Coupling factor 1 from Escherichia coli lacking subunits delta and epsilon: preparation and specific binding to depleted membranes, mediated by subunits delta or epsilon.
A procedure for the preparation of coupling factor 1 (F1) from Escherichia coli lacking subunits delta and epsilon is described. Using chloroform and dimethyl sulfoxide, we can isolate F1 containing only subunits alpha, beta, and gamma [F1(alpha beta gamma)] directly from membrane vesicles in 10-mg quantities. Pure and active subunits delta and epsilon were prepared from five-subunit F1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After addition of these subunits, F1(alpha beta gamma) is as active in reconstituting ATP-dependent transhydrogenase as five-subunit F1. The ATPase activity of F1 (alpha beta gamma) is inhibited by subunit epsilon in a 1:1 stoichiometry to the same extent (approximately equal to 90%) and with the same affinity (Ki = 0.2-0.8 nM) as reported earlier [Dunn, S.D. (1982) J. Biol. Chem. 257, 7354-7359]. In the presence of either delta or epsilon, F1(alpha beta gamma) binds to F1-depleted membrane vesicles and to liposomes containing the membrane sector (F0) of the ATP synthase to an extent commensurate with the F0 content. The binding ratios epsilon/F1 (alpha beta gamma) and probably also delta/F1 (alpha beta gamma) are close to unity. The specific, delta- or epsilon-deficient F1.F0 complexes presumably formed show ATPase activities sensitive to subunit epsilon but not to dicyclohexylcarbodiimide, and no energy-transfer capabilities. Binding studies at different pH values suggest that F1-F0 interactions in the presence of both subunits delta and epsilon are similar to a combination of those mediated by delta or epsilon alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Tuttas-Dörschug
R
R
Institute of Physiological Chemistry, Ruhr University, Bochum, West Germany.
Hanstein
W G
WG
D001665:Binding Sites; D002462:Cell Membrane / Q000032:analysis; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000032:analysis; D008433:Mathematics; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
D008081:Liposomes; D012967:Sodium Dodecyl Sulfate; D006180:Proton-Translocating ATPases