Mitochondrial adenosine triphosphatase from human placenta—Purification and catalytic properties

Mitochondrial adenosine triphosphatase from human placenta—Purification and catalytic properties

Inr J Biochrm., 0 Pergamon I I. pp. 0020.711X/80/0201-OI65$02.0010 Vol. 165 to 175 Press Ltd 1980 Prmted I” Great Britam MITOCHONDRIAL ADENOSINE T...

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Inr J Biochrm., 0 Pergamon

I I. pp.


Vol. 165 to 175 Press Ltd 1980 Prmted I” Great Britam


ZENONALEKSANDROWICZ I.B.M., Medical School, 80-211 Gdahsk, ul. Debinki 1, Poland (Received 2 July 1979)

Abstract-l. The purification of ATPaset (EC from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATPS hydrolysis was 85 pmol per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP > ITP > ATP z E-ATP > UTP > CTP in Tris-HCI buffer (pH 8.0), and in the order: ATP > GTP > ITP z E-ATP > UTP > CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (K, = 0.2 mM, 0.09 mM) and monophasic in Tris-bicarbonate medium (K, = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.


It is clear that the membrane-associated Mg* +-stimulated adenosinetriphosphatase (EC from mitochondria is involved in the synthesis of ATP during the terminal steps of oxidative phosphorylation (Fessenden & Racker, 1966). This enzyme also participates in energy-linked reactions in mitochondria involving the uptake of divalent cations, the reduction of NADP+ and reverse electron flow. The soluble part of the membrane ATPase complex-the coupling factor 1 (F+has been isolated from bovine heart (Pullman et a[., 1960; Knowles & Penefsky, 1972; Senior & Brooks, 1970), rat liver (Catterall & Pedersen, 1971; Lambeth & Lardy, 1971), yeast (Takeshige et al., 1976) and a number of bacterial and plant systems (Adolfsen & Moudrianakis, 1971; Yoshida et al.,

*This work has been supported by the Ministry of Higher Education, Science and Technology within the project R.1.9-02, 02. t Enzymes: ATPase, adenosine triphosphatase (EC, ATP phosphohydrolase; Lactaie ddhydroge&e, L-lactate: NAD+ oxidoreductase (EC Pvruvate kinase, ATP: pyruvate 2-0-pGosphotransfk;asd (EC 3 Abbreviations: E-ATP, 1,N6-ethenoadenosine 5’-triphosphate; GTP, guanosine triphosphate; ITP, inosine triphosphate; ATP, adenosine tiiphdsphate; UTP, uridine triphosphate; CTP, cytosine triphosuhate: P;. inorganic phosphate; Tris, N:tris(hydroxymeihyl)-aminomet;ane; Tricine, N-[tris(hydroxymethyl)-methyllglycine; EDTA, ethylene-diamine-tetraacetic acid; F,, soluble ATPase or coupling factor 1. B.C.


1975; Vogel 8~ Steinhart, 1976; Younis et al., 1977; Grubmeyer et al., 1977). Several extensive reviews have appeared (Senior, 1973; Penefsky, 1974; Pedersen, 1975), which suggest that F,-ATPases play analogous structural and functional roles in each of the rather different membrane systems. Despite the extensive studies on the physiological role of mitochondrial ATPase, its participation in the catalytic mechanism of oxidative phosphorylation remains unclear. No work had been performed so far on the mitochondrial ATPase of human tissues. The mitochondrial F,-ATPase has a complex structure and contains five different types of subunits (Knowles & Penefsky, 1972). However, the subunit composition of mitochondrial ATPase is still somewhat controversial and oligomers of the type A2B,C2D,E2 (Senior, 1975), A3B3CDE (Catterall & Pedersen, 1974) and A2B2C2DXEZ (Verschoor et al., 1977) have been proposed. The soluble enzyme is cold-labile and insensitive to inhibition by oligomycin (Pullman et al., 1960). The kinetic properties of the membrane-bound and soluble enzymes from mammalian tissues in bicarbonate buffer, are very similar, however differ markedly in Tris-HCl buffer (Pedersen, 1976a). Pedersen (1976a) suggested that the membrane-bound and soluble ATPase may have similar conformations in Trisbicarbonate but different conformations in Tris-HCl. Several methods for the preparation of a soluble coupling factor ATPase from various sources have been described (Pullman et al., 1960; Senior & Brooks, 1970; Selwyn, 1967; Catterall & Pedersen, 1971; Lambeth & Lardy, 1971). Usually, the enzyme 165



present in membrane particles has been solubilized by drastic sonication. This method is laborious, timeconsuming and its application to a peculiar type of tissue requires specified conditions. Recently an easy and rapid method of releasing Fi-ATPase from heart submitochondrial particles by treatment with chloroform has been reported by Beechey et al. (1975). This technique is adapted at present for the isolation of mitochondrial Fi from human term placenta-the only human tissue accessible easily at the amounts needed for isolation of the enzyme. In this report studies on the catalytic properties of the soluble ATPase isolated by this procedure from human placental mitochondria are presented. The results obtained are compared with the properties of ATPase from other sources. MATERIALS AND METHODS Glass

redistilled and deionized water was used for the preparation of all solutions. Sucrose solution was deionized by passing through a mixed-bed ion-exchange resin (Amberlit MB-3, BDH). l,N’-Ethenoadenosine 5’-triphosphate @ATP) was purchased from ICN Pharmaceuticals, Inc.; quercetin from Calbiochem; Trizma base, Tricine, oligomycin, phosphoenolpyruvate, nucleotides and auxiliary enzymes were obtained from Sigma Chemical Co. DEAE-Sephadex A-50 and Sephadex G-10 were purchased from Pharmacia Fine Chemicals, Inc.; salts of divalent cations from Ventron Corporation, Alfa products. Aurovertin (Pitman-Moore Co., Division of Dow Chemicals) was a generous gift from Dr E. J. Davis (Indiana University). Chloroform analytical grade, and other chemicals were of the highest purity obtainable and were used without further purification. Assay of ATPase activity

