Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.
F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.
Tsunoda
S P
SP
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama, Japan.
Aggeler
R
R
Noji
H
H
Kinosita
K
K
Yoshida
M
M
Capaldi
R A
RA
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D016477:Artifacts; D001704:Biopolymers / Q000378:metabolism; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D015335:Molecular Probes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012399:Rotation; D012995:Solubility / Q000187:drug effects; D014475:Uncoupling Agents / Q000494:pharmacology; D014698:Venturicidins / Q000494:pharmacology
D000199:Actins; D001704:Biopolymers; D003902:Detergents; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D014475:Uncoupling Agents; D014698:Venturicidins; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases