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Inorganic Pyrophosphatedriven Atpsynthesis in Liposomes Volume 155, number 1 FEBS LETTERS May 1983 Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and Fo-Fl complex from Rhodospirillum rubrum Pal NyrCn and Margareta Baltscheffsky Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden Received 14 March 1983 PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence ofADP,Mg ‘+ , Pi and PP, the system catalyzed phosphorylation by up to 25 nmol ATP formed x mg protein-’ x min-‘, at 2O”C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N’-dicyclohexylcarbodiimide. Pyrophosphatase Liposome Reconstitution A TPase A TP synthesis Proton pump 1. INTRODUCTION We have developed a method for obtaining a highly purified membrane-bound PPase from R. Inorganic pyrophosphatase is a product of rubrum chromatophore [lo]. This pure membrane- photophosphorylation in Rhodospirillum rubrum bound PPase, incorporated into phospholipid chromatophores [l] and is an energy donor for vesicles in the presence of PPi and Mg’+, can func- several energy-linked reactions in R. rubrum tion as a H+-pump. PPi can drive the synthesis of chromatophores including cytochrome reduction ATP from ADP and Pi in R. rubrum [2,3], transhydrogenation [4,5], NAD+ reduction chromatophores [ 111. [6] and the carotenoid shift [7]. The utilization of Here, we have obtained phosphorylation of PPi apparently involves the formation of an ADP to ATP at reasonable rates, by incorporation energized state, also indicated by the fact: that of the membrane-bound inorganic PPase (func- PPi-energized chromatophores of R. rubrum tioning as a proton pump) with the DCCD- generate a membrane potential (A$) and sensitive ATPase complex from R. rubrum into transmembrane pH difference (ApH) [8]; and that pre-formed liposomes. a partly purified membrane-bound PPase from R. rubrum, incorporated in a phospholipid mem- 2. MATERIALS AND METHODS brane, acts as a PPi-dependent electric generator 191. Triton X-100, DTE, FCCP, IDP, MDP, valinomycin, oligomycin, nigericin, Trizma-base, Abbreviations: DCCD, N,N’-dicyclohexylcarbodiimi- cholic acid and asolectin (crude soybean de; DTE, dithioerythritol; FCCP, carbonyl cyanide phospholipids used after an acetone wash [12]) p-trifluoromethoxyphenylhydrazone; IDP, imidodi- were purchased from Sigma. DCCD from Huka phosphate; MDP, methylenediphosphonate AG (Bucks). Luciferin/luciferase assay kit was Enzymes: Membrane-bound inorganic pyrophosphatase from LKB. Efrapeptin D was a kind gift of Pro- (EC 3.6.1.1) and adenosine 5 ’ -triphosphatase (EC fessor B. Beechey (Shell Research, Sittingbourne, 3.6.1.4) Kent). Published by Elsevier Science Publishers B. V. 00145793/83/$3.00 0 Federation of European Biochemical Societies 125 Volume 155. number 1 FEBS LETTERS May 1983 2.1. Preparation of chromatophores Tris-HCl (pH 7.5) and Hz0 in 2 ml total vol. The Rhodospirillum rubrum strain Sl was grown assay mixture was incubated at 30°C and the reac- anaerobically in the medium described in [13]. tion was terminated after 10 min by addition of After 40 h of growth, cells were harvested, washed 1 ml 10% trichloroacetic acid. Blanks were run and chromatophores were prepared as in [14]. where trichloroacetic acid was added before the sample. Supernatant (1.6 ml, after centrifugation) 2.2. Preparation of the DCCD-sensitive ATPase was used for Pi determination. Pi was assayed col- complex orimetrically as in [ 171. The PPase activity was The ATPase complex was prepared from R. assayed as the ATPase activity with the exception rubrum chromatophores as in [25]. Isolated that 0.5 mM PPi was used instead of ATP. chromatophores were solubilized with final con- centrations of 0.25% (w/v) sodium cholate plus 2.6. ATP-synthesis 15 mM octylglucoside at -8 mg protein/ml for The amount of ATP synthesized was measured 20 min at 4°C. The purification on the sucrose gra- with the luciferin/luciferase technique, 15-50 ~1 of dient was performed in the presence of 0.1% soy- reconstituted ATPase, PPase liposomes were add- bean phospholipids and 0.2% Triton X-100 [15]. ed directly to a test solution containing 200~1 The most active fraction for ATPase activity was luciferin/luciferase assay (LKB kit), 1 ml 0.2 M quickly frozen and was stored at -70°C. Protein glycylglycine (pH 7.8), 20 ~1 100 mM sodium was 0.5 mg/ml. phosphate, 10~1 10 mM ADP and 50~1 10 mM sodium pyrophosphate. The final concentration of 2.3. Preparation of the membrane-bound PPase MgCl2 was 10 mM as the luciferin/luciferase assay The preparation of the membrane-bound PPase contains a high [MgClz]. Addition