Large-scale purification and phosphorylation of a detergent-treated adenosine triphosphatase complex from plasma membrane of Saccharomyces cerevisiae

A new procedure for large-scale preparation of plasma-membrane-bound ATPase from Saccharomyces cerevisiae is described. The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.25 mg/mg protein), Triton X-100 (0.25%) and lysophosphatidylcholine (1 mg/mg protein). These treatments extract the mitochondria and strip the plasma membrane. From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2--3 days. Plasma-membrane-bound ATPase of specific activity of 10--13 mumol Pi x min-1 x mg protein-1 is obtained with a yield estimated to 60%. Dodecylsulfate/polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr = 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of Mr = 95000 identified as the ATPase subunit is phosphorylated by millimolar concentrations of ATP. The phosphorylated intermediate reaches the steady-state level in less than 100 ms and turns over very rapidly. It is hydrolyzed by hydroxylamine. Its formation is prevented by the ATPase inhibitors vanadate and Dio-9, a plasma-membrane ATPase inhibitor of unknown structure. At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein-kinase(s).     

Mitochondrial H+-ATPase activation by an amine oxide detergent

Lauryl dimethylamine oxide activates ATP hydrolysis by the mitochondrial H+-ATPase. Activation is observed in systems with a high content of inhibitor protein as described by Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769), i.e. Mg-ATP submitochondrial particles and a Triton X-100-solubilized H+-ATPase from the same particles. Detergent activation of ATP hydrolysis is also present in inhibitor-reconstituted systems, i.e. submitochondrial particles, Triton extracts, and soluble F1-ATPase. In submitochondrial particles depleted of inhibitor protein, lauryl dimethylamine oxide induced a biphasic response which is characterized by a drop-in activity induced by relatively low concentrations of LDAO; at higher concentrations the detergent activates to an extent never greater than the initial activity. In inhibitor protein-depleted oligomycin-sensitive Triton extracts, lauryl dimethylamine oxide stimulates ATP hydrolysis to very high values (30 mumol min-1 mg-1). These findings suggest that in addition to the inhibitor protein ATP hydrolysis is controlled by other subunit interactions.     

F0F1-ATPase of plant mitochondria: isolation and polypeptide composition

A simple and high yield purification procedure for the isolation of F0F1-ATPase from spinach leaf mitochondria has been developed. This is the first report concerning purification and composition of the plant mitochondrial F0F1-ATPase. The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-[(3-cholamidopropyl) dimethyl ammonio]-1- propane sulfonate (CHAPS). The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 mumol.min-1.mg-1). SDS-PAGE of the purified F0F1-ATPase complex reveals protein bands of molecular masses of 54 kDa (F1 alpha,beta), 33 kDa (F1 gamma), 28 kDa, 23 kDa, 21 kDa (F1 delta), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (F1 epsilon) and 8.5 kDa. All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100. Five polypeptides could be identified as subunits of F1. Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F0 part of the complex. Our results show that polypeptide composition of the plant mitochondrial F0 differs from other eukaryotic F0 of yeast, mammals and chloroplasts.     

Cleavage of type 2 adenovirus DNA BY HaeIII endonuclease. II. Map of HaeIII sites in EcoRI fragments C and E

1. A new method for the isolation of the oliogomycin-sensitive ATPase from beef-heart mitochondria is described. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 mumol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 mumol for the oligomycin-insensitive complex. 6. The preparation of the oligomycin-sensitive complex contains at least 6--7 polypeptides in addition to those derived from F1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. Some of these polypeptides have been identified and isolated.     

Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column.

The (Ca2+-Mg2+)-ATPase from human erythrocyte membranes has been solubilized in Triton X-100 and purified on a calmodulin affinity chromatography column in the presence of phosphatidylserine, to limit the inactivation of the enzyme. The enzyme was purified at least 150 times when compared with the original ghosts and showed a specific activity of 3.8 mumol.mg-1.min-1. In sodium dodecyl sulfate-polyacrylamide gels, a single major band was visible at a position corresponding to a molecular weight of about 125,000; a minor band (11% of the total protein) was present at a position corresponding to Mr = 205,000. Upon incubation of the purified preparation with [32P]ATP, both bands were phosphorylated in proportion to their mass, suggesting that both were active forms of purified ATPase.     

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.     

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes

Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.     

Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and characterization of an oligomycin and N,N'-dicyclohexylcarbodiimide-sensitive (Ca+ + Mg2+)-ATPase.

An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin has been solubilized from Rhodospirillum rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified RrFo . F1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-sensitive, water-soluble RrF1 ATPase. The ATPase activity of RrF0 . F1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and from detailed kinetic studies it is concluded that the divalent cation-ATP complex is the substrate for both ATPase complexes. Free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 muM, respectively. The subunit composition of the purified RrFo . F1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggests that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.     