ATPase activity of F, was measured in the presence of an ATP regenerating system either by calorimetric assay of Pi released from ATP, or by the spectrophotometric method as described by Pullman et al. (1960) and modified by Ebel & Lardy (1975). The buffers used in this study, TrisHCl and Tris-bicarbonate, had no effect on the reaction rates catalyzed by these coupled enzyme systems. Initial velocity experiments were performed at 30°C in a total volume of 1.0 ml containing 50 mM Tris-HCI, pH 8.0, 1 mM free magnesium as MgCII, 2 mM phosphoenolpyruvate (K+), 0.2 mM NADH (0.3 mM when using ITP, GTP, UTP or CTP) 8 units of pyruvate kinase (24 units when using UTP or CTP) 15 units of lactate dehydrogenase and nucleotides as designated in figure and table legends. The nonspecificity of pyruvate kinase for nucleotide diphosphates has been described by Kayne (1973). When submitochondrial particles ATPase activity was measured, 2 PM rotenone was added to inhibit the respiratory chain-linked NADH oxidation. Immediately before conducting the assay, the isolated ATPase was centrifuged at 12,OOOgfor 4min in microcentrifuge at room temperature to remove the (NH,),SO, and ATP that were included in the storage medium,~‘-and the sediment resuspended in 50mM Tris-HCl, pH 8.0, containing 30% (v/v) glycerol at 25°C. This solution was recentrifuged for 4 min to remove a small amount of insoluble material before assay. The reaction were initiated bv the addition of the ATPase preparation. In order to check whether ADP formation is the only rate-limiting step in the spectrophotometric assay for ATP hydrolysis, the following test was performed for each preparation of assay medium. Hexokinase and glucose were added to make a rate of absorbance change equal to or greater than that of the fastest ATP hydrolysis activity

to be measured. The amount of hexokinase was then doubled, and the assay medium was considered adequate if the rate of absorbance change doubled. The reaction, during kinetic studies, was followed by monitoring the disappearance of NADH absorbance at 340nm in a Specord UV-VIS spectrophotometer at the rapid chart speed (12 cm/min) recording system. The medium for determination of ATPase activity by Pi released was essentially the same as that described for the spectrophotometric method, except that NADH and lactic dehydrogenase were omitted. The reaction was started by the addition of enzyme (0.5-1.0 pg of protein) to the incubation medium (final vol 0.3 ml). It was terminated by the addition of 25 ~1 of 50% (w/v) trichloroacetic acid after the incubation had been carried out for 5 min at 30°C. Then 0.1 ml aliquots of the solutions were analysed for Pi. Blank values of phosphate were determined by reversing the order of addition of trichloroacetic acid and the enzyme. Measuring of ATPase activity by Pi released in the absence of ATP regenerating system was used only when the effect of metal ions, inhibitors or ADP on ATPase activity was aimed to be determined. The procedure was essentially the same as that described-in the presence of the regenerating system, except that phosphoenolpyruvate and pyruvate kinase were omitted. Pi was measured by the method of Parvin & Smith (1969). The commercial pyruvate kinase and lactic dehydrogenase preparations were centrifuged before use to remove the ammonium sulfate that was included in the storage medium, the sediment was resuspended in 25 mM Tris-HCI, pH 8.0 and then dialysed against 25 mM Tris-HCI, pH 8.0. Purification of ATP, ITP and GTP

Commercial preparations of NTP contain significant amounts of Pi. For this reason they were purified prior to use to remove this contaminant. The purification procedure consisted of chromatographing 1 ml of a 200 mM NTP solution on a Sephadex G-10 column (1 x lOcm), eluting with water, and adjusting pH of the effluent to 7.0 with 1 N Tris. The concentrations of ATP, GTP and ITP were determined spectrophotometrically at 259 (pH 7.0), 252 (pH 7.0) and 248.5 (pH 6.0) nm, assuming millimolar extinction coefficients of 15.4, 13.6 and 12.2, respectively (Beaven et al., 1955; Bock et al., 1956). Particulate protein was solubilized with deoxycholate and determined by the biuret method (Jacobs et al., 1956). Soluble protein was measured by the Coomassie blue method as described by Spector (1978). Crystalline bovine serum albumin was used as standard (E:&, = 6.67). Isolation of mitochondria All operations were performed at 2-5°C. Human placental mitochondria were isolated as described previously (Swierczynski et al., 1976). The mitochondria obtained were suspended in a buffer containing: 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5 and 1 mM EDTA and stored at a concentration of 50mg protein per ml at -26°C for at least 2 days. Preparation of submitochondrial particles

