Purirication and characterization of two forms of DNA-dependent ATPase from yeast

Two forms of DNA-dependent ATPase activity have been purified from yeast extracts. The two ATPases differ from each other in chromatographic properties and heat stabilities but have similar molecular weight and reaction properties. DNA-dependent ATPase I has been purified to near homogeneity, while DNA-dependent ATPase II is only partially purified. The two ATPases from yeast are related structurally since antiserum raised against ATPase I cross-react against ATPase II. Yeast DNA-dependent ATPase I has a native molecular weight of about 68,000 and consists of a single polypeptide chain. ATPase II also sediments on sucrose gradient as a 68,000-dalton protein. Both yeast DNA-dependent ATPases hydrolyze dNTPs and rNTPs to their corresponding nucleoside diphosphates and orthophosphate, but dATP and ATP are preferred substrates. In addition to nucleoside triphosphates, both enzymes require a divalent cation and a polynucleotide for activity. Single-stranded DNAs and polydeoxynucleotides are the most effective co-substrates for yeast DNA-dependent ATPases. Addition of yeast DNA-dependent ATPases to DNA synthesis system containing yeast DNA polymerases does not significantly stimulate the rate of DNA synthesis.     

An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.     

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

Properties of a DNA-dependent ATPase from rat mitochondria.

A DNA-dependent ATPase has been highly purified from rat liver mitochondria and characterized. The enzyme catalyzes the hydrolysis of ATP or dATP in the presence of single-stranded DNA cofactor and a divalent cation. The Km values for ATP and dATP are 0.15 mM and 0.35 mM, respectively. The enzyme activity is highly sensitive to N-ethylmaleimide. The sedimentation coefficient of the enzyme is 8.3 S in a glycerol gradient. From this and data on Sephadex G-200 gel filtration, the molecular weight of the native enzyme was calculated to be about 190,000. All the natural single-stranded DNAs tested were equally effective for the ATPase activity, but synthetic deoxyhomopolymer poly(dC) was found to be more effective than natural single-stranded DNAs. Synthetic and natural RNAs had no effect on the activity.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.     

Multiple deoxyribonucleic acid dependent adenosinetriphosphatases in FM3A cells. Characterization of an adenosinetriphosphatase that prefers poly [d(A-T)] as cofactor

Four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, have been partially purified from mouse FM3A cell extracts. These ATPases are distinguished from each other by their physical and enzymological properties. DNA-dependent ATPases B, C1, C2, and C3 have sedimentation coefficients in 250 mM KCl of 5.5, 5.3, 7.3, and 3.4 S, respectively. ATPases B, C2, and C3 hydrolyze dATP as efficiently as ATP, whereas C1 does not. ATPase B hydrolyzes other ribonucleoside triphosphates with relatively high efficiency as compared to the other three enzymes. ATPase C3 prefers poly[d(A-T)] to poly(dT) as cofactor, whereas the other three enzymes prefer poly(dT) to poly[d(A-T)]. Among the four ATPases, ATPase C3 has been highly purified and characterized in detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction of ATPase C3 showed two major bands corresponding to molecular weights of 66 000 and 63 000. The Km values of the enzyme for ATP and dATP are 0.53 and 0.86 mM, respectively. As cofactor, poly[d(A-T)] is the most effective among the DNAs tested. Heat-denatured DNA and native DNA are also effective but used with less efficiency. Almost no or very little activity has been detected with ribohomopolymers and oligonucleotides. The activity attained with poly(dT) and poly(dA) is 11 and 6% of that with heat-denatured DNA, respectively. When both polymers were added at a molar ratio 1 to 1, very high activity was obtained with these polymers. On the other hand, little activity was observed by the combination of noncomplementary homopolymers such as poly(dT) and poly(dG).     

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.     

Mg2+ adenosine triphosphatase from cell envelopes of free-living and bacteroid forms of Rhizobium lupini strain NZP2257

A procedure for the purification of Mg2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described. The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose. The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients. Kinetic analysis of activity of the enzyme from free-living R. lupini showed it to be typical of F1-type Mg2+ ATPases from bacteria. Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition (K1 = 1.5 mM). Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5. ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme. Hydrolysis of glucose 6-phosphate was not observed. The Km for ATP at pH 7.0 was 0.67 and for GTP 1.4 mM. ATPase activity was inhibited by ADP, and competitive with ATP (KI = 0.18 mM). Azide also caused inhibition but fluoride and DCCD had no effect. Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R. lupini.     

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.     

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.     

