Characterization of three-subunit chloroplast coupling factor

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)

“…For the same reason, F^a/Sy) may also be useful for growing crystals suitable for X-ray crystallography. By analogy to the chloroplast system (Mitra & Hammes, 1988),…”

Section: Discussionmentioning

confidence: 99%

Coupling factor 1 from Escherichia coli lacking subunits .delta. and .epsilon.: preparation and specific binding to depleted membranes, mediated by subunits .delta. or .epsilon.

Tuttas-Dörschug¹,

Hanstein²

1989

“…The γ-containing CF 1 complexes are markedly stimulated by methanol and/or octyl glucoside as well as sulfite, and inhibited by azide, excess free Mg 2+ ions, and low concentrations of tentoxin. It has also been reported that the removal of the δand -polypeptides from CF 1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme (Mitra & Hammes, 1988). Our observations that further removal of the γ-polypeptide leads to destabilization of the hexameric structure of the remaining CF 1 (Rβ) complex, and to drastic changes in its catalytic properties, indicate that the CF 1 γ subunit is most important for maintaining the structural and functional properties of the CF 1 -ATPase.…”

Section: Discussionmentioning

confidence: 95%

“…CF 1 , like all other F 1 -ATPases, is composed of five kinds of subunits designated Rand has a subunit stoichiometry of R 3 β 3 γδ (McCarty & Moroney, 1985;Nalin & Nelson, 1987;Jagendorf et al 1991). The CF 1 -ATPase activity does not require the presence of the δ and subunits, since their complete removal (Patrie & McCarty, 1984) was found to leave a fully active CF 1 (-δ-)-ATPase (Mitra & Hammes, 1988). This CF 1 -R 3 β 3 γ has also been found to retain the sensitivity of the whole CF 1 to its specific inhibitor, tentoxin (Hu et al, 1993).…”

mentioning

confidence: 99%

Spinach Chloroplast Coupling Factor CF₁-α₃β₃ Core Complex: Structure, Stability, and Catalytic Properties

Sokolov

Gromet-Elhanan

1996

A minimal chloroplast coupling factor CF1 core complex, containing only alpha and beta subunits, has been isolated from spinach thylakoids [Avital, S., & Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072]. This CF1(alpha beta) exhibited a low MgATPase activity, which was stimulated but not inhibited by low concentrations of the species-specific CF1 effector tentoxin. As is reported here, the structure of CF1(alpha beta) could not be determined due to its instability. However, its pretreatment with high tentoxin concentrations resulted in a remarkable 50-fold stimulation of the MgATPase activity as well as stabilization of its hexameric structure, thus enabling the isolation of a more active CF1-alpha 3 beta 3 complex by size-exclusion chromatography. A detailed characterization of the MgATPase activity of this tentoxin-stabilized CF1-alpha 3 beta 3 hexamer, as compared to the activity of a CF1 complex lacking the epsilon subunit, revealed similar apparent Km values and a similar stimulation by the presence of 100 microM tentoxin in the assay medium, but drastic differences in all other tested assays. Most pronounced were their different temperature profiles and different responses to all added inhibitors and stimulators of the CF1 MgATPase activity and to excess free Mg2+ ions. The specific properties of the stable CF1-alpha 3 beta 3 hexamer are identical to those earlier reported for its parent-unstable CF1(alpha beta). These results indicate that, although the CF1 gamma subunit is not required for the low CF1(alpha beta) ATPase activity nor for the higher activity of the tentoxin-stabilized CF1-alpha 3 beta 3, it plays a central role in obtaining the typical functional properties of the CF1-ATPase. Kinetic cooperativity could not be critically tested as yet with any F1-alpha 3 beta 3. However, tentoxin, as azide, has been shown to inhibit multisite but not unisite catalysis. Therefore, the observation that CF1-alpha 3 beta 3 is only stimulated by tentoxin suggests that the required presence of CF1-gamma for obtaining inhibition by tentoxin reflects the role of this subunit in cooperative interactions between the catalytic sites.

“…This enzyme preparation has variable amounts of the -disulfide reduced. In order to label specifically the -disulfide and to avoid labeling the exposed sulfhydryl of the -polypeptide ("dark" site), the enzyme was oxidized with CuCl2 for 1 h (Mitra & Hammes, 1988), and 1 mM EDTA then was added to chelate the Cu2+. This results in oxidation of the -disulfide.…”

Section: Methodsmentioning

confidence: 99%

Structural map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer

Mitra¹,

Hammes²

1989

Self Cite

Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. The vesicle surface was labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine. Steady-state energy transfer between the fluorescent-labeled enzyme (energy donor) and varying concentrations of the ethanolamine derivative (energy acceptor) indicated that the distance of closest approach between the energy donor and the outer vesicle surface is 16-24 A. Two specific sites on the gamma-polypeptide were reacted with a coumarinylmaleimide derivative; one is a sulfhydryl that can be labeled only on the thylakoids under energized conditions (the "light" site), while the other is the disulfide site that regulates enzymatic activity. Energy-transfer measurements utilizing steady-state fluorescence and fluorescence lifetime methods indicated that the dicyclohexylcarbodiimide site is approximately 41 A from the light site and approximately 50 A from the gamma-disulfide site. These distances are used to extend the current structural model of the chloroplast coupling factor.