Functional size analysis of F-ATPase from Escherichia coli by radiation inactivation.
A radiation inactivation technique was employed to determine the functional size of adenosine triphosphatase from Escherichia coli (EF0EF1-ATPase). Functional units of the membrane-bound and the soluble ATPases were estimated to be 300 +/- 39 and 295 +/- 32 kDa, respectively. The presence of the free radical scavenger dithiothreitol was crucial in measuring the radiation inactivation size of ATPase. When gramicidin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were added, an increase in the functional mass of membrane-bound ATPase was observed. In contrast, valinomycin and KCl had hardly any effect on the functional size of ATPase. We also determined a functional unit of 355 +/- 33 kDa for proton translocation by a fluorescence quenching technique. A reconstitution study using irradiated coupling factor 1 (EF1)-depleted membrane revealed that the functional mass of the proton channel was 96 +/- 11 kDa. A similar functional size for ATP-Pi exchange and ATP hydrolysis implies that both reactions might utilize identical machinery. Furthermore, functional units of soluble EF1 for unisite (nonsteady state) and multisite (steady state) ATP hydrolysis were calculated as 200 +/- 32 and 298 +/- 32 kDa, respectively. A working hypothesis was proposed from radiation inactivation analysis to elucidate the structure and mechanism of F1-ATPase.
Ma
J T
JT
Institute of Radiation Biology, College of Nuclear Science, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Wu
J J
JJ
Tzeng
C M
CM
Pan
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003037:Cobalt Radioisotopes; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000201:enzymology*; D006096:Gramicidin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013816:Thermodynamics; D014634:Valinomycin / Q000494:pharmacology
D003037:Cobalt Radioisotopes; D007476:Ionophores; D006096:Gramicidin; D014634:Valinomycin; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases