Characterization of the inhibitory (epsilon) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli.
The inhibitory subunit (epsilon) of the F1 adenosine triphosphatase (ATPase) was purified to homogeneity from the ML 308-225 and K12 (lambda) strains of Escherichia coli. No tryptophan or cysteine was detected in the subunit from either strain. The highly active epsilon from both strains was found to be a globular protein with a Stokes' radius of 18--19 A. Circular dichroism spectra suggested an alpha-helix content of approximately 40%. The molecular weight of epsilon was approximately 15000--16000 by sedimentation equilibrium centrifugation in the presence and absence of guanidinium hydrochloride, molecular sieve chromatography, and gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea. The s20,w of epsilon was approximately 1.6 s-1. Inhibition of the purified F1 ATPase by epsilon displayed noncompetitive kinetics with a Ki of approximately 10 nM. The inhibition of the ATPase was rapidly reversed by diluting the enzyme--epsilon mixture. [125I]epsilon which was incorporated into ECF1 was readily displaced by unlabeled epsilon. epsilon had no significant effect on the ATPase activity of "native" or reconstituted everted membrane vesicles under a variety of assay conditions. Combining the epsilon-inhibited F1 ATPase with its hydrophobic portion in everted membrane vesicles reconstituted the reversible proton-translocating ATPase and restored nearly full ATPase activity. These results suggest that epsilon inhibits the enzyme only when the F1 ATPase becomes detached from its hydrophobic subunits.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D011506:Proteins; D013056:Spectrophotometry, Ultraviolet; D000097795:ATPase Inhibitory Protein
D000251:Adenosine Triphosphatases; D000596:Amino Acids; D001426:Bacterial Proteins; D046911:Macromolecular Substances; D011506:Proteins; D000097795:ATPase Inhibitory Protein