Thermus thermophilus membrane-associated ATPase. Indication of a eubacterial V-type ATPase

An ATPase with Mr of 360,000 was purified from plasma membranes of a thermophilic eubacterium Thermus thermophilus, and was characterized. ATP hydrolytic activity of the purified enzyme was extremely low, 0.07 mumol of Pi released mg-1 min-1, and it was stimulated up to 30-fold by bisulfite. The following properties of the enzyme indicate that it is not a usual F1-ATPase but that it belongs to the V-type ATPase family, another class of ATPases found in membranes of archaebacteria and eukaryotic endomembranes. Among its four kinds of subunits with approximate Mr values of 66,000 (alpha), 55,000 (beta), 30,000 (gamma), and 12,000 (delta), the alpha subunit had a similar molecular size to the catalytic subunits of the V-type ATPases but was significantly larger than the alpha subunit of F1-ATPases. ATP hydrolytic activity was not affected by azide, an inhibitor of F1-ATPases, but was inhibited by nitrate, an inhibitor of the V-type ATPase. N-terminal amino acid sequences determined for the purified alpha and beta subunits showed much higher similarity to those of the V-type ATPases than those of F1-ATPases. Thus the distribution of the V-type ATPase in the prokaryotic kingdom may not be restricted to archaebacteria.     

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

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.     

Purification and biochemical characterization of the F1-ATPase from Acidithiobacillus ferrooxidans NASF-1 and analysis of the atp operon

ATPase was purified 51-fold from a chemoautotrophic, obligately acidophilic iron-oxidizing bacterium, Acidithiobacillus ferrooxidans NASF-1. The purified ATPase showed the typical subunit pattern of the F1-ATPase on a polyacrylamide gel containing sodium dodecyl sulfate, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa. The enzyme hydrolyzed ATP, GTP, and ITP, but neither UTP nor ADP. The K(m) value for ATP was 1.8 mM. ATPase activity was optimum at pH 8.5 at 45 degrees C, and was activated by sulfite. Azide strongly inhibited the enzyme activity, whereas the enzyme was relatively resistant to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide. The genes encoding the subunits for the F1F(O)-ATPase from A. ferrooxidans NASF-1 were cloned as three overlapping fragments by PCR cloning and sequenced. The molecular masses of the alpha, beta, gamma, delta, and epsilon subunits of the F1 portion were deduced from the amino acid sequences to be 55.5, 50.5, 33.1, 19.2, and 15.1 kDa, respectively.     

Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius

Electron transport phosphorylation has been demonstrated to drive ATP synthesis for the methanogenic archaebacterium Methanolobus tindarius: Protonophores evoked uncoupler effects and lowered the membrane potential delta psi. Under the influence of N,N'-dicyclohexylcarbodiimide [(cHxN)2C] the membrane potential increased while methanol turnover was inhibited. 2-Bromoethanesulfonate, an inhibitor of methanogenesis, had no effect on the membrane potential but, like (cHxN)2C and protonophores, decreased the intracellular ATP concentration. Labeling experiments with (cHxN)2(14)C showed membranes to contain a proteolipid, with a molecular mass of 5.5 kDa, that resembles known (cHxN)2C-binding proteins of F0-F1 ATPases. The (cHxN)2-sensitive membrane ATPase hydrolysed Mg.ATP at a pH optimum of 5.0 with a Km (ATP) of 2.5 mM (V = 77 mU/mg). It was inhibited competitively by ADP; Ki (ADP) = 0.65 mM. Azide or vanadate caused no significant loss in ATPase activity, but millimolar concentrations of nitrate showed an inhibitory effect, suggesting a relationship to ATPases from vacuolar membranes. In contrast, no inhibition occurred in the presence of bafilomycin A1. The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, alpha (67 kDa), beta (52 kDa), gamma (20 kDa) and beta (less than 10 kDa), as determined from SDS gel electrophoresis.     

Membrane ATPase from the aceticlastic methanogen Methanothrix thermophila.

A new isolate of the aceticlastic methanogen Methanothrix thermophila utilizes only acetate as the sole carbon and energy source for methanogenesis (Y. Kamagata and E. Mikami, Int. J. Syst. Bacteriol. 41:191-196, 1991). ATPase activity in its membrane was found, and ATP hydrolysis activity in the pH range of 5.5 to 8.0 in the presence of Mg2+ was observed. It had maximum activity at around 70 degrees C and was specifically stimulated up to sixfold by 50 mM NaHSO3. The proton ATPase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the membrane ATPase activity, but azide, a potent inhibitor of F0F1 ATPase (H(+)-translocating ATPase of oxidative phosphorylation), did not. Since the enzyme was tightly bound to the membranes and could not be solubilized with dilute buffer containing EDTA, the nonionic detergent nonanoyl-N-methylglucamide (0.5%) was used to solubilize it from the membranes. The purified ATPase complex in the presence of the detergent was also sensitive to N,N'-dicyclohexylcarbodiimide, and other properties were almost the same as those in the membrane-associated form. The purified enzyme revealed at least five kinds of subunits on a sodium dodecyl sulfate-polyacrylamide gel, and their molecular masses were estimated to be 67, 52, 37, 28, and 22 kDa, respectively. The N-terminal amino acid sequences of the 67- and 52-kDa subunits had much higher similarity with those of the 64 (alpha)- and 50 (beta)-kDa subunits of the Methanosarcina barkeri ATPase and were also similar to those of the corresponding subunits of other archaeal ATPases. The alpha beta complex of the M. barkeri ATPase has ATP-hydrolyzing activity, suggesting that a catalytic part of the Methanothrix ATPase contains at least the 67- and 52-kDa subunits.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.     