The frozen mitochondria were thawed at 25°C in a water bath, and diluted with 1 vol of 10 mM Tris-HCl buffer, pH 7.5, containing 0.25 M sucrose. The diluted mitochondria were centrifuged at 19,OOOn for 20min, and the pellet resuspended i; a buffer containing 0.25 M sucrose, 10 mM Tris-HCI. PH 7.7 and 2mM EDTA (15 mg protein/ml). Aliquots bi the suspension were added to i5ml beakers placed in an ice-water bath and sonicated in an MSE 100 W ultrasonic disintegrator for 3 min at maximum power. The treated material was pooled and centrifuged at 20,000 g at 2% for 3 min. The pellet was discarded and to the supernatant solution were added KCI, mercapto-


F, of human placenta ethanol, and glycerol to adjust the concentrations to 100mM, 2.5mM and 20% (v/v) respectively. The suspension was then centrifuged at 105,000 g and 22°C for 90 min. The resulting supernatant was decanted and discarded. The entire pellet (which contained submit~hondrial particles) was suspended in 0.25 M sucrose solution at room temperature. The yield of submitochondrial particles was usually about 40%. Solubilization of ATPase The small-scale preparation was performed essentially as described by Beechey et at. (1975). Submitochon~ial particles were suspended in a solution containing 0.1 M sucrose, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2.5 mM mercaptoethanol and 35% (v/v) glycerol to give a final protein concentration about 15 mg/ml. To this suspension l/2 vol of chloroform was added at room temperature (22°C) and the mixture was stirred vigorously with a magnetic stirrer for 20 sec. The chloroform/water emulsion was broken by low speed centrifugation (3OOg) for 2min at room temperature. The upper aqueous layer was removed, and diluted with 1 vol of 35mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 4 mM ATP and then recentrifuged at 20,000 g and 22°C for 30 min. The slightly yellow supernatant solution, which contained the ATPase, was brought to pH 8.0 with diluted Tris. Purificationof F, A soluble, Mg 2+-dependent ATPase was prepared by passing the crude aqueous supernatant (after chloroform extraction of submitochondrial particles) through a column (2cm dia x 4cm high) of DEAE-Sephadex A-50, equilibrated with buffer A (20 mM Tris-HCl, 2 mM ATP, 1 mM EDTA, 1 mM mercapt~thanol), pH 8.0 at 22°C. ATPase solution was applied slowly to the column, and washed first with 60ml of buffer A, and then with 1OOml of the same buffer containing 75mM Na2S04. F, was then sharply eluted from the column with 200 mM Na2S04 in buffer A at a flow rate of 200 ml per hr at 22°C. Fractions (5 ml) were collected. 8-9 fractions containing about 80% of the eluted activity were pooled, and then Tris-HCl, pH 8.0, ATP and EDTA were added to bring the concentrations to 50,4 and 2 mM respectively (TAE buffer). The enzyme solution was further concentrated to about 1 ml in a Schleicher and Schuell collodion bag apparatus. The preparation was stored at 4°C as a suspension in 70% saturated ammonium sulfate solution containing TAE buffer. Under these conditions. F, was stable for several months. One unit of ATPase activity is defined as the amount of enzyme that catalvses the hvdrolvsis of 1 Ltmol of ATP Der min at 30°C. Spedific activity is expressed as units per mg of protein.

RESULTS AND DISCUSSION Pur$cation of placental ATPase Mitochondriai ATPase complex is tightly bound to the inner mitochon~ial membrane. In its intact form,

the complete enzyme complex contains an ATPase inhibitor peptide (Pullman & Monroy, 1963). During isolation of the submitochondrial particles, total activity of submitochondrial enzyme increased 3.4-fold (Table l), apparently due to the removal of protein inhibitor. The membrane associated ATPase of human placenta was released in a soluble form by extraction submitochondrial particles with chloroform. This procedure typically results in solubilization of 3%38% of the ATPase activity. The soluble enzyme was then purified by ion-exchange chromatography on DEAESephadex A-50 as described above. The results of a typical purification procedure are summarized in Table 1. The isolated F,-ATPase represented about 0.2% of the mitochondrial proteins, and displayed a final specific activity of 34 units per mg of protein. Specific activity increased 380-fold during isolation and about 70% of the total original enzyme activity was recovered. The attempts to calculate the percent yield based on activity at each stage are confusing, since activation of the ATPase occurs both upon disruption of mitochondria and upon the release of the enzyme from the submitochondrial particles (Pullman et al., 1960). The purified enzyme was stable at room temperature in Tris-HCl buffer containing 30% (v/v) glycerol, for up 48 hr without loss of enzyme activity. The enzyme activity was lost rapidly in Tris-HCl buffer at O’C, as reported previously for other ATPase preparations (Pullman et al., 1960; Takeshige et al., 1976; Selwyn, 1967). The assay conditions of Table 1 (Tris-HCl, pH 8.0) were not optimal for ATPase activity (see below). Specific activity of purified ATPase was higher when measured in the presence of bicarbonate ions, as demonstrated also for other F,-ATPase preparations. Under optimal conditions (in the presence of 30 mM bicarbonate) activities in the range of 85 units per mg of protein were obtained. The specific activity of the placental ATPase was comparable with the rat liver enzyme (Pedersen, 1976a; Ebei & Lardy, 1975) and was about IS-fold lower than the enzyme isolated from beef heart (Senior & Brooks, 1970).

