due to stabilization of inactive E*.ADP complex formed during ATP hydrolysis; the activation of ATPase by sulphite is also realized through the equilibrium between intermediate active E.ADP complex and inactive E*.ADP complex.     

A novel method for the isolation of calsequestrin from porcine skeletal muscle sarcoplasmic reticulum

Bovine heart submitochondrial particles depleted of F1 by treatment with urea ("F1-depleted particles') were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH4+, K+, Rb+, Na+ and Li+ promoted reconstitution maximally at 40-74 mM, guanidinium+ and Tris+ at 20-30 mM, and Ca2+ and Mg2+ at 3-5 mM. The particles exhibited a negative zeta-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.     

F1-ATPase from different submitochondrial particles.

1. F1-ATPase has been extracted by the diphosphatidylglycerol procedure from mitochondrial ATPase complexes that differ in ATPase activity, cold stability, ATPase inhibitor and magnesium content. 2. The ATPase activity of the isolated enzymes was dependent upon the activity of the original particles. In this respect, F1-ATPase extracted from submitochondrial particles prepared in ammonia (pH 9.2) and filtered through Sephadex G-50 was comparable to the enzyme purified by conventional procedures (Horstman, L.L. and Racker, E. (1970) J. Biol. Chem. 245, 1336--1344), whereas F1-ATPase extracted from submitochondrial particles prepared in the presence of magnesium and ATP at neutral pH was similar to factor A (Andreoli, T.E., Lam, K.W. and Sanadi, D.R. (1965) J. Biol. Chem. 240, 2644--2653). 3. No systematic relationship has been found in these F1-ATPase preparations between their ATPase inhibitor content and ATPase activity. Rather, a relationship has been observed between this activity and the efficiency of the ATPase inhibitor-F1-ATPase association within the membrane. 4. It is concluded that the ATPase activity of isolated F1-ATPase reflects the properties of original ATPase complex provided a rapid and not denaturing procedure of isolation is employed.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with epsilon-ATP.

1. The use of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied. 2. Direct [3H]adenine nucleotide uptake studies with isolated mitochondria, indicate the epsilon-[3H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F1-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, epsilon-ATP is hydrolyzed by a Mg2+-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria. 3. epsilon-ATP can be utilized quite well by the exposed F1-ATPase of sonic submitochondrial particles. This epsilon-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F1-ATPase displays similar Km values for both ATP and epsilon-ATP; however, the V with ATP is approximately six times greater than with epsilon-ATP. 4. Since epsilon-ATP is a capable substrate for the submitochondrial particle F1-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F1-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Spermine binding to submitochondrial particles and activation of adenosine triphosphatase.

Studies on the effects of polyamines on oligomycin-sensitive ATPase activity of ox heart submitochondrial particles showed that, of the polyamines tested, only spermine affected the enzyme activity. Spermine within the physiological concentration range increased the Vmax. of the enzyme, but the Km for ATP was virtually unaffected. Binding studies of [14C]spermine to submitochondrial particles, under the same conditions as used for the ATPase assay, showed that the spermine binds to submitochondrial particles in a co-operative way; Hill plots of the data gave a Hill coefficient of 2 and a Kd of 8 microM. When submitochondrial particles were treated with trypsin, ATPase was not stimulated by spermine and the amount of spermine bound concomitantly was drastically decreased. The ATPase activity of isolated F1-ATPase was not affected by spermine. Removal of the natural protein ATPase inhibitor did not suppress either the stimulation of the ATPase activity by spermine or the spermine binding to the particles. The results obtained suggested that the polyamine binds and acts at the level of the liaison between the coupling factor F1 and the membrane sector F0 of the ATPase complex.     

Changes in activity and F1 content of mitochondrial H+-ATPase in regenerating rat liver

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS-PAGE. Use of an antibody directed against the F1 portion of the H+-ATPase complex demonstrated that there is a definite decrease in the amount of beta-subunit of F1 in both submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy.     

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

The use of aurovertin to determine the F1 content of submitochondrial particles and the ATPase complex.

(1) The concentration of aurovertin-binding sites calculated from fluorimetric titrations of submitochondrial particles is equal to the F1 concentration, calculated from the concentration of F1-binding sites in stripped particles. (2) Direct binding experiments show that the fluorescence enhancement of aurovertin bound to submitochondrial particles and the isolated ATPase complex is less (or absent) at higher concentrations than at lower concentrations. The binding data can be described by 'specific' and 'non-specific' binding. The concentration of the 'specific' sites is twice that derived from fluorimetric titrations. (3) After dissociation of the bound F1 with LiCl, fluorimetric titrations with aurovertin yield linear Scatchard plots. The fluorescence enhancement and KD are equal to those of the beta-subunit-aurovertin complex. The concentration of beta-subunits is double the concentration of F1. (4) It is concluded that both for submitochondrial particles and the isolated ATPase complex the most reliable and simple way to determine the F1 content is to dissociate the F1 with LiCl, spin down the insoluble material and titrate the supernatant (containing free beta-subunit) with aurovertin.     

