The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane.

The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon. Properties of delta-deficient ECF1 have previously been described. F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E. coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit. The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles. Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation. Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex. Neither four-subunit enzyme binds to depleted membranes. The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits. Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits. A role for gamma in the binding of epsilon to F1 is suggested. epsilon does not bind to ECF1-depleted membranes. Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment. Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule.     

Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion

Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cleaved near the C-terminus, the gamma subunit was cleaved near Ser202, and the delta and epsilon subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECF1 enhanced the ATPase activity between 6- and 8-fold in different preparations, in a time course that followed the cleavage of the epsilon subunit. This removal of the epsilon subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the epsilon subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis and also increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the alpha and beta subunits along with two fragments of the gamma subunit. All of the subunits of ECF1 were cleaved by trypsin in preparations of ECF1F0 at the same sites as in isolated ECF1. Two subunits, the beta and epsilon subunits, were cleaved at the same rate in ECF1F0 as in ECF1 alone. The alpha, gamma, and delta subunits were cleaved significantly more slowly in ECF1F0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids.

We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes. We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light. The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon. Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective. Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma. The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel. The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.     

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

This review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of F1 have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An alpha 3 beta 3 gamma delta epsilon stoichiometry is now accepted for MF1 and BF1; the alpha 2 beta 2 gamma 2 delta 2 epsilon 2 stoichiometry for CF1 remains as matter of debate. The major subunits alpha, beta and gamma are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits delta and epsilon. The delta subunit of MF1 corresponds to the epsilon subunit of BF1 and CF1, whereas the mitochondrial subunit equivalent to the delta subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The alpha beta gamma assembly is endowed with ATPase activity, beta being considered as the catalytic subunit and gamma as a proton gate. On the other hand, the delta and epsilon subunits of BF1 and CF1 most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondrial ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the epsilon subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is beta comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The alpha subunit also plays a central role in catalysis, since structural alteration of alpha by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase

Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.     

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.     

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)     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

N,N'-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different beta subunits of the F1 ATPase of Escherichia coli

The fluorescent thiol reagent 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the gamma, delta, and one of the three beta subunits of the F1 ATPase from Escherichia coli (ECF1). This is the same beta subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) [H. Stan-Lotter and P. D. Bragg (1986) Eur. J. Biochem. 154, 321-327]. After inactivation of ECF1 with N,N'-dicyclohexylcarbodiimide (DCCD), IAANS labels in addition to the beta, gamma, and delta subunits also the alpha subunit. This suggests a conformational change of ECF1 upon binding of DCCD. The beta subunit which incorporates DCCD does does not bind IAANS. Likewise, IAANS-modified ECF1 does not incorporate DCCD into the same beta subunit. It is concluded that DCCD and Nbf bind to different beta subunits. Since neither of these reagents binds to that beta subunit which can be crosslinked to to the epsilon subunit by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, these data show that there is a difference in the chemical reactivity of each of the three beta subunits of ECF1, despite their identical primary structures. This suggests that there is an asymmetry in the F1 molecule.     

Chemical cross-linking studies of chloroplast coupling factor 1.

Cross-linking reagents have been used to link covalently adjacent subunits of solubilized spinach chloroplast coupling factor 1, which is a latent ATPase. 1,5-Difluoro-2,4-dinitrobenzene, dimethyl-3,3'-dithiobispropionimidate, and dimethylsuberimidate are able to form bridges of 3 to 11 A between amino groups, and hydrogen peroxide and the o-phenanthroline-cupric ion complex catalyze the oxidation of intrinsic sulfhydryl groups. The five individual subunit bands (alpha, beta, gamma, delta, and epsilon) and several new aggregate bands can be separated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same four fastest moving aggregate bands, as characterized by their mobilities, migrate more slowly than the heaviest subunit band and appear with all of the cross-linkers employed. The subunit composition of the aggregate bands has been determined through the use of the reversible cross-linkers, dimethyldithiobispropionimidate, (o-phenanthroline)2Cu(II), and H2O2, and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which aggregates are separated in the first dimension, the disulfide cross-links are cleaved, and the individual subunits present in the aggregates are separated in the second dimension. The subunits are detected by Coomassie brilliant blue staining and by labeling some of the sulfhydryl groups of the gamma and epsilon subunits with radioactive N-ethylmaleimide. The results obtained indicate that the alpha and beta subunits can cross-link directly with each of the other subunits, that two beta subunits are adjacent, and that gamma epsilon, gamma epsilon 2, alpha delta, and beta delta aggregates are present. A minimal subunit stoichiometry consistent with these results is alpha 2 beta 2 gamma delta epsilon 2. A possible structural model of the coupling factor is derived from the data. Similar, but less extensive, experiments have been carried out with the heat-activated coupling factor (which is an ATPase); no differences in the spatial arrangement of subunits are detected from the two-dimensional gel electrophoresis analysis of the cross-linked aggregates.     