Table 1. Summary of the purification of human placenta1 ATPase

Fraction 1. Mitochondria 2. Submitochondrial particles 3. Chloroform extract

4. DEAE-Sephadex eluate (concentrated) 5. Ammonium sulfate precipitate

Volume (ml)

Total protein (mg)

Total activity (W

Specific activity (Wmg)

Yield (%)






24 24

350 28

245 85.7

0.7 3.1













*This assay was carried out after the mitochondria described under Materials and Methods.

had been frozen and thawed as







25 [KHCO$


50 (mM)









I 15 05 I/KHC03 (mM-')


Fig. 1. A. Effect of bicarbonate on ATPase activity at pH 8.0. Rate of ATP hydrolysis determined by estimating inorganic phosphate. The experiment was carried out at 5 mM Mg-ATP in the presence= of ATP regenerating system as described under Materials and Methods. B. Activation of ATPase by KHCO, in the presence of 1 mM Mg-ATP. Rate of ATP hydrolysis determined by spectrophotometric method as described under Materials and Methods. v, = velocity in the presence and u,, = velocity in the absence of bicarbonate. Velocities are expressed as pmols.min-’ per mg of enzyme protein. The specific activity of the ATPase was measured as a function of enzyme concentration (not shown). For the range 0.4-3.5 pg/ml the specific activity of placental enzyme was independent of enzyme concentration. All experiments were carried out at the enzyme concentration within this range. InjIuence of bicarbonate on ATPase activity Lambeth & Lardy (1971) have shown that soluble mitochondrial ATPase of rat liver is activated by various anions. They proposed the existence of an anion-binding site on the ATPase. Ebel & Lardy (1975) suggested that anions activate ATPase by inducing conformational changes in the enzyme such that the rate of the product release is increased. Pedersen (1976a) reported that the purified ATPase exhibits identical kinetic properties to the membranebound enzyme when assays are carried out in Tris bicarbonate buffer. He suggested that both forms of the enzyme may have similar conformations in Tris bicarbonate. The kinetics of hydrolysis of Mg-ATP by placental ATPase, was similar to that of rat liver enzyme (Ebel & Lardy, 1975), the rate being higher in the presence of bicarbonate. Data illustrating the effect of bicarbonate is shown in Fig. lA, in which the release of Pi is plotted against KHC03 concentration. Bicarbonate concentration required to achieve the maximum activation was about 30mM; higher concentrations of the anion inhibited the ATPase activity. The fold activation and the activation constant value for bicarbonate were determined using plots of l/(u, - uO) versus l/concentration of bicarbonate (Fig. 1B). Bicarbonate produced a 2.5-fold activation of placental ATPase with a K, of about 2.4mM. The degree of bicarbonate activation is similar to that obtained by Pedersen et al. (1974) with mitochondrial ATPase of rat liver. However, the values reported here differ (about 2-fold lower) from those reported by Ebel &

Lardy (1975) for the enzyme from rat liver mitochondria. The bicarbonate effect was highly dependent on pH. Maximum activation was observed at pH 7.4 (Fig. 2). At pH 8.5 little stimulation of the ATPase activity by bicarbonate was observed. As can be seen in Fig. 3, the degree of stimulation due to 30mM bicarbonate was not markedly dependent on NTP concentrations. The marked activation by bicarbonate of Mg-ATP hydrolysis by placental ATPase suggests the existence of an anion-binding site on the enzyme. Previously, a competitive interaction between the modulatory anions, which compete among themselves for binding sites on the ATPase of rat liver, as reported by Lambeth & Lardy (1971). Recktenwald & Hess (1977) suggested that in addition


0-O _/ \ \



____________; I----

o_Q "h

Fig. 2. pH dependent stimulation of ATPase activity by bicarbonate. Rate of MP-ATP hvdrolvsis in the uresence of ATP regenerating system determinei by anal&in* inorganic phosphate as described under Materials and Methods. v, = velocity in the presence and v0 = velocity in the absence of bicarbonate.

F, of human





-owD GTP _o-~e-“a-

1, , , , , -8






Fig. 3. The degree of bicarbonate activation of placental ATPase at varying Mg-NTP concentrations. Rate of NTP hydrolysis determined by spectrophotometric method as described under Materials and Methods.

to ATP, an anion is binding to a regulatory site on the enzyme from yeast. The experiments presented in Fig. 3 show that the effect of bicarbonate was not dependent significantly on ATP concentration. This data suggests that bicarbonate is binding to a separate regulatory site different from ATP-binding site. Substrate specijcity The placental ATPase was examined for substrate specificity at 5 mM concentration of Mg-nucleotides at pH 8.0, each test being made in the absence and in the presence of 30 mM KHCO,. It may be seen from Table 2 that the placental enzyme catalyzed preferentially the hydrolysis of purine nucleotides. Although all four purine nucleotides tested were readily hydrolysed by the ATPase, GTP was clearly the best of the four substrates in Tris-HCl buffer. The nucleoside triphosphate specificity of the enzyme was in the order GTP > ITP > ATP > E-ATP > UTP > CTP when tested in Tris-HCl buffer. Similar to beef heart F1 (Pullman et al., 1960) and rat liver ATPase (Ebel & Lardy, 1975), placental enzyme catalyzed the hydrolysis of the pyrimidine nucleotides at a lower rate. In addition, the anionic composition of the assay medium had a significant effect on the relative rates of

Table 2. Substrate


specificity Relative Tris-HCl medium

100 131.8(153.3) 152 (168.3) 80.1 I 2.2

of placental


reaction rate in Tris-bicarbonate medium 100 12 (83.8) 79.1 (88.7) 36.1 2.9 0.9

The incubation was carried out for 5 min at pH 8.0 in the presence of the ATP regenerating system as described under Materials and Methods. The incubation mixture contained 5 mM Mg-nucleotides, and 30 mM KHCOJ as indicated. ATPase activity was measured by estimating the release of Pi at 30°C.



NTPase activity. In the presence of bicarbonate, the nucleoside triphosphate specificity of placental ATPase was in the order ATP > ITP > GTP > E-ATP > UTP > CTP. In this case the rates of GTP and ITP hydrolysis was almost equal. Similar to rat liver enzyme (Ebel & Lardy, 1975) bicarbonate had little or no effect on the hydrolysis of nucleoside triphosphates other than ATP (see also Fig. 3). Similar results have been obtained from spectrophotometric (initial velocity) experiments (the values in parentheses, Table 2). Neither the nucleoside diphosphates nor the monophosphates were split (not shown). The results presented here indicate that the placental enzyme shows a broad nucleotide specificity. The substrate specificity of placental enzyme is very similar to the specificity of soluble rat liver F,-ATPase, since V,,, values for the GTPase and ITPase of that enzyme were two-fold greater than V,,, values for ATPase activity in Tris-HCl buffer (Pedersen, 1976a; Ebel & Lardy, 1975). However, the specificity of beef heart F, appears to be different. The results reported by Pullman et al. (1960) indicate that ATPase, ITPase, and GTPase activities were about equal. Metal ions activation The purified enzyme showed very little activity in the absence of an added divalent metal cation as reported previously for other ATPase preparations (Selwyn, 1967; Pedersen et al., 1974; Yeates, 1974; Tyler & Webb, 1979; Adolfsen 8~ Moudrianakis, 1973). The addition of Mg’+ ions stimulated the placental ATPase activity (Fig. 4A). In the presence of 5 mM ATP, the maximal stimulation of the enzyme required a 3 mM Mg2+ ions concentration (molar ratio of ATP to Mg’+ equal 1.6) and the activity was half-maximal in 0.24 mM Mg2+ ions. Higher concentration of Mg *+ ions inhibited this activity significantly both in the presence and in the absence of bicarbonate. The results obtained were similar to those reported by Pullman et al. (1960) with the ATPase from beef heart mitochondria. The concentrations up to 10mM Mg2+ ions were found to have no effect on the bicarbonate activation. Bicarbonate accelerated the reaction by 1.9-fold, at each concentration of Mg ‘+ ions under these conditions (in the absence of ATP regenerating system). To elucidate the mode of action of free Mg’+ ions on the ATPase activity, some kinetic parameters were examined. ATPase activity of human placental mitochondria was measured with varying concentrations of Mg-ATP complex in the presence of either 1 mM or 5 mM free Mg ’ + ions by spectrophotometric method. The results were analyzed by linear regression and fitted to double-reciprocal plot (Fig. 4B). In the presence of 5 mM free Mg*+ ions, reciprocal plots of initial velocities against varying Mg-ATP concentrations were curved and placental enzyme exhibited apparent negative cooperativity (Hill coefficient approx 0.5). In the presence of 1 mM free Mg2+ ions the reciprocal plot was linear with respect to the Mg-ATP concentration at the range 0.1-5 mM (Hill coefficient 0.75). Eadie-Hofstee plots (Eadie, 1952; Hofstee, 1952) (used to examine deviations from the MichaelisMenten kinetics and to obtain kinetic constants) is presented in the insert of Fig. 4B. The







Fig. 4. A. Optimal magnesium ion concentration for placental ATPase activity in the presence (W----O) and in the absence (A-A) of 30mM KHC03. ATPase activity was assayed with various concentrations of magnesium ions by estimating the release of Pi at 30°C in the absence of ATP regenerating system as described under Materials and Methods. B. Effect of free Mg *+ ions on ATPase activity. All measurements were performed in Tris-HCl buffer, pH 8.0 in the presence of ATP regenerating system at 30°C. Rate of ATP hydrolysis determined by spectrophotometric method as described under Materials and Methods. Determinations were carried out in the presence of 1 mM (0-O) or 5 mM (C--O) free Mg z + ions. The numbers in parentheses are the Hill coefficients. The insert (b) show Eadie-Hofstee plots. C. Inhibition by free Mg *’ ions of ATPase activity at 5 mM Mg-ATP. Assays were carried out as

described in Fig. 4A. Eadie-Hofstee plot of Fig. 4B shows that in the presence of 5 mM free Mg2+ ions a biphasic slope was obtained indicating at least two different states of the enzyme. It should be noted that Catterall & Pedersen (1974) suggested that purified mitochondrial ATPase from rat liver contains a functionally important divalent metal binding site that may play a role in regulating enzyme activity. The data presented in Fig. 4B suggests that free Mg 2+ ions might be a potent competitive inhibitor of ATPase when Mg-ATP was used as substrate. It is apparent from this figure that intercepts of the plots for two various concentrations of free Mg 2+ ions share an identical point on the reciprocal velocity axis. The competitive nature of the inhibition of ATPase activity by the free divalent cations suggested requirement for the formation of a Mg-ATP complex for catalysis. As can be seen in Fig. 4C, the inhibition produced by the free Mg2+ ions was linear and the Ki, estimated from the point at which the extrapolated curve has a value equal to

