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/) 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. Synthesis of ATP continued almost linearly 0 0 o-C 0 until oleoylphosphate was depleted; then the ATP level again x 10 4'- decreased due to hydrolysis by the Fl-ATPase. The specific E x I,4 0 activity for the ATP synthesis reactions was 1.5 ,tmol of ATP E0. a, 8 ,. formed/min per mg of enzyme with the assay conditions used. 0 3 .Ca, 0 6 0- 04 0 21 I 2I0L 1 4 \\ 2 CL < .02 Z E 4 8 12 16 20 24 L .01I o Fraction FIG. 2. Conversion of ADP to ATP by oligomycin- I Or- - catalyzed 0 2 4 6 8 10 12 sensitive ATPase in the presence of [32Pjphosphate. The reaction was Min as in the legend ofFig. 1 except that 2.5 nCi of [32P]Na2HPO4 per ml was included in the assay with a final Na2HPO4 concentration of 1 FIG. 3. Time course of ATP synthesis catalyzed by F1. The ATP mM. The reaction was stopped after 2.5 min and the reaction mixture synthase reaction with F1 was run with limiting amounts of oleoyl- placed on a Sephadex ion exchange column. The column was eluted phosphate. At various times the reaction was stopped by heating for with a linear gradient from 0.0375 to 0.24 M NH4Cl in 0.1 M Tris-HCl, 5 min at 900. The amount of ATP present at each time was deter- pH 8.1. Radioactivity of fractions was determined in Bray's scintil- mined by the luciferin-luciferase reaction. The initial reaction mixture lation fluid. The elution profiles show [32P]HP04= elution (0) and contained 1.5 units of FI-ATPase, 0.2 mM ADP, and approximately [I4C]adenine nucleotide elution (0). 0.05 jmol of oleoylphosphate. Downloaded by guest on September 29, 2021 Biochemistry: Johnston and Criddle Proc. Natl. Acad. Sci. USA 74 (1977) 4921 Table 2. Effect of oxidative phosphorylation inhibitors on ATP synthesis by oligomycin-sensitive ATPase Luciferase assay [I4C]ATP formation Counts, gmol cpm, 11mol Inhibitor added X10-3 ATP/5 min X10-3 ATP/5 min Control 4.2 0.23 39.4 0.22 Oligomycin (400 Mg/mg protein) 28.73 0.18 1799 (20 MM) 0.38 0.020 C Dnp (20 MM) 0.40 0.021 6.5 0.03 0 0.3 Antimycin A 4.2 0.23 39.4 0.22 0.2 Assay conditions for the reactions assayed by the luciferase method were as in the legend for Table 1 except that the reaction contained 1.26 units of oligomycin-sensitive ATPase per ml. [14C]ATP assay was 0.I as described in Materials and Methods and synthesis of ATP was determined from the total cpm eluted with ATP from the ion ex- 0 change column. -, not determined. The data in this table represent 0 2 3 4 5 6 7 8 two separate ATP synthase reactions, one assayed by the luciferase cm assay and the other by [14C]ATP formation, rather than two methods of same reaction mixture. FIG. 4. Sodium dodecyl sulfate gel analysis of purified F1- of analysis the ATPase. A 50-Mg sample of F1 protein was electrophoresed on 10% acrylamide gel using the buffer system of Weber and Osborn (15). The Dnp causes a change in apparent Km to 67 MM. By way of samples were stained with Coomassie blue, destained, and scanned comparison, Kayalar et al. (16) have recently reported apparent at 600 nm using a Gilford gel scanner. Subunit identifications were Km values of 13 AuM for ADP in the entire process of oxidative based on the molecular weights of standard proteins electrophoresed phosphorylation. Interpolation of their values indicates that the in identical gels. apparent Km is increased to 39 ,uM by addition of 40MuM Dnp. of added Dnp on the ATP synthase This is about 1/20 the specific activity of the enzyme assayed as Thus, the relative effects an ATPase. Fig. 4 indicates a densitometer scan of the protein reaction and on oxidative phosphorylation are nearly identi- subunits of the FI-ATPase sample separated by electrophoresis cal. in sodium dodecyl sulfate/acrylamide gels. The results of Table 3, when coupled with assays of ATP Fl-ATPase activity is not inhibited by oligomycin; similarly, formation, indicate that one ATP is formed per oleoylphosphate ATP synthesis catalyzed by F1 is not blocked by this antibiotic. used (reaction mixture 3). When oligomycin-sensitive ATPase complex catalysis of the synthase reaction was studied, little inhibition occurred at 400 DISCUSSION ,ug of oligomycin per mg of protein (Table 2). In contrast, These studies clearly show that FI-ATPase from yeast mito- ATPase activity of this complex was more than 50% inhibited chondria catalyzes formation of ATP from oleoylphosphate and at 10 ,ug of oligomycin per mg