The calcium-binding ATPase inhibitor protein from bovine heart mitochondria. Purification and properties

Two ATPase inhibitor proteins were isolated together from bovine heart mitochondria by a new procedure; each was purified further. The one inhibitor is a Ca2+-binding protein. It was found to contain 2 cysteine residues/mol as well as threonine and proline residues, all of which the other inhibitor (first isolated by Pullman and Monroy (Pullman, M.E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769] lacks. Its minimal molecular weight was 6390 with 62 amino acid residues/mol, and its isoelectric point was 4.6. Besides differences in size, composition, and response to Ca2+, the two inhibitor proteins also differed in response to sulfhydryl compounds, pH, KCl, and cardiolipin. Inhibition by the two inhibitor proteins was additive. Both cross-reacted with mitochondrial ATPase from rat skeletal muscle. Calmodulin, with or without Ca2+, had no effect on the activity of either inhibitor protein. Antibody to the Ca2+-binding inhibitor protein did not interact with the Pullman-Monroy inhibitor or have any effect on its activity. The antibody interacted with intact submitochondrial particles that contained both inhibitor proteins but not with particles from which only the Ca2+-binding inhibitor had been removed. Clearly, the two inhibitors are distinct immunologically as well as in other properties. The two types of inhibitor protein were also isolated from rat skeletal muscle mitochondria by the new procedure.     

Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria

Summary Phosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a gt11 expression library with antibody raised against the mitochondrial Ca²⁺-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498–11503, 1988). Similar clones were obtained by the immunoscreening of a rat heart expression library. In agreement with others, the open reading frames of the cDNAs from the two species coded for the same amino acid sequence. No difference in S6 of the human neoplastic cells compared to that of non-neoplastic cells was found. However, common antigenic determinants in S6 and CaBI were indicated. Accordingly, S6 was purified from rat liver ribosomes and antiserum prepared. Immuno-dot blot and Western blot analyses showed high specific reactivity between S6, the cloned chimeric -galactosidase fusion protein from a cDNA clone, and CaBI with anti-S6 and anti-CaBI antibodies. The antibodies also showed a high degree of discrimination for S6 and CaBI. Neither interacted with the other ribosomal proteins nor with another ATPase inhibitor protein from bovine heart mitochondria. Neither interacted with the Ca²⁺-binding proteins, calmodulin, oncomodulin, Protein C, or Factor X. Prothrombin was weakly reactive with anti-CaBI but not with anti-S6. Thus, the results fulfill the specific criteria for the concept and operational definition of common protein epitopes in S6 and CaBI. However, neither prothrombin nor S6 fusion protein inhibited mitochondrial ATPase activity even at 20 times the concentrations at which CaBI gave 97% inhibition.Abbreviations CaBI the Ca²⁺-binding mitochondrial ATPase inhibitor protein - PMI the mitochondrial ATPase inhibitor protein of Pullman and Monroy [31]     

Inactive to active transitions of the mitochondrial ATPase complex as controlled by the ATPase inhibitor.

The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, of particles that are in an 'active' state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 microM ATP giving 50% inhibition of the proton-motive force-induced transition.     

Inactive to active transitions of the mitochondrial ATPase complex as controlled by the ATPase inhibitor

The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, or particles that are in an ‘active’ state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 μM ATP giving 50% inhibition of the proton-motive force-induced transition.     

Regulation of the synthesis and hydrolysis of ATP by mitochondrial ATPase. Role of the natural ATPase inhibitor protein

The action of the natural ATPase inhibitor protein of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) on the mechanisms of energy conservation of heart mitochondria has been explored. The synthesis and hydrolysis of ATP and the Pi-ATP exchange reaction were studied in submitochondrial particles that possess the ATPase-inhibitor protein complex in two distinguishable states. In addition to their different rates of hydrolysis, the two states of the complex have been identified from their different accessibility to antibodies directed against the inhibitor protein, and from the different action of antibodies and trypsin on the ATPase activity of the two types of particles studied. The steady state rates of hydrolysis and of the Pi-ATP exchange reaction of the particles are determined by the state in which the ATPase-inhibitor complex exists. Apparently by modifying the rate of one of the steps involved in the catalytic reaction of the ATPase, the inhibitor protein determines the extent to which the enzyme is able to catalyze ATP hydrolysis and the Pi-ATP exchange reaction. This action of the inhibitor protein also reflects the rate at which the particles carry out oxidative phosphorylation.     

