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)     

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.     

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.     

Chemical crosslinking of alpha subunits in the F1 adenosine triphosphatase of Escherichia coli

The arrangement of the subunits in the F1 adenosine triphosphatase of Escherichia coli has been investigated using bifunctional chemical crosslinking agents to covalently link adjacent subunits in the enzyme molecule. The synthesis of the new cleavable crosslinking agent 2,2'-dithiobis(succinimidyl propionate) is described. The crosslinked products resulting from the reaction of the enzyme with 2,2'- and 3,3'-dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), disuccinimidyl tartrate, dimethyl adipimidate, 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, and 1,2:3,4-diepoxybutane were analyzed by "three-dimensional" polyacrylamide gel electrophoresis in which they were resolved first in a two-dimensional system. Following cleavage of the crosslinking bridge in the separated products, the constituent subunits were identified by a further one-dimensional gel electrophoresis step. This procedure greatly improved the precision with which crosslinked subunits could be identified. It largely overcame problems due to abnormal migration of crosslinked species on gel electrophoresis and to the formation of multiple species of the same crosslinked subunit dimers. The following crosslinked subunit dimers were identified: alpha alpha, alpha beta, beta gamma, alpha delta, beta epsilon, and gamma epsilon. The trimer alpha alpha delta was recognized. The formation of alpha alpha over alpha beta dimers was favored when more polar crosslinking agents were used. The constraints placed by the finding of adjacent alpha subunits upon current models for the arrangement of the subunits in the F1 ATPase are discussed.     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

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)     

Mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus: purification, characterization, and kinetic studies.

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.     

ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits.

A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit.     

Guanidine-induced dissociation of mitochondrial F1-ATPase

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, and the fluorescence emission intensity of mitochondrial F1-ATPase was examined. Purified F1 from bovine heart mitochondria was slowly inactivated at low denaturant concentration, and inactivation was associated with delta and epsilon subunit dissociation. delta and epsilon subunits were bound together to form a stable and soluble heterodimer. In parallel, appearance of turbidity was observed. This was caused by the formation of alpha3beta3gamma non-covalent aggregates, as analyzed by SDS-PAGE. Short periods of exposition of the F1 complex to high concentrations of guanidine hydrochloride (0.8-3 M) again induced deltaepsilon dissociation as a heterodimer and the formation of an inactive alpha3beta3gamma subcomplex. This eventually dissociated progressively into single subunits caused by partial unfolding, as evidenced through changes of the protein intrinsic fluorescence emission. Our results suggest that the delta and epsilon subunits are loosely bound to alpha3beta3gamma , and play an important role in determining structural stability to isolated mitochondrial F1-ATPase.     

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)     

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.     

Complementation in vitro of mutant and wild-type ATPase of Escherichia coli using isolated subunits.

1. The inactive ATPases of four different mutant strains of Escherichia coli have been purified to homogeneity. 2. Molecular weights, subunit patterns in sodium dodecylsulfate electrophoresis and immunological properties of mutant and wild-type proteins are identical. The mutant enzymes compete with the wild-type enzyme for the binding sites on the membrane. 3. On freezing and thawing in salt solutions, the ATPase is split into subunits IA (alpha, gamma, epsilon), IB (delta; alpha, gamma, epsilon), and II (beta). By complementation in vitro of the isolated subunits, it is shown that subcomplex IA (alpha, gamma, epsilon) is altered in the mutant strains described here.     

Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.     

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.     

Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.

The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.     

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%.     

Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer

The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids.     

F1-ATPase from Micrococcus sp. ATCC 398. Purification by ion-exchange chromatography and further characterization. (Auto)proteolysis and dissociative effects.

The preparation of highly purified F1-ATPase from Micrococcus sp. ATCC 398 by application of DEAE-Sepharose CL-6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: alpha (65000), beta (55000),gamma (35000), delta (20000), and epsilon (17000). Disc electrophoresis on 5% polyacrylamide gels removes the epsilon-polypeptide yielding an active ATPase complex with four different subunits: alpha, beta, gamma, delta. Additionally, by variation of the ionic strength delta can (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits alpha, beta, and gamma. If the DEAE-Sepharose chromatography is carried out in the absence of diisopropyl phosphofluoridate (auto)proteolysis yields both an active ATPase with the subunits alpha+ (mol. wt 61000), beta, gamma, and delta and an inactive protein complex with the subunits alpha+, beta, gamma, delta, and two additional polypeptides a (mol. wt 38000) and b (mol. wt 23000). The latter two polypeptides are supposedly fragments of alpha+-chains which have become partially cleaved by (auto)proteolysis.     

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)     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.