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F1-Atpase-Catalyzed Synthesis of ATP from Oleoylphosphate and ADP (Mitochondria/Adenosine Triphosphate) RICHARD JOHNSTON and RICHARD S

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F1-Atpase-Catalyzed Synthesis of ATP from Oleoylphosphate and ADP (Mitochondria/Adenosine Triphosphate) RICHARD JOHNSTON and RICHARD S Proc. Natl. Acad. Sci. USA Vol. 74, No. 11, pp. 4919-4923, November 1977 Biochemistry F1-ATPase-catalyzed synthesis of ATP from oleoylphosphate and ADP (mitochondria/adenosine triphosphate) RICHARD JOHNSTON AND RICHARD S. CRIDDLE* Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Communicated by Paul D. Boyer, August 29,1977 ABSTRACT Purified preparations of F1-ATPase (ATP The specific activities of purified preparations of oligomy- phos hohydrolase; EC 3.6.1.3) isolated from yeast mitochondria cin-sensitive ATPase were in the range of 15-20 ,qmole of ATP cata yze the reaction of oleoylphosphate with ADP to yield ATP and oleic acid. Formation of ATP is specifically inhibited by hydrolyzed/min per mg of protein. Specific activities of F1 the F1-ATPase inhibitor 1799 and by dinitrophenol. In the preparations were near 33 umol of ATP hydrolyzed/min per presence of Fi, dinitrophenol "uncouples" the synthase reaction mg. ATPase assays were performed by the coupled spectro- by causing rapid hydrolysis of oleoylphosphate without ATP photometric method of Monroy and Pullman (8) in pH 7.4 formation. It is propse that this Fl-catalyzed ATP synthesis Tris-HCI buffer containing 6 mM MgCI2. Oligomycin, dini- reaction corresponds to the terminal chemical step in oxidative trophenol, and 1799 were added as methanolic solutions. phosphorylation. Oleoylphosphate was prepared by both the methods de- D. Griffiths and coworkers have recently reported ATP syn- scribed by Lehninger (9) and by Hildebrand and Spector (10). thesis catalyzed by preparations of submitochondrial particles Alternatively, oleoylphosphate was prepared by a combination and by the oligomycin-sensitive ATPase from beef heart or of these two methods. To 0.2 ml of 92% phosphoric acid (Mal- yeast mitochondria (1-4). They demonstrated that dihydroli- linckrodt reagent grade), 1 ml of oleoylchloride (Sigma, 99%) poate may serve as a component of an ATP synthesizing com- was added at 00. After 15-30 min, the mixture was extracted plex and proposed that both oleoyl-S-lipoate and oleoylphos- twice with 5 volumes of diethylether. The combined ether ex- phate may serve as intermediates in the synthesis reactions. The tracts were rotary evaporated under a partial vacuum at room analogous reactions of substrate level phosphorylation led us temperature. The remaining clear oil was used immediately to postulate that oleoylphosphate could be involved in the ter- or stored at -70° under argon until needed. Hydroxylamine minal step of ATP synthesis. We found that a purified prepa- analysis (11) and thin-layer chromatography indicated a yield ration of FI-ATPase (ATP phosphohydrolase; EC 3.6.1.3) from of 80-90%. No di- or trioleoylphosphates were.observed on yeast mitochondria efficiently catalyzed an oligomycin-in- chromatographic analysis examined by phosphate staining (12). sensitive ATP hydrolysis and also catalyzed formation of ATP Oleoylphosphate was dissolved in dimethylformamide, gen- in the presence of added oleoylphosphate and ADP. The ATP erally at concentrations near 10 Mmol/ml before addition to the synthase reaction was sensitive to the F1 inhibitors 1799 reaction mixture. [bis(hexafluoracetonyl)-acetone] and to dinitrophenol (Dnp) ATP synthase reactions were followed by two methods. (a) at levels commonly used for blocking oxidative phosphorylation [14C]ADP (New England Nuclear, 50 mCi/mmol) was added in mitochondria. to a reaction mixture containing, in a total volume of 2 ml, 1.5 units of ATPase enzyme preparation, 0.4 ,imol of ADP, and 1.0 MATERIALS AND METHODS ,Amol of oleoylphosphate in 40 mM Tris-HCl buffer (pH 7.5) containing 6 mM MgCl2 and 1 mM Na2HPO4. Conversion to Saccharomyces cerevssiae strain D243-4A (a,ade,lys) was used [14C]ATP was measured by stopping the reaction at various in the following studies. Cells were grown in batch cultures to times with 5% trichloroacetic acid, neutralizing with 1 M late logarithmic phase in 1% Difco Peptone, 1% Difco yeast NaOH, and separating the nucleotides by chromatography on extract, and 2% glucose or 2% ethanol media (wt/wt). Cells Sephadex A-25 (13). A 0.5 X 10-cm column was used and elu-- were then harvested and broken using a Braun glass bead ho- tion was effected by a 100-ml linear gradient varied from 0.075 mogenizer. Mitochondria were prepared by differential sedi- to 0.24 M NH4CI in 0.1 M Tris-HC1 at pH 8.1. Elution of nu- mentation as described by Enns and Criddle (5). Submito- cleotides was monitored by observing A20of added nucleotide chondrial particles and oligomycin-sensitive ATPase solubilized and by measurement of radioactivity in a Beckman scintillation by Triton X-100 were prepared