Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum

被引:18
作者
Das, A
Ivey, DM
Ljungdahl, LG
机构
[1] UNIV GEORGIA, DEPT BIOCHEM & MOL BIOL, ATHENS, GA 30602 USA
[2] UNIV GEORGIA, CTR BIOL RESOURCE RECOVERY, ATHENS, GA 30602 USA
关键词
D O I
10.1128/jb.179.5.1714-1720.1997
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 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 of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F-1-ATPase purified from the latter bacterium. The delta subunit 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 delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N',N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-P-i exchange, carbonylcyanide p-trifluoromethoxyphenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F-1 subunits from both clostridia and the beta subunit of F-1 from E. coli.
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页码:1714 / 1720
页数:7
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