V,,,, is approx 1.4 mM. The fact that the Ki value for free Mg2+ ions was higher (about 7-fold) than the K, value for Mg-ATP could possibly indicate that in the presence of Mg-ATP bound to the catalytic site the conformation of the enzyme does not allow effective competition between the free cation and the cationATP complex at the catalytic site of the enzyme. The placental enzyme was not highly specific with regard to metal ion activators and showed similar specificity in its metal requirement to F1 isolated from beef heart or rat liver (Selwyn, 1967; Pedersen et al., 1974; Tyler & Webb, 1979). The data presented in Table 3 shows that divalent ions such as Zn2’, Mn2+, Co’+, CdZ+ and Ca’+ could be substituted for Mg2* with varying degrees of effectiveness in activating the enzyme. The degree of activation of placental enzyme was not dependent significantly on the presence of monovalent cations (K+), unlike the activation of the bacterial ATPase from Alcaligenes faecalis and bovine heart F1 (Adolfsen & Moudrianakis, 1973). The selec-

Table 3. Effect of various divalent cations on the placental ATPase activity

Metal added Mg2+ Zn2+ Mn” co*+* Cd2 + Ca’+ Ni2+ None

10 mM Tricine + 50 mM sucrose (U/mg) (%) 21.5 24.0 12.0 10.1 10.1 7.6 0.0 3.6

100 111.6 55.8 47.0 47.0 35.3 0.0 16.7

10 mM Tricine +25mM KCI (U/mg) (%) 19.8 22.0 11.4 9.5 6.9 4.5 0.0 3.9

100 111.1 57.6 48.0 34.8 22.7 0.0 19.5

10 mM Tricine + 25 mM KHCOS (Umg) (%) 36.1 19.6 22.0 25.9 4.5 5.1 2.4 4.5

100 54.3 60.9 71.7 12.5 14.1 6.6 12.3

v KHC03 v KC1 1.82 0.89 1.93 2.73 0.65 1.13 1.15

* Nitrate. The assay system contained 5 AM metal chloride, 5 mM ATP (Tris salt), and buffers (pH 8.0) as noted. Incubation were carried out for 5 min at 30°C in the absence of the ATP regenerating system, and Pi was determined as described under Materials and Methods.

F1 of human placenta


Fig. 5. A. Effect of pH on ATPase activity with Mg-ATP (O-Cl), Mg-GTP (A-A) or Mg-ITP as substrates. B. Effect of pH on the rate of Mg-ATP hydrolysis in the presence (O---O) or in the absence (M) of added 30 mM bicarbonate. The experiments were carried out in the presence of Tris-acetate and Tris-ammonia buffers at the different pH values. The buffers were prepared according to Meyers & Slater (1957) and used at a final concentration of 50 mM. The enzyme activities were assayed at 3o”C, the incubations were carried out for 10min in the presence of the ATP regenerating system.


of placental ATPase by divalent cations was markedly different in the presence of bicarbonate. In Tris-bicarbonate buffer, the specificity of the activating metal ions was higher; Zn’+ was about half as effective as Mg*+ in stimulating the enzyme activity; Cd*+ and Ca*+ ions were significantly less effective under these conditions. On the other hand, the effect of bicarbonate was depending very much on the activating metal ion present in the incubation medium (Table 3). tivity of the activation

Eflect of pH

The effect of pH on the hydrolytic activity of the placental enzyme with Mg-ATP, Mg-GTP or Mg-ITP as substrate is illustrated in Fig. 5A. In the presence of Mg-GTP or Mg-ITP the enzyme showed a broad pH activity curve. The activity gradually increased from pH 7.0 to a maximal activity between pH 8.0 and 8.5 and 80% of the maximal activity at pH 7.25 and pH 8.9. The enzyme activity measured in the presence of Mg-ATP was sharply dependent on pH. Maximum activity was observed at pH 8.5 with approx. 25% decrease in activity at 0.2 pH unit below and 0.5 pH unit above the optimum. A similar pH optimum for Mg-ATP hydrolysis by beef heart F1 has been reported by Pullman et al. (1960). As can be seen in Fig. 5A the specificity of the placental ATPase at pH 8.0 followed the order GTP > ITP > ATP. The results of experiments performed at pH 8.5 were quite different from those at pH 8.0; the substrate specificity of the placental enzyme decreased in the order ATP 2 GTP > ITP. Additional studies showed that the optimum pH for placental ATPase was markedly affected by the presence of bicarbonate. The effect of pH on Mg-ATP hydrolysis at two different media is presented in Fig. 5B. With 30mM KHC03 in the reaction mixture, 7.4 was found to be the optimum pH for ATPase activity. Kinetic properties of placental ATPase

The general kinetics of the purified placental ATPase were tested in the presence and in the