of inhibitors is from R. rubrum chromatophores was performed as indicated in the text and figures. The reaction as in [lo]. The purified PPase was concentrated by was carried out at room temperature. The resulting ultrafiltration through an Amincon XMlOOA luminescence was measured in an LKB lumino- membrane and was kept frozen at - 70°C. Protein meter 1250. The light output was calibrated by ad- was 0.1-0.2 mg/ml. dition of a known amount of ATP. Protein was determined according to the 2.4. Preparation of ATPase and PPase liposomes modified Lowry method [18]. The freeze-thaw technique of [16] was used to reconstitute the PPase and ATPase into liposomes; 3. RESULTS AND DISCUSSION 40 mg soybean phospholipids were supplemented with 1 ml medium, containing 10 mM Tris-HCl 3.1. PP,-driven A TP formation (pH 7.5), 0.5 mM DTE, 0.5 mM EDTA and The time course of ATP formation in liposomes 0.05% Na-cholate. The suspension was flushed is illustrated in fig.1. With 0.1 pmol PPi the reac- with nitrogen and sonicated in a covered test tube tion was complete in 15 min. At least 50 nmol PPi for 20-25 min at 20°C in a bath-type sonicator must be added to detect phosphorylation. This model G 112 SP lT, Lab. Supplies (Hicksville amount of PPi might give the approx. K,,, for NY). The PPase (0.1 ml desalted preparation [lo], PPase and, thereby, a low rate of PPi hydrolysis. containing lo-20 pg protein) and ATPase (0.1 ml, No reaction was observed if only PPi without containing 50 pg protein) preparations were then liposomes or liposomes without PPi was added to added to 0.5 ml liposomes, sonicated for a few the ATP synthesis assay. ATP hydrolysis was seconds and frozen at -70°C. After thawing at observed with no added PPi (fig.2). The hydrolyz- room temperature, the preparation was stored on ed ATP originated from the ATP contamination ice with only minor losses in activity for at least of the ADP in the sample (0.5-l%). ATP 3 h. hydrolysis could be abolished with oligomycin (fig.2). With ADP, PPi and liposomes present, 2.5. A TPase activity ATP was synthesized at 25 nmol . mg This was assayed in a reaction mixture contain- protein-’ . min-‘. This value can be compared with ing 10 mM MgC12, 1 mM ATP, 1 ml 0.1 M 23 pmol . mg Bchl-’ . h-’ for the chromatophore 126 Volume 155, number 1 FEBS LETTERS May 1983 14Opmol ATP T80pmoL Fig.1. Time course of ATP synthesis in a suspension of PPase,ATPase liposomes; 25~1 liposomes (-0.07 mg Fig.3. Time course of ATP synthesis as a function of the protein/ml) were suspended in a medium containing amount of liposomes added; (---+ ) 25 ,uI liposomes 1 ml 0.2 M glycylglycine (pH 7.8), 0.2 ml luciferin/luci- added to the same medium as in fig.1 except that 50 ~1 ferase assay, 20~1 100 mM sodium phosphate, 10 pl 10 mM sodium pyrophosphate was present. 10 mM ADP and 504 10 mM sodium pyrophosphate; final [MgClz] was 10 mM; ( -) 25 pl liposomes -30,~M PPi and a corrected value of added. The resulting luminescence was measured in an LKB luminometer 1250. The light output was calibrated 130 PPi/ATP is obtained. by addition of a known amount of ATP. (2) Determining PPi hydrolysis calorimetrically, and ATP synthesis by the luciferase technique: lmbn This was done under similar conditions, ex- H cept that with the calorimetric assay no phosphate was added; the two rates were com- pared. This method gave 100 PPi/ATP. About 10 PPi/ATP was obtained for the 4Opmol ATP f chromatophore reaction in [lo]. The dif- ference can be explained by assuming that not all liposomes contain both enzymes, so some Fig.2. Time course of ATP hydrolysis in a suspension of of the energy will be wasted. PPase,ATPase liposomes; 50 ,uI liposomes were suspended in the same medium as in fig. 1 except that no 3.2. Effects of energy-transfer inhibitors sodium pyrophosphate was added. At the first arrow The energy-transfer inhibitor DCCD inhibits the 50 ~1 liposomes was added. At the second arrow 50 ~1 membrane-bound PPase of chromatophores to oligomycin (2 mg/ml) was added. -75% [ 191. The titration curves with added DCCP were similar for both PPase and ATPase activities reaction [ll]. If we assume that 1 mg Bchl cor- of chromatophores. However, after purification responds to -40 mg protein, the rate becomes and reconstitution the PPase, but not the ATPase, 9.6 nmol.mg protein-‘.min-‘. As yet, no at- lost the DCCD-sensitivity [14]. So the 50% inhibi- tempts have been made to optimize the conditions tion of phosphorylation in fig.4 might be due to for the phosphorylation reaction; the reaction with the inhibition of the ATPase complex.
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