Kinetic properties and solubilization of microsomal cholesterol ester hydrolase from rat liver

Some kinetic properties of the microsomal cholesterol ester hydrolase (CEH) have been examined in rat liver. The reaction was linear with time up to 60 min and with enzyme concentration up to 0.3 mg/mL, and a pH optimum of 6.7 for enzyme activity was observed. Cholesterol esterase exhibited the following apparent kinetic constants: Km, 68.88 microM and Vmax, 33 Units/mg protein. Cholesteryl palmitate was hydrolyzed to a much greater extent than cholesteryl oleate by the enzyme. Product inhibition with cholesterol and palmitic acid was not apparent; however, oleic acid added to the system reduced markedly microsomal CEH activity. The present paper also reports the solubilization of cholesteryl palmitate hydrolase from the microsomal fraction by pretreating it with Triton X-100, sodium deoxycholate, and sodium dodecylsulfate. All ionic and non-ionic detergents tested are capable of making the enzyme soluble, and maximal effects were found at higher concentrations of detergents although the esterase activity was strongly inhibited. Triton X-100 was found to be more effective than sodium deoxycholate and sodium dodecylsulfate in enzyme and protein solubilization. When the direct effects of detergents on CEH activity were studied, progressive concentration-dependent inhibitions were observed.     

Rapid purification of membrane extrinsic F1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization.

A new chromatographic procedure for purification of the membrane extrinsic F1-domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca(2+)-dependent ATPase activity of 1.0 mumol.mg-1 min-1 and a Mg(2+)-dependent activity of 4.4 mumol.mg-1 .min-1. Both activities are increased up to 8-10-fold after dithiothreitol activation. Analysis of the purified F1-complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose-1,5-bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1-complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1-complex could be crystallized by MicroBatch.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

Effect of clofibrate on the adenosine triphosphatase activity of rat liver mitochondria.

The antihypercholesterolemic drug clofibrate (ethyl-α-p-chlorophenoxyisobutyrate) stimulated the latent ATPase activity and “superstimulated” the uncoupler-induced ATPase activity of rat-liver mitochondria. Addition of clofibrate decreased the turbidity of mitochondrial suspensions and released considerable amount of mitochondrial protein into solution. In these properties it closely resembled detergents like Triton X-100 and deoxycholate. However, unlike the detergents, clofibrate required the presence of a permeant cation for its disruptive action. Also, it was without any such effect on sonic submitochondrial particles. The drug enhanced the uptake of both Mg² and Cl^? by mitochondria suggesting that osmotic swelling precedes lysis. Sonic submitochondrial particles prepared in the presence of clofibrate showed a greater yield and comparable ATPase activity.     

Induction of a novel long-chain acyl-CoA hydrolase in rat liver by administration of peroxisome proliferators

The activity of long-chain acyl-CoA hydrolase in rat liver was increased by the administration of peroxisome proliferators, such as ethyl p-chlorophenoxyisobutyrate, di(2-ethylhexyl)phthalate or acetylsalicylic acid. The induced activity was mainly confined in the soluble fluid after the subcellular fractionation. The enzyme was purified nearly to homogeneity from livers of rats treated with di(2-ethylhexyl)phthalate. The specific activity of the final preparation was 247 mumol palmitoyl-CoA hydrolyzed min-1 mg protein-1. The molecular weight of the native enzyme was estimated to be 150 000 by gel filtration and that of the subunits was 41 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The activity of the enzyme was not increased but inhibited by bovine serum albumin or Triton X-100. The molecular and catalytic properties of the enzyme suggest that the induced enzyme was different from mitochondrial and microsomal long-chain acyl-CoA hydrolyses in liver.     

The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum

Membrane vesicles capable of energized Ca2+ pumping have been reconstituted from cardiac sarcoplasmic reticulum (SR). Cardiac SR was solubilized with Triton X-100 in a detergent to protein weight ratio of 0.8, and membranous vesicles were reconstituted by removal of detergent with Bio-Beads SM-2 (a neutral porous styrene-divinylbenzene copolymer). The reconstituted vesicles exhibited ATP-dependent oxalate-facilitated Ca2+ accumulation with rates and efficiency comparable to the best reconstituted skeletal muscle preparation (Ca2+-loading rate = 1.65 +/- 0.31 mumol mg-1 min-1, Ca2+-activated ATPase activity = 2.39 +/- 0.25 mumol mg-1 min-1, efficiency (Ca2+/ATP) = 0.69 +/- 0.09). Phospholamban in the reconstituted vesicles was phosphorylated with added catalytic subunit of cAMP-dependent protein kinase to almost the same extent as that in original vesicles. However, phosphorylation of phospholamban had no effect on the Ca2+ accumulation of the reconstituted vesicles. This is to be contrasted with a decrease in the half-maximal concentration of Ca2+ for Ca2+ accumulation (KCa) in the original vesicles from 1.35 +/- 0.08 microM to 0.75 +/- 0.12 microM by cAMP-dependent phosphorylation of phospholamban. On the other hand KCa for the reconstituted vesicles was about 0.5 microM and remained unchanged by phosphorylation, indicating that the Ca2+ pump in the reconstituted vesicles is already fully activated. These results suggest that in normal cardiac SR, phospholamban in the dephosphorylated state acts as a suppressor of the Ca2+ pump and that phosphorylation of phospholamban serves to reverse the suppression.     

ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.     

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.     

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

(i) The method of preparing the oligomycin-insensitive F₁-ATPase by chloroform treatment of mitochondrial membranes (Beechey et al., 1975, Biochem. J.148, 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p-aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, (ii) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F₁-F₀ ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F₁ sector. The complex retains oligomycin- and uncoupler-sensitive ATP-³²P_i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. (iii) F₁-ATPase is released from the F₁-F₀ complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F₁ sector can be isolated containing either four or five subunits depending on whether the starting F₁-F₀ complex contained the 42,000 and 29,000 molecular weight polypeptides. (iv) Sensitivity of the F₁-F₀ ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.     

Purification and properties of the F1-ATPase from liver mitochondria of Gallus gallus.

1. This paper is the first detailed report of the purification of a mitochondrial ATPase from an avian species. 2. The Gallus gallus liver mitochondrial F1-ATPase was purified by chloroform extraction and ion-exchange chromatography. 3. The enzyme shows the five alpha, beta, tau, delta, and epsilon subunits characteristic of mitochondrial F1-ATPases. 4. The Km for ATP is 1 mM and for Mg 0.5 mM with a specific activity of 25.2 mu moles of ATP hydrolyzed x min-1 x mg-1. 5. Unlike mammals enzymes the chicken mitochondrial ATPase shows maximal activity with ITP as substrate, and is strongly inhibited by Cu.     

The native mitochondrial F1-inhibitor protein complex carries out uni- and multisite ATP hydrolysis

The rate of ATP hydrolysis under multi- and unisite conditions was determined in the native F1-inhibitor protein complex of bovine heart mitochondria (Adolfsen, R., MacClung, J.A., and Moudrianakis, E.N. (1975) Biochemistry 14, 1727-1735). Aurovertin was used to distinguish between hydrolytic activity catalyzed by the F1-ATPase or the F1-inhibitor protein (F1.I) complex. We found that incubation of aurovertin with the F1.I complex, prior to the addition of substrate, results in a stimulation of the hydrolytic activity from 1 to 8-10 mumol min-1 mg-1. The addition of aurovertin to a F1.I complex simultaneously with ATP results in a 30% inhibition with respect to the untreated F1.I. In contrast, if the F1.I complex is activated up to a hydrolytic activity of 80 mumol min-1 mg-1, aurovertin inhibits the enzyme in a manner similar to that described for F1-ATPase devoid of the inhibitor protein. The native F1.I complex catalyzes the hydrolysis of ATP under conditions for single catalytic site, liberating 0.16-0.18 mol of Pi/mol of enzyme. Preincubation with aurovertin before the addition of substrate had no effect under these conditions. On the other hand, if the F1.I ATPase was allowed to hydrolyze ATP at a single catalytic site, catalysis was inhibited by 98% by aurovertin. In F1-ATPase, the hydrolysis of [gamma-32P]ATP bound to the first catalytic site is promoted by the addition of excess ATP, in the presence or absence of aurovertin. Under conditions for single site catalysis, hydrolysis of [gamma-32P]ATP in the F1.I complex was not promoted by excess ATP. We conclude that the endogenous inhibitor protein regulates catalysis by promoting the entrapment of adenine nucleotides at the high affinity catalytic site, thus hindering cooperative ATP hydrolysis.     

Immunological and reconstitution studies on the adenosine triphosphatase complex from Rhodospirillum rubrum.

Studies on restoration of membrane-bound adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from Rhodospirillum rubrum show that the delta-subunit is capable of binding to the F1 factor or to the F0 moiety of the F0-F1 ATPase complex. This subunit is thus likely involved in linking the F0 and F1 factor. During solubilization of the oligomycin-sensitive F0-F1 ATPase complex with Triton X-100 the detergent becomes specifically associated with the lipophilic F0 part of the enzyme complex. Crossed immunoelectrophoresis, agglutination tests, and kinetic studies with anti-F1 ATPase antibodies reveal a reaction of immunological identity of membrane-bound ATPase, F0-F1 ATPase, and F1 ATPase.