absence of bicarbonate at pH 8.0. The results are presented in Fig. 6. The rates of NTP hydrolysis by mitochondrial ATPase from human placenta were a timedependent phenomenon. In the case of Mg-ATP hydrolysis catalysed by F1 in Tris-HCl buffer, the initial linear rate of reaction was observed which then leveled off to a slower linear rate. Initial rates of Mg-ATP hydrolysis were used in all calculations. In the cases of Mg-ITP or Mg-GTP hydrolysis the rates of reaction were biphasic, an initial nonlinear rate was observed which then (after 2min) leveled off to a higher linear rate. In these cases the rates used to obtain the data for the figures presented were these from the linear, steady state nucleoside triphosphatase activity. Lineweaver-Burk plots of the kinetic data for Mg-ATP hydrolysis in the absence of bicarbonate deviated considerably from typical Michaelis-Menten behaviour (Fig. 6A). Eadie-Hofstee plots were used to obtain kinetic constants. This plot is presented in the insert of Fig. 6A. Such plots are biphasic with two distinct slopes (K, A’TP0.2 mM and 0.09 mM). In contrast to Mg-ATP hydrolysis, Lineweaver-Burk plots of the kinetic data for the hydrolysis of Mg-ITP and Mg-GTP are monophasic and of the classical (K,ITP = 0.92 mM; MichaelisMenten type K ,,,GTP= 0.79 mM) (Figs 6B and 6C). When the assays were carried out in the presence of bicarbonate, Lineweaver-Burk plots representing the hydrolysis of Mg-ITP and Mg-GTP were not markedly different from those observed in TrisHCl buffer. Reciprocal plots representing the hydrolysis of Mg-ATP, however, under these conditions showed a monophasic linear relationship with a K,ATP of 0.16 mM. Recently, non-linear kinetics of mitochondrial ATPase from beef heart (Schuster et al., 1975), rat liver (Ebel & Lardy, 1975; Pedersen, 1976a) and yeast (Takeshige et al., 1976) have been reported in various laboratories. In all cases, the existence of separate catalytic and regulatory sites for the binding of ATP were suggested. In Tris-HCl medium, placental ATPase exhibited apparent negative cooperativity with ATP as the sub-









t 3























Fig. 6. Nucleosidetriphosphatase activity of placental ATPase as a function of nucleoside triphosphate concentration in the presence (o-_o) or absence (M) of 30 mM bicarbonate. Spectrophotometric assays were car-

ried out as described under Materials and Methods. Velocities are expressed as @mols.min- 1 per mg of enzyme protein. A. ATPase activity as a function of Mg-ATP concentration. The insert (a) shows Eadie-Hofstee plot. B. ITPase activity as a function of Mg-ITP concentration. C. GTPase activity as a function of Mg-GTP concentration.

strate just as does the beef heart Fr (Schuster et al., 1975) and the rat liver enzyme (Ebel & Lardy, 1975). Higher concentrations of bicarbonate eliminated the negative cooperativity. At 20mM KHC03 concentration linear plots were observed, the maximum velocity increased 2.35-fold and the Hill coefficient of 0.75 was brought to approx 1. Negative cooperativity could not be shown when Mg-ITP or Mg-GTP were the substrates. In these cases the bicarbonate did not affect the Hill coefficients (1.0 in all cases). The extent of bicarbonate activation was much less with ITP or GTP than with ATP (1.2X-fold, 1.24-fold, 2.35fold, respectively). Similar results have been reported by Ebel & Lardy (1975), with rat liver ATPase. The kinetic parameters are summarized in Table 4. In Tris-HCl medium, the maximum velocities for the hydrolysis of Mg-GTP and Mg-ITP were higher than the maximum velocity for the hydrolysis of Mg-ATP. The maximum velocity for Mg-ATP hydrolysis in the presence of bicarbonate was about 2.35-fold higher (84.5 pmols-min-’ .mg- ‘), as compared with the maximum velocity observed without added bicarbonate (36 pmols . min - ’ per mg). In the presence of KHC03 the V,,, of Mg-ITP and Mg-GTP hydrolysis were only about 70.8 and 75 ~mols.rnin- ’ per mg, respectively. Inhibition by ADP Early work with beef heart mitochondrial Ft (Pullman et al., 1960) indicated that ADP strongly inhibits the ATPase as well as the ITPase activity of the enzyme. Hammes & Hilborn (1971), Philo & Selwyn (1973) reported that in the case of the mitochondrial Fr from beef heart, ADP is a competitive inhibitor of the ATPase and ITPase activity. Similar results have been reported for the mitochondrial ATPase of rat liver (Catterall & Pedersen, 1974; Pedersen et af., 1974). These workers found that IDP and GDP have little effect on the ATPase activity. Since IDP is not a competitive inhibitor of ITPase activity the progress curves have an appreciable linear region and accurate calculation of initial rates can be done graphically. The inhibition of the placental ITPase reaction by ADP was investigated at pH 8.0. The results are summarized in Fig. 7A. Inhjbition by ADP appears to be a result of competition with ITP for the hydrolytic site since Lineweaver-Burk plots characteristic of competitive inhibition were obtained. Also shown in Fig. 7A (insert) is the replot of slopes of the ADP effect on ITPase activity. The slope is a non-linear function of Mg-ADP concentration. This parabolic inhibition could result from the combination of at least two ADP molecules with one molecule of the enzyme (Cleland, 1963). The Kj for ADP measured in this system was 23 PM (Fig. 7B). In this case the value of& for ADP was about 38-fold smaller than the K, value for ITP (0.9mM). The Ki value obtained is similar to that reported by Philo & Selwyn (1973) for the ATPase of ox heart mitochondria. However, these authors failed to observe curvature at the Dixon plot perhaps because data for Dixon plots was collected only at ADP concentrations up to 40 PM. From the mode of inhibition by ADP of Mg-ITP hydrolysis, the existence of two catalytic sites acting independent is proposed.


F, of human placenta Table 4. Kinetic parameters of human placenta m~tochondria~ ATPase Tr~~B~carbonate medium

Trk-WC1 medium Ymax (fimol - min- 1-mg- ‘) Iz.i,


E.7 55.2 60.6





70.8 75.0

0.947 0.680

3.28 I.24

(~rno~.rn~~~ .rng-‘)

0.2cJO 0.087 0.920 0.790



stimulation by 30 mM KHCOa (-fold)

Kinetic constants were calculated from the data presented in Rg. 6. V,,, values reported here are extra~lated from the ordinates divided by the rn~~~~grarnsof ATPase protein in the assay. They represent tberefore the maximum specific activities of the NTPase reactions under assay conditions described in Fig. 6.