The epsilon subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli

The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor [M. E. Pullman and G. C. Monroy (1963) J. Biol. Chem. 238, 3762-3769] distinct from the epsilon subunit of E. coli ATPase. Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step. An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the epsilon subunit of E. coli ATPase. The other inhibitory fraction observed in the purification procedure consisted of aggregated epsilon subunits.     

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.     

Overexpression, Purification, and Characterization of Human and Bovine Mitochondrial ATPase Inhibitors: Comparison of the Properties of Mammalian and Yeast ATPase Inhibitors

Mitochondrial ATP synthase (F₁F₀-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F₁F₀-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F₁F₀-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested.     

Diphosphatidylglycerol-induced changes in the organization of mitochondrial ATPase

1. On submitochondrial particles from bovine heart, diphosphatidylglycerol produced a selective solubilization of ATPase. The solubilized enzyme was purified further by ammonium sulfate fractionation and shown to have the same reconstitutive activity as coupling factor F₁ (Pullman, M.E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322–3329).

2. Diphosphatidylglycerol-treated submitochondrial particles retained large amounts of the phospholipid and showed a decreased ATPase activity. Once the excess of phospholipid was removed, soluble ATPase could be again reincorporated in an oligomycin-sensitive complex.

3. On Mg-ATP particles the solubilization of ATPase induced by diphosphatidylglycerol was preceded by a stimulation of oligomycin-sensitive ATPase which indicated a dissociation of F₁ from the ATPase inhibitor (Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762–3769). Magnesium was required to obtain the oligomycin-sensitive stimulation whereas in the absence of magnesium the solubilization of ATPase was prevalent.

4. It is concluded that the decreased association of F₁ with the ATPase inhibitor produced by diphosphatidylglycerol, causes a labilization of ATPase-membrane interaction. Under particular conditions, e.g. a high amount of phospholipid and a low concentration of magnesium, this is followed by the detachment of ATPase.     

Evidence for an endogenous ATPase inhibitor protein in plant mitochondria. Purification and characterization

An endogenous ATPase inhibitor protein has been identified and isolated for the first time from plant mitochondria. The inhibitor protein was isolated from potato (Solanum tuberosum) tuber mitochondria and purified to homogeneity. The isolated inhibitor is a heat-stable, trypsin-sensitive, basic protein, with a molecular mass approximately 8.3 kDa. Amino acid analysis reveals a high content of glutamic acid, lysine and arginine and the absence of proline; threonine and leucine. The interaction of the inhibitor with F1-ATPase requires the presence of Mg2(+)-ATP in the incubation medium. The ATPase activity of isolated F1 is inhibited to 50% in the presence of 14 micrograms inhibitor/mg F1. A stoichiometry of 1.3 mol inhibitor/mol F1 for complete inhibition can be calculated from this value. The potato ATPase inhibitor is also a potent inhibitor of the ATPase activity of the isolated yeast F1. The inhibitor resembles the ATPase inhibitors of yeast and mammalian mitochondria, and does not seem to be related to the inhibitory peptide, epsilon subunit, of chloroplast ATPase.     

Regulation of the ATP-supported Ca2+ uptake by heart and liver mitochondria

The ATP-supported Ca²⁺ uptake of heart and liver mitochondria preincubated in conditions in which electron transport had either been prevented by rotenone or antimycin, or induced by oxidizable substrates, has been studied. Mitochondria preincubated with respiratory inhibitors accumulate Ca²⁺ less efficiently than mitochondria preincubated with oxidizable substrates. The difference correlates with the degree of activation of the oligomycin-sensitive ATPase. The results indicate that the rate at which mitochondria take up Ca²⁺ in the ATP-supported system may be controlled by the reversible asociation of the inhibiting peptide (Pullman,. and Monroy, J. Biol. Chem., 238, 3762–3769) with the ATPase complex. Since this process appears to be modulated by the transmembrane electrochemical gradient, the latter may regulate the uptake of Ca²⁺ in a hitherto undescribed way.     

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.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor.

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Purification of the mitochondrial citrate transporter in yeast.