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli

We have partially purified active delta and epsilon subunits of the E. coli membranebound Mg²⁺ -ATPase (ECF₁). Treating purified ECF₁ with 50% pyridine precipitates the major subunits (α, β, and γ) of the enzyme, but the two minor subunits (δ and ϵ), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF₁. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the δ subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the α and β subunits, was insensitive to the ATPase inhibitor, suggesting that the γ subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem. Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF₁ restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF₁.     

Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.     

The structure of the central stalk in bovine F(1)-ATPase at 2.4 A resolution

The central stalk in ATP synthase, made of gamma, delta and epsilon subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The gamma subunit penetrates the catalytic (alpha beta)(3) domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F(1)-ATPase, the protrusion was disordered, but with crystals of F(1)-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The delta and epsilon subunits interact with a Rossmann fold in the gamma subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (alpha beta)(3) domain.     

Cryoelectron microscopy of Escherichia coli F1 adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex

Monoclonal antibodies directed against epitopes on each of the five subunits (alpha, beta, gamma, delta, and epsilon) of the Escherichia coli F1 ATPase (ECF1) have been prepared and used to localize the subunits in the enzyme complex. Fab' fragments, prepared by pepsin digestion of the antibodies, were bound to ECF1 and visualized by cryoelectron microscopy of the unstained, frozen hydrated ECF1-Fab' complexes. Besides aiding in the identification of the ECF1 subunits, addition of Fab's to the specimen fortuitously offers additional advantages in this technique. ECF1 labeled with anti-alpha Fab' is uniformly oriented in the amorphous ice layer, in contrast to unlabeled ECF1, which exhibits a multitude of projection views when examined in ice. Almost all complexes display a triangular projection, which image averaging reveals to be a hexagonal view of ECF1 with Fab' fragments labeling every other peripheral subunit, confirming the alternating arrangement of alpha and beta subunits in the enzyme. A density in the interior of the structure is positioned asymmetrically, adjacent to an unlabeled peripheral mass, indicating that its primary linkage is to a beta rather than an alpha subunit. The composition of the asymmetric density was explored by examining the trypsin-treated ECF1, taking advantage of the unique orientation induced by the binding of anti-alpha Fab'. Trypsin treatment releases the delta and epsilon subunits and cleaves the gamma subunit; the internal density is reduced but not eliminated, showing the contribution of the gamma subunit to the residual structure, and suggesting that the loss of the delta and epsilon subunits, or a structural rearrangement of the gamma subunit, is responsible for its smaller size.(ABSTRACT TRUNCATED AT 250 WORDS)     

Large-scale purification and characterization of the five subunits of F1-ATPase from pig heart mitochondria.

A large-scale purification procedure was developed to isolate the five subunits of F1-ATPase from pig heart mitochondria. The previously described procedure (Williams, N. and Pedersen, P.L. (1986) Methods Enzymol. 126, 484-489) to dissociate the rat liver F1-ATPase by cold treatment followed by warming at 37 degrees C has been adapted for the pig heart enzyme. Removal of endogenous nucleotides from that enzyme before dissociation led to the efficient separation of the alpha and gamma subunits from beta, delta and epsilon subunits. The beta subunit was purified in the hundred-milligram range by anion-exchange chromatography in the absence of any denaturing agent. This subunit was free from any bound nucleotide and almost no ATPase and adenylate kinase-like activities were detected. The delta and epsilon subunits were purified by reversed-phase chromatography (RP-HPLC) in the milligram range. As recently reported (Penin, F., Deléage, G., Gagliardi, D., Roux, B. and Gautheron, D.C. (1990) Biochemistry 29, 9358-9364), these purified subunits kept biophysical features of folded proteins and their ability to reconstitute the tight delta epsilon complex. The alpha and gamma subunits remained poorly soluble and required dissociation by 8 M guanidinium chloride prior to their purification by RP-HPLC. In addition, characterizations of the five subunits by IEF and SDS-polyacrylamide gel electrophoresis are reported, as well as ultraviolet spectra and solubility properties of the beta, delta and epsilon subunits.     

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.     

Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits.

1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.