as described by Tzagaloff and counter using Bray's reagent. (b) ATP was also analyzed by the Meagher (6). Oligomycin-sensitive ATPase was then purified luciferin-luciferase method (13) with ATP-dependent light by chromatography on Sepharose 6B gel columns (5). F1- production monitored directly in a scintillation counter. The ATPase was prepared by the method of Tzagaloff (7) except relation between counts observed and ATP concentration was Sepharose 6B was substituted for Sephadex G-25 to remove determined by preparation of a standard curve with known ammonium sulfate and simultaneously purify protein before amounts of added ATP. chromatography on DEAE-cellulose. Purified FI-ATPase was Thin layer chromatographic analysis of oleoylphosphate was precipitated by addition of 70% ammonium sulfate, centri- carried out on Baker silica gel 1B plates. Ascending chroma- fuged, and stored as a slurry at room temperature under argon. tography was in chloroform/methanol/acetic acid/water Enzyme stored this way was stable for at least 3 weeks. (85:15:10:4). Samples were visualized on the plates by exposure The costs of publication of this article were defrayed in part by the to iodine vapor, then eluted and measured by colorimetric payment of page charges. This article must therefore be hereby marked phosphate analysis (14). "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 4919 Downloaded by guest on September 29, 2021 4920 Biochemistry: Johnston and Criddle Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Synthesis of ATP by oligomycin-sensitive ATPase Counts 4mol/ Reaction mixture measured 5 min Complete 8800 0.19 -ADP 350 -Oleoylphosphate 360 16 - -Oleoylphosphate + acetylphosphate 300 x ATP E 2 Xl -Enzyme 280 Heat-denatured enzyme 275 8- The complete reaction mixture contained 1.5 units ofATPase per mol, 0.2 mM ADP, 1.0 ,tmol ofoleoylphosphate, 40 mM Tris.HCl (pH 7.5), and 6 mM MgCl2 in a volume of 2 ml. One unit ofATPase activity 4- corresponds to the amount of enzyme required to hydrolyze 1 ,mol of ATP per min. The reaction was stopped after 5 min by heating for 5 min at 900. The ATP produced was determined by the luc ferin- 0 4812 16 O 0 4 8 12 1620 0 4 8 12 16 20 procedure in a scintillation counter. Fraction number luciferase assay FIG. 1. Conversion of [14C]ADP to [14C]ATP catalyzed by oligomycin-sensitive ATPase, and by F1. [14C]ADP and ATPase preparations. (A) Yeast submitochondrial particles (1.5 units of ATPase) were added to an assay mixture containing oleoylphos- [14C]ATP in the reaction mixture were separated by ion ex- phate and [14C]ATP. The reaction was terminated after 0, 5, and 10 change chromatography on Sephadex A-25. The elution posi- min and the reaction products were separated by chromatography tions of the nucleotides were verified by chromatography of in Sephadex A-25. The elution profiles show the amounts of 14C in standard solutions. The zero time reactions, in which enzyme ADP and ATP after reaction for 0 (0), 5 (0), and 10 (X) min. Stan- was inactivated with trichloroacetic acid immediately after dard AMP preparations eluted at the position indicated by the arrow. addition to the reaction mixture, show all the label to be in ADP. (B) Purified oligomycin-sensitive ATPase (1.5 units) was used in the ADP was to ATP formation of [14C]ATP as in A. Elution profiles following reaction for In each case, more than 90% of the converted 0 (0) and 10 (X) min are shown. (C) Soluble F1-ATPase (1.5 units) during the 10-min reaction. No indication of AMP formation was used for [14C]ATP synthesis. Reaction times of 0 (0) and 10 (X) was noted, eliminating the possibility of ATP synthesis via min are shown. dismutation of ADP to ATP + AMP. Fig. 2 illustrates the ad- dition of [32P]phosphate to a reaction mixture containing Oligomycin and luciferin were obtained from Calbiochem; [14C]ADP, oleoylphosphate, and oligomycin-sensitive ATPase. dinitrophenol from Matheson, Coleman and Bell; luciferase No transfer of labeled phosphate into ATP was observed. from Boehringer; and acetyl phosphate and oleoylchloride from Table 1 illustrates that ATP synthase, assayed by the lucif- Sigma. [32P]Phosphate, carrier free, was obtained from New erin-luciferase assay method, is dependent on active enzyme, England Nuclear. 1799 was a gift from Walter Hanstein. ADP, and oleoylphosphate. No synthesis was observed when acetylphosphate was substituted for oleoylphosphate. The rate RESULTS of the reaction is dependent upon oleoylphosphate concentra- As shown in Fig. 1, ATP was formed from oleoylphosphate and tion, yielding an apparent Km for oleoylphosphate of about 50 ADP in reactions catalyzed by submitochondrial particles, by ,tM. This value may change with further study because of the micellar nature of the substrate. The yield of oleoylphosphate from chemical synthesis must also be determined accurately 0~ in order to make this value more precise. The time course of ATP synthesis catalyzed by pure F1 is 12 llt 0 shown in Fig. 3.
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