We present a method of partial purification of mitochondrial citrate transporter of the yeast Saccharomyces cerevisiae. Based on functional evidence of interaction between citrate transport and citrate synthase, we have used an affinity column containing pig heart citrate synthase (PHCS) for the purification. The purified preparation shows two protein components whose Mr is approximately 50K and 60K. The specific activity of our purest fractions is 2.6 mumoles/min which compares favorably to that of purified beef liver enzyme and purified rat liver enzyme.     

Purification and immunological properties of proton-ATPase complexes from yeast and rat liver mitochondria

Proton-ATPase complexes from yeast and rat liver mitochondria were isolated by a simple method previously employed for the purification of the proton-ATPase complex from chloroplasts. After reconstitution into liposomes, the purified complexes were active in the ATP-Pi exchange reaction, the rate of which was 120 and at least 200 nmol/mg of protein/min for the rat liver and yeast mitochondria ATPases, respectively. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, each complex exhibited 11 to 12 different polypeptides. The isolated ATPase complexes from rat liver and yeast mitochondria, from Swiss chard chloroplasts, and Escherichia coli membranes were reacted with antibodies prepared against the various subunits of ATPase complexes. From all the combinations of antigen-antibody examined, only the antibodies against beta subunit cross-reacted with the corresponding subunit of all the ATPase complexes tested. These results indicate that certain amino acid sequences in the beta subunit have been preserved in all of the proton-ATPase complexes.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

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.     

A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae.

A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1.     

Alpha-methylisocitrate. A selective inhibitor of TPN-linked isocitrate dehydrogenase from bovine heart and rat liver.

Alpha-Methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) is a potent inhibitor, competitive with isocitrate (1-hydroxy-1,2,3-propanetricarboxylate), of the TPN-linked isocitrate dehydrogenase from bovine heart and rat liver; it does not inhibit the DPN-specific enzyme from these tissues. In the presence of magnesium ion, values of Kis for DL-alpha-methylisocitrate for purified bovine heart enzyme, rat liver cytosol, and rat liver mitochondrial extract were in the range of 0.1 muM to 0.3 muM. This compared to values of apparent Km for DL-isocitrate for the same tissue preparations of 14 muM to 20 muM. One of the DL isomer pairs of alpha-methylisocitrate was inactive; the observations suggest that it is threo-alpha-methylisocitrate which inhibits TPN-linked isocitrate dehydrogenase. A method of synthesis of DL-threo-alpha-methylisocitric lactone (2-methyl-5-oxo-2,3-furandicarboxylic acid) from dimethyl trans-epoxymethylsuccinate and dimethylmalonate is described.     

Cytochrome c1 of bakers' yeast. I. Isolation and properties.

Cytochrome c1 has been purified from mitochondria of the yeast Saccharomyces cerevisiae. The procedure involves solubilization withcholate, ammonium sulfate fractionation, disruption of the dytochrome b-c1 complex with mercaptoethanol and detergents, and chromatography on DEAE-cellulose. The final product is psectrally pure, contains up to 62 nmol of covalently bound heme per mg of protein and does not react with oxygen or carbon monoxide. Sodium dodecyl sulfate disaggregates the purified cytochrome into a single 31,000 dalton subunit carrying the covalently attached heme group. Many cytochrome c1 preparations contain in addition an 18,500 dalton polypeptide which is devoid of covalently bound heme. Since this polypeptide can be removed from the heme-carrying polypeptide by relatively mild procedures, it is probably not an essential subunit of cytochrome c1. Cytochrome c1 is extremely sensitive to proteolysis. If it si purified in the absence of protease inhibitors, a family of heme polypeptides with molecular weights of 29,000, 27,000, and 25,000 daltons is obtained. In the presence of the protease inhibitor phenylmethylsulfonylfluoride the purification yields predominantly a 31,000 dalton heme protein with only little contamination by a 29,000 dalton degradation product. In order to show that only the 31,000 dalton heme-polypeptide is the native species, yeast cells were labeled with the heme-precursor delta-amino[3H]levulinic acid, converted to protoplasts and directly lysed with dodecyl sulfate in the presence of protease inhibitors. Subsequent electrophoresis of the lysate in the presence of dodecyl sulfate reveals the covalently bound heme of cytochrome c1 as a single symmetrical peak at 31,000 daltons.