PROTON-TRANSLOCATING ADENOSINE-TRIPHOSPHATASE OF THE OBLIGATELY ANAEROBIC BACTERIUM CLOSTRIDIUM-PASTEURIANUM .2. ATP-SYNTHETASE ACTIVITY

1. Vesicles which demonstrated ATP‐dependent proton influx were produced from cell membranes of Clostridium pasteurianum by a cholate‐dialysis procedure which was also employed to introduce bacteriorhodopsin into such membrane vesicles and into artificial proteoliposomes. 2. ATP synthetase activity was assayed using illuminated bacteriorhodopsin‐containing crude membrane vesicles plus a glucose and hexokinase ‘ATP trap’. The membrane‐bound ATPase of vegetatively grown cells of Cl. pasteurianum displayed measurable ATP synthetase activity in this assay. 3. ATPase‐proteoliposomes constructed of purified ATPase (BF0F1) of Cl. pasteurianumw with bacteriorhodopsin and a mixture of phospholipids accomplished light‐dependent synthesis of ATP from ADP plus Pi. The reaction was inhibited by N,N′‐ dicyclohexylcarbodiimide and by proton conductors such as tetrachlorosalicylanilide. The specific ATP synthetase activity of the purified Cl. pasteurianum ATPase was significantly less than that of similarly purified ATPases (BF0F1) from Escherichia coli, Streptococcus faecalis and Streptococcus pleomorphus. The specific ATP synthetase activity of the ATPase of Clostridium formicoaceticum was greater when the enzyme complex was derived from fumarate‐grown cells then when it was purified from organisms grown on fructose. 4. The apparent Km value (for Mg2+‐ADP‐Pi) displayed by the ATPase of Cl. pasteurianum when acting as an ATP synthetase was much higher than the apparent Km, value (for ATP) in ATP phosphohydrolysis. A similar disposition to serve as an ATP phosphohydrolase was displayed by the ATPase of fructose‐grown Cl. formicoaceticum, but the ATPase from fumarate‐grown cells of this organism was substantially more effective in ATP synthesis. 5. The ATP synthetase activity of Cl. pasteurianum ATPase (BF0F1) was as susceptible as was its ATP phosphohydrolase activity to inhibition by dicyclohexylcarbodiimide, butyricin 7423, Dio‐9, 4‐chloro‐7‐nitrobenzofurazan, quercetin and citreoviridin and was similarly insensitive to inhibition by triethyl tin and tributyl tin. Copyright © 1979, Wiley Blackwell. All rights reserved