PURIFICATION AND RECONSTITUTION INTO PROTEOLIPOSOMES OF THE F1F0 ATP SYNTHASE FROM THE OBLIGATELY ANAEROBIC GRAM-POSITIVE BACTERIUM CLOSTRIDIUM-THERMOAUTOTROPHICUM

The proton-translocating F1F0 ATP synthase from Clostridium thermoauto trophicum was solubilized from cholate-washed membranes with Zwitterge nt 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12 .6 mu mol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed o f six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, eps ilon, and c subunits, respectively, as their N-terminal amino acid seq uences matched the deduced N-terminal amino acid sequences of the corr esponding genes of the atp operon sequenced from Clostridium thermoace ticum (GenBank accession no. U64318), demonstrating the close similari ty of the F1F0 complexes from C. thermoaceticum and C. thermoautotroph icum. Four of these subunits, alpha, beta, gamma, and epsilon, constit uted the F-1-ATPase purified from the latter bacterium. The delta subu nit could not be found in the purified F-1 although it was present in the F1F0 complex, indicating that the F-0 moiety consisted of the delt a and the c subunits and lacked the a and b subunits found in many aer obic bacteria. The c subunit was characterized as N',N'-dicyclohexylca rbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consis ting of subunits alpha, beta, gamma, delta, epsilon, and c was reconst ituted with phospholipids into proteoliposomes which had ATP-P-i excha nge, carbonylcyanide p-trifluoromethoxyphenylhydrazone-stimulated ATPa se, and ATP-dependent proton-pumping activities. Immunoblot analyses o f the subunits of ATP synthases from C. thermoautotrophicum, C. thermo aceticum, and Escherichia coli revealed antigenic similarities among t he F-1 subunits from both clostridia and the beta subunit of F-1 from E. coli.