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

Structure of an ATPase operon of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The nucleotide sequence of the operon of the ATPase complex of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, has been determined. In addition to the three previously reported genes for the alpha, beta, and c (proteolipid) subunits of the ATPase complex (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1989) J. Biol. Chem. 264, 7119-7121), the operon contained three other genes encoding hydrophilic proteins with molecular masses 25, 13, and 7 kDa. The 25-kDa protein is the third largest subunit (gamma), the 13-kDa protein is most likely the fourth subunit (delta), and the 7-kDa protein may correspond to an unknown subunit of the ATPase, tentatively named as epsilon subunit. They do not have significant sequence similarity to subunits in F0F1-ATPases and eukaryotic V-type ATPases, whereas the other three subunits, alpha, beta, and c, have homologous counterparts in F0F1- and V-type ATPases. The order of the genes in the operon was delta alpha beta gamma epsilon c. The S. acidocaldarius ATPase operon differed from the eucabacterial F0F1-ATPase operon in that the former contains only one gene for a hydrophobic subunit at the most downstream part of the operon whereas the latter has three hydrophobic F0 genes preceding five hydrophilic F1 genes.     

Characterization of a membrane-associated ATPase from Methanococcus voltae, a methanogenic member of the Archaea.

A membrane-associated ATPase with an M(r) of approximately 510,000 and containing subunits with M(r)s of 80,000 (alpha), 55,000 (beta), and 25,000 (gamma) was isolated from the methanogen Methanococcus voltae. Enzymatic activity was not affected by vanadate or azide, inhibitors of P- and F1-ATPase, respectively, but was inhibited by nitrate and bafilomycin A1, inhibitors of V1-type ATPases. Since dicyclohexylcarbodiimide inhibited the enzyme when it was present in membranes but not after the ATPase was solubilized, we suggest the presence of membrane-associated component analogous to the F0 and V0 components of both F-type and V-type ATPases. N-terminal amino acid sequence analysis of the alpha subunit showed a higher similarity to ATPases of the V-type family than to those of the F-type family.     

Subunit analysis of latent and non-latent F1-ATPase from Micrococcus lysodeikticus (luteus)

Limited trypsin and chymotrypsin digestions were performed on the latent F1-ATPase from M. lysodeikticus, and subsequent analysis on SDS-polyacrylamide gels revealed subunit profiles with degraded alpha and delta subunits similar to those of ATPase preparations with spontaneously occurring lower degrees of latency. The ATPase obtained from M. lysodeikticus membranes after n-butanol extraction was also non-latent with similar SDS-gel patterns to the aforementioned ATPases. In addition, the sensitive technique of crossed immunoelectrophoresis was used to show that all of the above ATPases contained alpha, beta, gamma, delta and epsilon subunits but some of them were in degraded forms. Although the delta subunit was the first to be cleaved, the loss of latency can be attributed to the degradation of the alpha subunit.     

Purification, characterization, and application of an acid urease from Arthrobacter mobilis

It has been shown that urea in fermented beverages and foods can serve as a precursor of ethylcarbamate, a potential carcinogen, and acid urease is an effective agent for removing urea in such products. We describe herein the purification and characterization of a novel acid urease from Arthrobacter mobilis SAM 0752 and show its unique application for the removal of urea from fermented beverages using the Japanese rice wine, sake, as an example. The purified acid urease showed an optimum pH for activity at pH 4.2. The enzyme exhibited an apparent K(m) for urea of 3.0 mM and a Vmax of 2370 mumol of urea per mg and min at 37 degrees C and pH 4.2. Gel permeation chromatographic and sodium dodecyl sulfate gel electrophoretic analyses showed that the enzyme has an apparent native molecular weight (M(r)) of 290,000 and consisted of three types of subunit proteins (M(r), 67,000, 16,600, 14,100) denoted by alpha, beta, and gamma. The most probable stoichiometry of the subunits was estimated to be alpha: beta: gamma = 1:1:1, suggesting the enzyme subunit structure of (alpha beta gamma)3. The enzyme also existed as an aggregated form with an M(r) of 580,000. The purified enzyme contained 2 g-atom of nickel per alpha beta gamma unit of the enzyme. Enzyme activity was inhibited by acetohydroxamic acid, HgCl2, and CuCl2. The isoelectric point of the native enzyme was estimated by gel electrofocusing to be 6.8. Urea (50 ppm), which was exogenously added to sake (pH 4.4, 17 +/- 1% (v/v) ethanol), was completely decomposed by incubation with the enzyme (0.09 U ml-1) at 15 degrees C for 13 days. The enzyme was unstable at temperatures higher than 65 degrees C and pHs lower than 4, and was completely inactivated under the conditions of a pasteurization step involved in the traditional sake-making processes. These results indicate that the enzyme is applicable to the elimination of urea in fermented beverages with minimal modification to the conventional process.     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