I&V A 50








Fig. 7. A, ~nbibjt~on of Mg-XTP bydro~ys~s by Mg-ADP at the indicated micromolar cuncentrations. (a). Sope replots. B. Dixon plot to determine the K< for ADP acting as a competitive inhibitor of the hydrolysis of MY by placental ATPase, Mg-ITP was used at the indicated mjl~irno~ar con~ntrat~ans. ITPase activity was assayed in the absence of rcgenerat~og system by extimating the release of PI at 30°C after inditing the sample For 5 min as described under Materials and Methods. Velocities are expressed as pmols ’ min- ’ per mg of enzyme protein.


Temp. feCl




33 34 104/T (K-l) -c


Fig. 8. Arrhenius plots for placental ATPase. Enzyme assays were carried out at 5mM-ATP in the presence of ATP regenerating system and Y values determined by s~trophotometric method as described under Materials and Methods. The plots represent the ATPase activity in the presence {A--A) and in the absence (u---0) of 30 mM b~c~bonat~. TWO ~nde~ndent measurements of V were performed at each temperature. The specific activities are expressed in U/mg.


ZENONALEKSANDROWICZ Table 5. Effect of inhibitors of oxidative phosphorylation ATPase

Compound tested None Oligomycin Aurovertin Quercetin Azide pCMS

Concentration 1 pglml 0.5 pg/ml 30 FM 50 /IM 0.5 mM

on purified mitochondrial

Percent of original activity - HCO; + HCO; 100 100 41 41 50 102

100 100 45 30 67 71

+ HCO; - HCO; 1.9 1.9 2.1 1.38 2.55 1.35

The incubation mixture contained 1 mM Mg-ATP in 50mM Tris-HCI, pH 8.0 or in 25 mM Tris-HCI + 25 mM KHCO,, pH 8.0. ATPase activity was assayed by following the release of Pi as described under Materials and Methods. Temperature dependence

The activities of the ATPase in the presence and in the absence of bicarbonate were studied under maximum velocity conditions in the range of temperatures from 10 to 45°C. The values of specific activity obtained are presented as a function of temperature in the form of Arrhenius plots in Fig. 8. Discontinuities were observed in the presence of bicarbonate as well as in the absence of bicarbonate, and the transition temperatures were 21 and 25”C, respectively. Both forms of enzyme showed higher energies of activation below the transition temperature. As can be seen in Fig. 8 the energies of activation for the enzyme suspended in the medium containing 30 mM bicarbonate were higher as compared with the system without bicarbonate. The placental enzyme revealed values of activation energies of 4.1, 12.5 kcal/mol and 2.6, 8.7 kcal/mol in the presence and in the absence of bicarbonate, respectively. The existence of the discontinuity in the Arrhenius plot of the ATPase suggests that the enzyme-substrate complex formation is taking place at two different catalytic sites. A second, more probable, possibility is that a temperaturedependent change in the quaternary structure of the enzyme does occur, which causes the conformation change of the same active site. E$xt

of inhibitors

The effect of various compounds on the ATPase activity is summarized in Table 5. In all of these experiments the agents were added directly to the assay system, with 10min preincubation with the enzyme. The reaction was initiated by the addition of the 1 mM Mg-ATP. Table 5 shows that the placental ATPase activity, like soluble enzymes from other sources, was not affected by oligomycin. It was, however, inhibited by aurovertin, quercetin, azide and p-chloromercuriphenylsulfate @CMS). Azide inhibited the activity to a greater extent in the absence than in the presence of bicarbonate, with the result that the stimulation by KHCOJ was increased from 90 to 155%. The opposite effect was observed with querc&in. ATPase activity of placental enzyme was inhibited by pCMS, only when assayed in the presence of bicarbonate. This finding is entirely consistent with the earlier observations of Pullman et al. (1960X Senior (1975) and Pedersen (1976b) that the ATPase activity is insensitive to inhibition by sulfhydryl re-

agents when assayed in nonactivating buffers. Pedersen (1976b) suggested that mercurials inhibit bicarbonate-stimulated ATPase activity by blocking a site associated with the anion binding. Thus the human placental ATPase was shown to be reactive to inhibitors in the similar manner as the F1 preparations from other mitochondria (Pullman et al., 1960; Beechey et al., 1975; Pedersen et al., 1974). CONCLUSIONS

The many similarities between the properties of mitochondrial ATPase from human placenta and other F1 preparations including cold lability, substrate specificity, stimulation by bicarbonate, inhibition patterns by ADP, divalent cation requirements and the insensitivity of the soluble ATPase to inhibition by oligomycin all point to these enzymes being closely related. Results of experiments summarized here show that the mitochondrial ATPase activity of human placenta can be modulated by a variety of agents e.g. ATP, bicarbonate, free Mg2+ ions. While using Mg-ATP as substrate in the absence of bicarbonate, a curvature of the reciprocal plot was obtained, which suggests that two kinetically distinct classes of ATP-binding site must be present on mitochondrial ATPase of human placenta. Further studies are in progress to confirm the existence of two substrate sites for Mg-ATP and to determine whether they both are catalytic sites or whether one of them is a regulatory site without hydrolytic activity. REFERENCES

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