of protein. Neither the reaction ADP. The protein specificity seems quite clear. Only with catalyzed by F, nor the oligomycin-sensitive ATPase complex preparations containing active FI-ATPase has it been possible was inhibited by antimycin A but both were sensitive to 1799 to catalyze the ATP-forming reaction. Submitochondrial par- and to Dnp. Fig 5 shows inhibition of oligomycin-sensitive ticles containing ATPase and Triton X-100-solubilized olig- ATPase by various levels of 1799 and by Dnp. ATP synthesis omycin-sensitive ATPase both work well, but heat-denatured was inhibited by 50% at inhibitor concentrations of 3 ,uM 1799 ATPase or various added non-ATPase proteins fail to catalyze and 14 MM Dnp. ATP synthesis. Still these observations leave some questions The effect of Dnp on ATP synthesis was examined to de- termine whether this simply inhibited enzyme ac- - I010: tivity or had an effect on hydrolysis of oleoylphosphate. The ,x 30 assay reactions were run with various amounts of Dnp and ADP, 8 the reaction was quenched, and the products were chromato- graphed on thin layer silica gels. Because a quantitative assay ¶, 6 20- for oleoylphosphate in the reaction solutions was not available, 0 hydrolysis of oleoylphosphate was determined by the disap- T- x 0Ex4 - x - E pearance of the phosphate-containing moiety migrating with -1 IC CL 0.43. This indicates the Dnp RF assay clearly participation of x=- - in an enzyme-catalyzed hydrolysis of oleoylphosphate (Table E - -- =- 3). Neither enzyme alone nor Dnp alone catalyzes hydrolysis of oleoylphosphate. Enzyme plus Dnp rapidly hydrolyzes the 4 8 12 16 20 50 high energy phosphate. An interesting effect noted is that in- IMM17,99 creased levels of ADP decrease the rate of oleoylphosphate 20 40 60 80 100 160 hydrolysis (reactions 4-6) as the rate of ATP synthesis is slower MM Dnp than the enzyme-catalyzed, Dnp-induced oleoylphosphate FIG. 5. Inhibition of ATP synthesis by Dnp and 1799. Dinitro- hydrolysis. phenol (X) or 1799 (0) was added to 1-ml reaction mixtures con- taining F1, 50MuM oleoylphosphate, and 500MgM ADP. After 5 min of The effects of Dnp on the Km for ADP in the ATP synthase reaction time, trichloroacetic acid was added to stop the reaction, the reaction are consistent with this observation. Without added sample was neutralized, and ATP was assayed by the luciferin-lu- Dnp, the apparent Km for ADP is near 22 MM. Adding 40,uM ciferase technique in a scintillation counter. Downloaded by guest on September 29, 2021 4922 Biochemistry: Johnston and Criddle Proc. Natl. Acad. Sci. USA 74 (1977) Table 3. Thin-layer chromatographic analysis of residual the breakdown of oleoylphosphate. Kinetic studies illustrating oleoylphosphate following reaction the effects of.Dnp on oxidative phosphorylation show that Dnp Oleoylphosphate competes for the ADP-binding site on ATPase (18) and causes Reaction mixture detected, Mmol a marked increase in the apparent Km of ADP during oxidative phosphorylation (16). Examination of the change in apparent 1. OP alone 0.17 Km for ADP, caused by addition of Dnp to the ATP synthase 2. OP+enzyme 0.11 reaction, indicates a change nearly identical to that noted by 3. OP + enzyme + 200 ,uM ADP 0.023 Kayalar et al. for measurements of the Km for oxidative phos- phorylation (16). These results suggest that not only does Dnp 4. OP + enzyme + 200 ,uM ADP + 20MM Dnp 0.056 block the ATP synthase reaction, but also that this is the primary 5. OP + enzyme + 50,uM ADP + 20AM Dnp 0.032 site of action of this uncoupler on in vivo oxidative phospho- 6. OP + enzyme + 5 MM ADP + 20 MM Dnp 0.014 rylation. The question of whether oleoylphosphate or some other 7. OP + enzyme + 20 AM ADP + 50guM Dnp 0.009 in vivo 8. OP + enzyme + 20MM ADP + 5 MM Dnp 0.018 acylphosphate is the high-energy phosphate form for 9. OP + enzyme + 20MM ADP + 0.5 MM Dnp 0.014 mitochondrial ATP synthesis has not been resolved completely. Acetylphosphate did not serve as a substrate for ATP synthesis 10. OP + enzyme + 50MM Dnp 0.024 catalyzed by F1 though it may be used in certain bacterial substrate level ATP synthesis reactions (19). Other acylphos- The ATP synthase reaction was carried out in a 2-ml reaction phates have not yet been tested. Several studies do show that mixture with 1.7 units of ATPase per ml, a total reaction volume of unsaturated fatty acid is required for oxidative metabolism in 0.5 ml in 40 mM Tris-HCl buffer (pH 7.5), 6 mM MgCl2, and levels of reactants as indicated. After the 2-min reaction samples were E. coli and that unsaturated fatty acid auxotrophs of yeast will heated at 800 for 1.5 min to stop the reaction, oleoylphosphate was grow on oxidizable substrates only when unsaturated fatty acids extracted with chloroform/methanol (2:1) and chromatographed on are added (20, 21). A high degree of specificity for cis, A9 Silica gel 1B plates. The developing solvent was chloroform/metha- monoenoic acids is reported (22). Similarly, ATP synthesis in nol/glacial acetic acid/water (85:15:10:4). Oleoylphosphate-containing unsaturated fatty acid-deficient promitochondria from an- spots (Rf 0.43) were collected by scraping them off the silica gel and aerobically grown yeast is restored with oleate, but is inhibited phosphate was determined by the method of Rockstein and Herron saturated acids these suggest (14). OP, oleoylphosphate; enzyme, purified F1-ATPase. The lower by fatty (23). Together findings limits of sensitivity of this method are near 0.015-0.020 Amol of that an oleate derivative such as oleoylphosphate may actually oleoylphosphate. Thus, values for reactions 6-10 indicate essentially be the high-energy phosphate intermediate involved. complete hydrolysis of oleoylphosphate. The data of Fig. 1 establish a stoichiometric relationship between loss of ADP and formation of ATP. No AMP was de- tectable as a product of this reaction. If an adenylate kinase type reaction was responsible for ATP formation in these studies, equal molar amounts of [14C]AMP and [14C]ATP would be concerning the biological significance of the reaction studied. observed. Fig. 2 shows that inorganic phosphate in the assay (a) Does the ATP synthase reaction using oleoylphosphate and solution does not get incorporated into ATP during the reaction. ADP correspond to the terminal chemical step of ATP synthesis Finally, there is a one-to-one stoichiometry between oleoyl- via oxidative phosphorylation; or (b) does ATP synthesis via this phosphate utilized and ATP formed. These findings establish reaction reflect some property of the actual enzymes of the that the acylphosphate is the donor of the y-phosphate of ATP oxidative phosphorylation pathway but perhaps not represent in the enzyme-mediated reaction. the normal physiological reaction pathway and substrate in- The assembled data on reaction requirements and inhibitor termediates; or (c) does this reaction represent an additional and reagent specificity support a conclusion that ATP formation reaction of the ATPase complex not related to oxidative phos- from oleoylphosphate and ADP is the terminal chemical step phorylation and having some unknown physiological func- in oxidative phosphorylation. The inhibition of this reaction by tion? Dnp then provides an interesting insight into the mechanism The terminal step in oxidative phosphorylation is generally of action of this uncoupler. In the presence of Dnp and enzyme, accepted to be associated with the FI-ATPase based on studies oleoylphosphate is rapidly hydrolyzed. This reaction is faster in many laboratories (for review see ref. 17). There are, how- than the ATP synthase reaction and is actually inhibited by ever, five subunits in the FI-ATPase complex and it is possible added ADP. Thus, the high-energy phosphate moiety is de- that one or more of these subunits could catalyze the ATP stroyed without ATP produciion. This suggests that hydrolysis synthase reaction described here while others catalyze the of the acylphosphate may be the primary mechanism for un- terminal steps of oxidative phosphorylation. This possibility coupling oxidative phosphorylation by Dnp. This does not rule seems unlikely when the effects of some inhibitors of oxidative out additional effects of Dnp on membrane potentials in mi- phosphorylation are considered. It was shown that 1799, a tochondria and other membrane systems. Such effects are well specific inhibitor of F1-ATPase which correspondingly blocks documented. However, there is a close correspondence between oxidative phosphorylation, also inhibits the ATP synthase re- concentration of Dnp required for uncoupling mitochondrial action. This finding does not agree with the preliminary results oxidative phosphorylation (23) (5-20 ,uM for 50% inhibition) of Griffiths (1), but does agree with his more recent studies. The and that required for inhibition of ATP synthesis via oleoyl- level of 1799 required to cause 50% inhibition of