Photoaffinity labeling of mitochondrial adenosine triphosphatase by an azido derivative of the natural adenosine triphosphate inhibitor

The natural mitochondrial ATPase inhibitor (IF1) was modified with a radioactivity labeled heterobifunctional and photosensitive reagent, methyl 4-azido(14C)benzimidate ((14C)MABI). Titration experiments of IF1 by (14C)MABI and tryptic maps of (14C)MABI-IF1 indicated that specific lysine residues in IF1 are preferentially labeled by (14C)MABI. Under appropriate conditions of labeling (1 to 2 lysine residues modified per IF1), MABI-IF1 exhibited the same inhibitory potency as native IF1 on the hydrolytic activity of the coupling factor 1 of mitochondrial ATPase (F1). The same conditions were required for inhibition of F1 by MABI-IF1 and IF1 (slightly acidic pH and presence of ATP and MgCl2). In photolabeling experiments, (14C)MABI-IF1 was used to investigate the localization of IF1 binding sites on F1. Upon photoirradiation, MABI-IF1 bound selectively to the beta subunit of soluble or membrane-bound F1. Adenylyl imidodiphosphate and quercetin, two compounds which partially mimic the inhibitory effect of IF1 on ATPase activity of F1, markedly prevented the binding of (14C)MABI-IF1 to F1; on the other hand, aurovertin, a specific ligand of the beta subunit of F1, did not affect the interaction between (14C)MABI-IF1 and F1. In the absence of light, (14C)MABI-IF1 was used as a reversible radiolabeled ligand with respect to membrane bound F1 to investigate F1-IF1 interactions to inside-out submitochondrial particles as a function of the energy state of the particles. Oxidation of NADH by submitochondrial particles resulted in a decrease of bound (14C)MABI-IF1; the effect was counteracted by antimycin. The data suggested that added (14C)MABI-IF1 is capable of exchanging with IF1 bound to F1 in submitochondrial particles and that the rate and extent of (14C)MABI-IF1 release are triggered by the proton-motive force developed by the particles.     

Regulatory role of the ATPase inhibitor protein on proton conduction by mitochondrial H+-ATPase complex

This study shows that the natural inhibitor protein of mitochondrial H+-ATPase complex (IF1) inhibits, in addition to the catalytic activity, the proton conductivity of the complex. The inhibition of ATPase activity by IF1 is less effective in the purified F1 than in submitochondrial particles where F1 is bound to F0. No inhibition of H+ conductivity by F0 is observed in F1-depleted particles.     

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.     

Effect of 2-hydroxy-5-nitrobenzyl bromide on proton translocation by the mitochondrial H+-ATPase

2-Hydroxy-5-nitrobenzyl bromide, a highly reactive reagent towards tryptophan residues in proteins, is shown to activate the passive proton flux through the inner mitochondrial membrane of bovine heart submitochondrial particles (ETPH). When added at low concentrations, the reagent increased both the ATPase activity of the particles and the passive proton transport rate through the membrane. The presence of oligomycin reduced the extent of the 2-Hydroxy-5-nitrobenzyl bromide action on the proton conductivity suggesting that it acted primarily on the H+-ATPase complex. Similar effects were observed on F1-depleted particles, whilst no effect was observed on the isolated F1-ATPase activity. The results suggest that polypeptides bearing tryptophan residues may be involved in the gating function of proton channels of the mitochondrial membrane and this is particularly evident for the F0F1-ATPase complex.     

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the FoF1 ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (delta mu H+) generated by succinate-driven respiration. In control rat-liver submitochondrial particles containing the FoF1 moiety of the ATPase complex, anaerobic relaxation of delta mu H+ showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin and N,N-dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1-Fo ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin-sensitivity-conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat-liver mitochondrial membranes. The results reported show that the structural and functional integrity of the Fo-F1 ATPase of rat liver is severely perturbed during regeneration.     

The oxidation of sulfhydryl groups in mitochondrial F1-ATPase decreases the rate of its inactivation by the natural protein inhibitor

The oxidants of the SH groups (o-iodozobenzoate, oxidized glutathione, etc.) and the divalent cations of some metals (Zn2+ and Cd2+) significantly slow down the rate of inactivation by the protein inhibitor of the isolated F1-ATPase and ATPase in submitochondrial particles. Modification of SH groups in the ATPase does not change the rate of inactivation but completely prevents the effect of oxidants.     

Assembly of F1-ATPase in isolated mitochondria

The assembly of the proton-translocating ATPase complex was studied in isolated mitochondria by incubating yeast mitochondria with radiolabeled precursors of mitochondrial proteins which had been made in a cell-free protein synthesis system. Following such an incubation, the ATPase complex (F1F0) was isolated. Newly assembled F1-ATPase was detected by autoradiography of the isolated enzyme, only peptide subunits which had been made in vitro and imported into the isolated mitochondria could be radioactive. Incorporation of radiolabeled ATPase subunits into the enzyme does not occur in the presence of an uncoupler of oxidative phosphorylation or of a divalent metal chelator, nor does it occur in submitochondrial particles rather than intact mitochondria. Incorporation of labeled ATPase subunits into the enzyme can be completed by unlabeled subunits, provided the unlabeled proteins are added before the mitochondria are incubated with radioactive precursors. These findings suggest that F1-ATPase is assembled from a pool of subunits in mitochondria.     

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.