A novel microtubule-associated protein from mammalian nerve shows ATP- sensitive binding to microtubules

We report the isolation of a protein from mammalian nerve which shows ATP-sensitive binding to microtubules and ATPase activity. This protein, which we have designated HMW4, was prepared from bovine spinal nerve roots by microtubule affinity and ATP-induced release, and was further purified by sucrose density gradient centrifugation. It is a high molecular weight protein with a denatured Mr of 315,000, a Stokes radius of 90 A, and a sedimentation value of approximately 19S. It can be resolved electrophoretically from the well-characterized bovine brain microtubule-associated proteins (MAPs) and also appears to be distinct from MAP 1C. HMW4 has a vanadate-sensitive and azide-insensitive ATPase activity which averages 20 nmol Pi/min per mg protein and is different from dynein and myosin ATPases. HMW4 prepared on sucrose gradients exhibits binding to MAP-free microtubules in the absence of ATP which is reduced by ATP addition. Assayed by darkfield microscopy, HMW4 causes bundling of MAP-free microtubules which is reversed by ATP addition.     

Quaternary structure of delta-aminolevulinic acid synthase from Rhodopseudomonas spheroides.

Delta-Aminolevulinic acid synthase (succinyl-CoA: glycine C-succinyltransferase (decarboxylating) EC 2.3.1.37) was purified from Rhodopseudomonas spheroides. The purity of the enzyme preparation was established by its behavior in disc electrophoresis in the presence and absence of sodium dodecyl sulfate and by analytical ultracentrifugation. The molecular weight of the enzyme as determined by sedimentation equilibrium was found to be about 80,300, a value similar to those obtained by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient centrifugation. The molecular weight of the enzyme, denatured with either sodium dodecyl sulfate or guanidine hydrochloride, was found to be about 45,000 and 41,000, respectively. The dimeric structure was supported by sedimentation in sucrose gradients. Further evidence for the dimetic nature of the enzyme was obtained by gel electrophoresis of the enzyme treated with dimethylsuberimidate and sodium dodecyl sulfate.     

DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme

We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.     

Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA

There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.     

Purification and characterization of a DNA-dependent ATPase from Bacillus cereus

A DNA-dependent ATPase (molecular weight 71 000) free of nuclease activity has been purified from Bacillus cereus. The enzyme shows similar characteristics as the enzyme isolated from Escherichia coli and Bacillus subtilis. Heat denatured DNA stimulates the rate of ATP hydrolysis to ADP and Pi to an extent about tenfold higher than the native DNA. Double stranded DNA without single stranded regions is not a suitable cofactor for the enzyme. The ATPase is inhibited by adenosine 5'-(beta, gamma-imino)-diphosphate, while another ATP analogue, adenosine 5'-(beta, gamma-methylene)-diphosphate has no effect on ATPase activity. KM for ATP is 0.38 mM, the apparent KM for nucleotide equivalent DNA is 1.2 microM. Evidence of the unwinding function of the enzyme is presented.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties.

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.     

RNA-dependent ATPase from Saccharomyces cerevisiae

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.     

Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

A novel ATPase was solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, with low ionic strength buffer containing EDTA. The enzyme was purified to homogeneity by hydrophobic chromatography and gel filtration. The molecular weight of the purified enzyme was estimated to be 360,000. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate revealed that it consisted of three kinds of subunits, alpha, beta, and gamma, whose molecular weights were approximately 69,000, 54,000, and 28,000, respectively, and the most probable subunit stoichiometry was alpha 3 beta 3 gamma 1. The purified ATPase hydrolyzed ATP, GTP, ITP, and CTP but not UTP, ADP, AMP, or p-nitrophenylphosphate. The enzyme was highly heat stable and showed an optimal temperature of 85 degrees C. It showed an optimal pH of around 5, very little activity at neutral pH, and another small activity peak at pH 8.5. The ATPase activity was significantly stimulated by bisulfite and bicarbonate ions, the optimal pH remaining unchanged. The Lineweaver-Burk plot was linear, and the Km for ATP and the Vmax were estimated to be 1.6 mM and 13 mumol Pi.mg.-1.min-1, respectively, at pH 5.2 at 60 degrees C in the presence of bisulfite. The chemical modification reagent, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, caused inactivation of the ATPase activity although the enzyme was not inhibited by N,N'-dicyclohexylcarbodiimide, N-ethyl-maleimide, azide or vanadate. These results suggest that the ATPase purified from membranes of S. acidocaldarius resembles other archaebacterial ATPases, although a counterpart of the gamma subunit has not been found in the latter. The relationship of the S. acidocaldarius ATPase to other ion-transporting ATPases, such as F0F1 type or E1E2 type ATPases, was discussed.