ATPase ac- phosphate using F1 (12 ,uM for 50% inhibition). In contrast, the tivity is identical to that blocking the ATP synthase (Fig. 5). levels of Dnp altering the conductivity of phospholipid mem- Either these reactions are carried out at the same active site or brane preparations appreciably is more than 10-fold higher 1799 simultaneously exerts identical effects at more than one (200-500 ,uM) (24). If these measurements reflect the corre- active site. sponding levels of Dnp required to significantly alter mito- Results with Dnp furnish another test of inhibitor specificity chondrial membrane permeability in vivo, then chemical un- in this reaction. Dnp prevents ATP formation and stimulates coupling at the terminal step of phosphorylation is the dominant Downloaded by guest on September 29, 2021 Biochemistry: Johnston and Criddle Proc. Natl. Acad. Sci. USA 74 (1977) 4923 effect on oxidative phosphorylation. This proposed chemical 10. Hildebrand, J. G. & Spector, L. B. (1969) J. Biol. Chem. 244, uncoupling by Dnp is consistent with the demonstration by 2606-2613. Hanstein and Hatefi (25) of a specific uncoupler binding site 11. Wolf, E. C. & Black, S. (1959) Arch. Biochem. Biophys. 80, with ATPase and with the preparation of 236-242. associated proteins 12. Rosenberg, H. (1974) in Data for Biochemical Research, eds. uncoupler-resistant mutants of yeast by Griffiths et al. (26). Dawson, R. M. C., Elliott, D. C., Elliott, W. H. & Jones, K. M. Neither of these observations fits well with the proposal that (Oxford Press, London), 2nd Ed., pp. 562-564. uncoupling was linked to membrane conductance changes. 13. Strehler, B. L. & Totter, J. R. (1954) in Methods ofBiochemistry The stoichiometric relation between ATP synthesis and ADP Analysis, ed. Glick, D. (Interscience, New York), Vol. I, pp. disappearance illustrated in Fig. 1 is noteworthy. In spite of the 341-56. fact that F1 is an active ATPase which would be expected to 14. Rockstein, M. & Herron, P. W. (1972) Anal. Chem. 23, 1500- hydrolyze ATP as it is formed, a greater than 90% conversion 1503. to ATP is shown. Also, as shown in Fig. 3, ATP hydrolysis does 15. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- not appear to be important until oleoylphosphate is depleted. 4412. 16. Kayalar, C., Rosing, J. & Boyer, P. D. (1976) Biochem. Biophys. This may be accounted for by our observation that preparations Res. Commun. 72, 1153-1159. of oleoylphosphate strongly inhibit ATPase activity in our 17. Racker, E. (1970) in Membranes of Mitochrondria and Chlo- standard assays (unpublished observations). This suggests sub- roplasts, ed. Racker, E. (Van Nostrand Reinhold Co., New York), strate-mediated control over the synthesis or hydrolysis of pp. 127-171. ATP. 18. Stockdale, M. & Selwyn, M. J. (1971) Eur. J. Biochem. 21, 416-423. 19. Racker, E. (1961) in Advances in Enzymology, ed. Nord, F. F. 1. Griffiths, D. E. (1976) Biochem. J. 160,809-812. (Interscience, New York), Vol. 22, pp. 336-339. 2. Cain, K. & Griffiths, D. E. (1977) Blochem. J. 162,575-580. 20. Silbert, D. F. & Vagelos, P. (1967) Proc. Natl. Acad. Sci. USA 58, 3. Griffiths, D. E. (1976) in Genetics and Biogenesis ofChloroplasts 1579-1586. and Mitochondria, eds. Bucher, Th., Neupert, W., Sebald W. 21. Walenga, R. W. & Lands, W. E. M. (1975) J. Biol. Chem. 250, & Werner, S. (North Holland Publ. Co., Amsterdam), pp. 9121-9129. 175-185. 22. Griffiths, D. E., Hyams, R. L. & Bertoli, E. (1977) FEBS Lett. 4. Griffiths, D. E., Cain, K. & Hyams, R. L. (1977) Biochem. Soc. 74,38-42. Trans. 5, 205-206. 23. Drysdale, G. R. & Cohn, M. (1958) J. Biol. Chem. 233, 1574- 5. Enns, R. E. & Criddle, R. S. (1977) Arch. Biochem. Biophys., 182, 1577. 587-600. 24. Skulachev, V. P., Sharaf, A. A., Yagujzinsky, L. S., Jasaitis, A. A., 6. Tzagaloff, A. & Meagher, P. (1971) J. Biol. Chem. 248, 7328- Lieberman, E. A. & Topali, V. P. (1968) Curr. MOD! Biol. 2, 7338. 98-105. 7. Tzagaloff, A. (1969) J. Biol. Chem. 244, 5020-5026. 25. Hanstein, W. & Hatefi, Y. (1974) J. Biol. Chem. 249, 1356- 8. Monroy, G. C. & Pullman, M. E. (1967) in Methods in Enzy- 1363. mology, eds. Estabrook, R. & Pullman, M. E. (Academic Press, 26. Griffiths, D. E., Houghton, R. L. & Lancashire, W. E. (1973) in New York), Vol. X, pp. 500-512. The Biogenesis ofMitochrondria, eds. Kroon, A. M. & Saccone, 9. Lehninger, A. L. (1946) J. Biol. Chem. 162, 333-342. C. (Academic Press, New York), pp. 215-224. Downloaded by guest on September 29, 2021