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.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor

Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A., Penin, F., Godinot, C. and Gautheron, D.C. (1980) Biochemistry 19, 5671-5678). It is shown here that inorganic phosphate (Pi) together with ADP during preincubation abolishes the time-dependence of the inhibition after the addition of the substrate Mg-ATP. This preincubation in the presence of both Pi and ADP slowly leads to a conformation of the enzyme immediately inhibited after the addition of the substrate Mg-ATP. The Pi effect is half-maximal at 35 microM and pH 6.6, whereas a limited effect is induced at pH 8.0. The preincubation of F1-ATPase with Pi and ADP must last long enough (t1/2 = 5 min). The effects can be correlated to the amount of Pi bound to the enzyme, 1 mol Pi per mol (apparent KD of 33 microM) at saturation. Pi neither modifies the ADP binding nor the final level of the concomitant inhibition. When Pi is not present in the preincubation, the final stable rate of ADP-induced hysteretic inhibition is always reached when a near-constant amount of Pi has been generated during Mg-ATP hydrolysis. Kinetic experiments indicate that preincubation with ADP and Pi decreases both Vmax and Km which would favor a conformational change of the enzyme. Taking into account the Pi effects, a more precise model of hysteretic inhibition is proposed. The natural protein inhibitor IF1 efficiently prevents the binding of Pi produced by ATP hydrolysis indicating that the hysteretic inhibition and the IF1-dependent inhibition obey different mechanisms.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of coupling factor 1 of Rhodospirillum rubrum from a Ca2+-ATPase into a Mg2+-ATPase

Isolation of F1-ATPase from Rhodospirillum rubrum by chloroform extraction of chromatophores, followed by purification on a glycerol gradient, results in a very pure enzyme preparation containing five subunits with high Ca2+-ATPase activity (15 mumol per min per mg protein). Furthermore, conditions are reported under which the purified F1 exhibits Mg2+-dependent ATPase activity of about 35 mumol per min per mg protein. NaHCO3 stimulates the Mg2+-activity from 1.5 mumol per min per mg protein to 5 mumol per min per mg protein giving a maximal activity at a concentration of about 60 mM NaHCO3. Lauryl dimethylamine oxide (LDAO), octyl glucoside and nonanoyl N-methylglucamide enhance the Mg2+-ATPase activity from 1.5 to 14, 22 and 35 mumol per min per mg protein, respectively, in the absence of NaHCO3, and from 5 to 34, 30 and 37 mumol per min per mg protein, respectively, in the presence of 50 mM NaHCO3. The Vmax is increased, but the Km for ATP remains the same, about 0.22 mM, both in the absence of activators and in the presence of NaHCO3, LDAO or NaHCO3 plus LDAO. Ca2+-dependent ATPase activity is slightly stimulated by NaHCO3 but strongly inhibited by octyl glucoside.     

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.     

Kinetic mechanism of Fo x F1 mitochondrial ATPase: Mg2+ requirement for Mg x ATP hydrolysis

The initial rates of ATP hydrolysis catalyzed by Fo x F1 (bovine heart submitochondrial particles) preincubated in the presence of Pi for complete activation of the oligomycin-sensitive ATPase were measured as a function of ATP, Mg2+, and Mg x ATP concentrations. The results suggest the mechanism in which Mg x ATP complex is the true substrate of the ATPase and the second Mg2+ bound at a specific pH-dependent site is needed for the catalysis. Simple hyperbolic Michaelis--Menten dependences of the reaction rate on the substrate (Mg x ATP) and activating Mg2+ were found. In contrast to the generally accepted view, no inhibition of ATPase by free Mg2+ was found. Inhibition of the reaction by free ATP is due to a decrease of free Mg2+ needed for the catalysis. In the presence of both Ca2+ and Mg2+ the kinetics of ATP hydrolysis suggest that the Ca x ATP complex is neither hydrolyzed nor competes with Mg x ATP, and free Ca2+ does not affect the hydrolysis of Mg x ATP complex. A crucial role of free Mg2+ in the time-dependent inhibition of ATPase by azide is shown. The dependence of apparent Km for Mg x ATP on saturation of the Mg2+-specific site suggests the formal ping-pong mechanism in which bound Mg2+ participates in the overall reaction after dissociation of one product (most likely Pi) thus promoting either release of ADP (catalytic turnover) or slow isomerization of the enzyme--product complex (formation of the dead-end ADP(Mg2+)-inhibited enzyme). The rate of Mg x ATP hydrolysis only slightly depends on pH at saturating Mg2+. In the presence of limited amounts of free Mg2+ the pH dependence of the initial rate corresponds to the titration of a single group with pKa = 7.5. The simple competition between H+ and activating Mg2+ was observed. The specific role of Mg2+ as a coupling cation for energy transduction in Fo x F1-ATPase is discussed.     

Amount and turnover rate of the F0F1-ATPase and the stoichiometry of its inhibition by oligomycin in Rhodospirillum rubrum chromatophores

The amount of F1-ATPase in chromatophores from Rhodospirillum rubrum was determined by Western blotting using anti-RrF1 rabbit antibodies. 9.1 mmol F1 (mol bacteriochlorophyll)-1 was obtained or 14% of the total protein content of the chromatophores. The turnover rate of the F0F1-ATPase was 17 molecules ATP s-1 during synthesis, 2 molecules ATP s-1 during hydrolysis under coupled conditions with Mg2+ as the divalent cation, and 7 molecules ATP s-1 during hydrolysis in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Binding of 1 mol oligomycin/mol F0F1-ATPase was found to inhibit the activities of the enzyme completely. A single binding site was found with a Kd of approximately 2 microM.     