The F1-type ATPase in anaerobic Lactobacillus casei

An ATPase from anaerobic Lactobacillus casei has been isolated and 100-times purified. The 400 kDa enzyme molecule was found to have a hexagonal structure 10 nm in diameter composed of at least six protein masses. SDS-electrophoresis reveals four or, under certain conditions, five types of subunit, of apparent molecular masses 57 (alpha), 55 (beta), 40 (gamma), 22 (delta) and 14 (epsilon) kDa with stoichiometry of 3 alpha, 3 beta, gamma, delta, epsilon. The following features resembling F1-ATPases from other sources were found to be inherent in the solubilized L. casei ATPase. (i) Detachment from the membrane desensitizes ATPase to low DCCD concentrations and sensitizes it to water-soluble carbodiimide. (ii) Soluble ATPase is inhibited by Nbf chloride and azide, is resistant to SH-modifiers and is activated by sulfite and octyl glucoside, the activating effect being much stronger than in the case of the membrane-bound ATPase. Substrate specificity of the enzyme is also similar to that of other factors F1. Divalent cations strongly activate the soluble enzyme when added at a concentration equal to that of ATP. An excess of Mn2+, Mg2+ or Co2+ inhibits ATPase activity of F1, whereas that of Ca2+ induces its further activation. No other F1-like ATPases are found in L. casei. It is concluded that this anaerobic bacterium possesses a typical F1-ATPase similar to those in mitochondria, chloroplasts, aerobic and photosynthetic eubacteria.     

Phosphorylated intermediate of the ATPase of plant plasma membranes

A partially purified preparation of the plant plasma membrane ATPase was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. The sensitivity of the phosphoenzyme bond to alkaline pH and to hydroxylamine indicates that it is an acylphosphate. Both the ATPase activity and the phosphorylation of the enzyme exhibited an apparent Km value of 0.3 mM ATP. When the phosphorylated enzyme was analyzed by electrophoresis in sodium dodecyl sulfate, only one major band with a molecular weight of about 105,000 contained radioactivity. These results indicate that the plant plasma membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal and fungal plasma membranes.     

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.     

Cross-linking of the gamma subunit of the Escherichia coli ATPase (ECF1) via cysteines introduced by site-directed mutagenesis.

The gamma subunit of the Escherichia coli F1 ATPase (ECF1) has been altered by site-directed mutagenesis to create five different mutants, gamma-S8C, gamma-S81C, gamma-T106C, gamma-S179C, and gamma-V286C, respectively. ECF1 isolated from four of these mutants had ATPase activities similar to that of a wild-type isogenic strain used as a control, the exception was enzyme isolated from mutant gamma-S81C, which had an ATPase activity of around 70-80% of the wild type. ECF1 isolated from each of the various mutants was reacted with N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide (CM). The fluorescent reagent was incorporated into Cys residues placed at positions 8, 106, 179, and 286, but not at 81, indicating which of these Cys residues are on the surface of the gamma subunit in the enzyme complex. Modification of the Cys at position 106 with CM activated the enzyme, and modification of the Cys at position 8 inhibited ATPase activity a small amount; however, modification of Cys at 179 or 286 had no effect on enzyme activity. The four mutants with a reactive Cys were reacted with tetrafluorophenylazide maleimides (TFPAMs), novel photoactivatable cross-linkers. In the mutant gamma-S8C, cross-links were formed between the introduced Cys on the gamma subunit and sites on the beta subunit. This cross-linking between gamma and beta depended on nucleotide conditions under which the photolysis was carried out, with differently migrating cross-linked products being obtained in ATP + EDTA compared with ATP + Mg2+ or ATP + Mg2+ Pi. Cross-linking between beta and gamma inhibited ATPase activity in proportion to the yield of cross-linked product. In the mutant gamma-V286C, cross-links were formed between the introduced Cys on gamma and the alpha subunit which were the same in all nucleotide conditions and which led to inhibition of ATPase activity.