Structural and functional differences in H+-ATPases with native and reconstituted inhibitor protein

Anti F1 antibodies that react with the alpha and beta subunits of the mitochondrial F0-F1 ATPase complex do not interfere with the natural inhibitor protein-ATPase interaction as revealed by inhibitor peptide titration curves. Submitochondrial particles with endogenous or added bound inhibitor protein show differences in immunoprecipitation. Submitochondrial particles which are partially depleted of inhibitor protein gave the same immunoprecipitation curve as the Mg-ATP particle. Anti F1 antibodies induce differential effects in ATP hydrolysis and ATP-Pi exchange. ATP hydrolysis is stimulated in Mg-ATP particles to 200%, while inhibitor depleted and inhibitor reconstituted particles are inhibited by the presence of the antibodies. ATP-Pi exchange is stimulated in inhibitor reconstituted particles and inhibited in Mg-ATP and inhibitor depleted particles. These results suggest that the inhibitor protein when endogenously bound confers a different conformation to the F1-ATPase than that of the F1 ATPase with added bound inhibitor protein.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Investigation of the substrate structure and metal cofactor requirements of the rat liver mitochondrial ATP synthase/ATPase complex

The F1 moiety of the rat liver mitochondrial ATP synthase/ATPase complex contains as isolated 2 mol Mg2+/mol F1, 1 mol of which is nonexchangeable and the other which is exchangeable (N. Williams, J. Hullihen, and P.L. Pedersen, (1987) Biochemistry 26, 162-169). In addition, the enzyme binds 1 mol ADP/mol F1 and 3 mol AMP.PNP, the latter of which can bind in complex formation with divalent cation and displace the Mg2+ at the exchangeable site. Thus, in terms of ligand binding sites the fully loaded rat liver F1 complex contains 3 mol MgAMP.PNP, 1 mol ADP, and 1 mol Mg2+. In this study we have used several metal ATP complexes or analogs thereof to gain further insight into the ligand binding domains of rat liver F1 and the mechanism by which it catalyzes ATP hydrolysis in soluble and membrane bound form. Studies with LaATP confirmed that MgATP is the most likely substrate for rat liver F1, and provided evidence that the enzyme may contain additional Mg2+ binding sites, undetected in previous studies of F1-ATPases, that are required for catalytic activity. Thus, F1 containing the thermodynamically stable LaATP complex in place of MgATP requires added Mg2+ to induce ATP hydrolysis. As Mg2+ cannot readily displace La2+ under these conditions there appears to be a catalytically important class of Mg2+ binding sites on rat liver F1, distinct from the nonexchangeable Mg2+ site and the sites involved in binding MgATP. Additional studies carried out with exchange inert metal-nucleotide complexes involving rhodium and the Mg2+ and Cd2+ complexes of ATP beta S and ATP alpha S imply that the rate-limiting step in the ATPase reaction pathway occurs subsequent to the P gamma-O-P beta bond cleavage steps, perhaps at the level of Mg(ADP)(Pi) hydrolysis or MgADP release. Evidence is presented that Mg2+ remains coordinated to the leaving group of the reaction, i.e., the beta phosphoryl group. Finally, in contrast to soluble F1, F1 bound to F0 in the inner mitochondrial membrane failed to discriminate between the Mg2+ complexes of the ATP beta S isomers. This indicates that a fundamental difference may exist between the catalytic or kinetic mechanism of F1 and the more physiologically intact F0F1 complex.     

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.     

Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.     

Loss of unisite and multisite catalyses by Escherichia coli F1 through modification with adenosine tri- or tetraphosphopyridoxal

Pyridoxal phosphate (PLP) and adenosine diphospho (AP2-PL)-, triphospho (AP3-PL)-, and tetraphospho (AP4-PL)-pyridoxals (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964) were tested as potential affinity probes for F1 ATPase of Escherichia coli. Both AP3-PL and AP4-PL bound and inhibited F1 ATPase, whereas PLP and AP2-PL were weak inhibitors. The concentrations of AP3-PL and AP4-PL for half-maximal inactivations of the multisite (steady state) ATPase activity were both 18 microM. The binding of these reagents to a reactive lysyl residue(s) was confirmed from the difference absorption spectra, and the stoichiometry of binding of [3H]AP3-PL to F1 at the saturating level was about 1 mol/mol F1. The analogue bound to both the alpha subunit (about two-thirds of the radioactivity) and the beta subunit (about one-third of the radioactivity). No inactivation of multisite ATPase activity or binding of AP3-PL was observed in the presence of ATP. F1 modified with about one mol of AP3-PL had essentially no uni- and multisite hydrolysis of ATP. The rate of binding of ATP decreased to 10(-2) of that of unmodified F1, and the rate of release of ATP was about two times faster. The equilibrium F1 X ATP in equilibrium F1 X ADP X Pi was shifted toward F1 X ATP, and no promotion of ATP hydrolysis at unisite was observed with excess ATP. These results suggest that the AP3-PL or AP4-PL bound to an active site, and catalysis by the two remaining sites was completely abolished.     

Energy-linked reactions catalyzed by the purified ATPase complex (F0F1) from Rhodospirillum rubrum chromatophores

1. The isolation of F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dye fluorescence and ATP-32Pi exchange.     

Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives.

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.     

Defective proton ATPase of uncA mutants of Escherichia coli. 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis

The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase. In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity. uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1). Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled. Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin. The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide. Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS)