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Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 ± 5 kDa. The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis-Menten kinetics with a Km of about 7 μM for PPi and a specific activity of about 1200 U/mg at 56°C. Surprisingly, maximum activity could be observed at 85 °C which is more than 20 °C above the temperature for optimal growth. Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross-reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum. [ABSTRACT FROM AUTHOR]

Plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate-hydrolyzing activity [M. Lübben & G. Schäfer (1987) Eur. J. Biochem. 164, 533–540]. In our present work, we solubilized and purified this pyrophosphatase to homogeneity. It consists of a single subunit with a molecular mass of 17–18 kDa, forming an oligomer of 70 kDa under native conditions. Edman degradation revealed 30 amino acids of the N-terminus. The enzyme cleaves phosphoric-acid-anhydride bonds independently of monovalent or divalent cations. Temperature and pH optima of 75°C and 3.5–3.7, respectively, characterize it as an ectoenzyme. Membrane lipids of Sulfolobus stimulate the activity. The dolichol-pyrophosphate-complexing peptide-antibiotic bacitracin inhibited growth of Sulfolobus. A possible function of the acid pyrophosphatase is the hydrolysis of dolichol pyrophosphate in connection with glycosylation reactions of membrane proteins. [ABSTRACT FROM AUTHOR]

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) extrudes protons when expending respiratory energy {Moll. R. & Schäfer, G. (1988) FEBS Lett. 359-363]. Cytochromes of the membrane electron-transport systems are assumed to represent the proton pumps. Only a and b-type cytochromes can be found; no-c-type cytochromes are present. Of the two terminal oxidases [Anemüller, S. & Schäfer, G. (1989) FEBS Lett. 244, 451-455] one shows an absorption band at 604-605 nm, typical of cytochromes of the aa3. This hemoprotein has been solubilized from the membrane and purified to homogeneity. It exhibits distinct differences from known aa3-type oxidases. (a) It consists of a single polypeptide subunit of 38-40 kDa apparent molecular mas with two heme-a molecules and two copper ions. (b) In the oxidized state, absorption maxima are found at 421 nim and 597 nim and in the reduced state at 439 nim and 601 nim; CO difference spectra suggest one heme to be a heme-a3 centre. (c) The redo potentials of the heme centres are + 220 ma and + 370 ma, respectively. (d) A high-spin heme signal at g = 6 is present in EPR spectra, which is more prominent than the low-spin heme signal at g = 3, the former already being present in the oxidized state. A signal at g = 2.1 may be due to one of the copper ions and is superimposed upon a minor free radical signal at g = 2. (e) Caldariella quinone was also isolated from the plasma membrane of Sulfolobus. Its redox midpoint potential at pH 6.5 was determined to be + 100 (±5) mV; spectral properties have also been determined. (f) the isolated aa3 preparation does not oxidize cytochrome c; however, it oxidizes N,N,N', N'-tetramethyl-1,4-phenylenediamine dihydrochloride as an artificial single-electron donor as well as reduced caldariella quinone, which is assumed to represent the natural substrate. The reaction is cyanide-sensitive an the product of oxygen reduction is water. (g) On the basis of the results obtained a novel type of cytochrome aa3 is postulated in this paper which oxidizes reduced quinones; its ability to act as a proton pump remains to be shown. [ABSTRACT FROM AUTHOR]

Electron transport phosphorylation has been demonstrated to drive ATP synthesis for the methanogenic archaebacterium Methanolobus tindarius: Protonophores evoked uncoupler effects and lowered the membrane potentia; Δψ. 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)214C 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: K1 (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 At. The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, α (67 kDa), β(52 kDa), γ(20 kDa) and δ( < 10 kDa), as determined From SDS gel electrophoresis. [ABSTRACT FROM AUTHOR]

Thermoacidophilic archaebacteria have gained much interest because of their phylogenetic distance to eubacteria and eukaryotes and also because of their unique living conditions. Investigation of the energyconverting system therefore offers a key for understanding the evolutionary position and environmental adaptation of these unusual bacteria. A plasma-membrane-associated adenosine triphosphatase with specific activities of 0.3-0.6 µmol min-1 (mg protein)-1 has been detected in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639). The enzyme exhibits two optima at pH 5.5 and 8.0, sulfite activation leads to only one optimum at pH 6.25. In the presence of the divalent cations Mg2+ or Mn2+ it hydrolyzes ATP with highest reactivity and also other purine and pyrimidine nucleotides, but not ADP and pyrophosphate. A specific stimulation by monovalent cations is not observed. The ATPase activity is not inhibited by N,N'-dicyclohexylcarbodiimide, azide or vanadate, but it is by the vacuolar ATPase inhibitor nitrate with an [I]50 of 8 mM. Linear Arrhenius plots up to 75°C reflect pronounced adaptation to the hot environment of the archaebacterium. The solubilized ATPase as localized by activity staining in non-denaturating gels and further analyzed by sodium dodecyl sulfate electrophoresis is composed of two major polypeptides of 65 and 51 kDa reminiscent of the α and β subunits of eubacterial and eukaryotic F0F1-ATPases. The ATPase is suggested as a probable candidate for a reversibly acting ATP synthase responsible for oxidative phosphorylation found in Sulfolobus acidocaldarius. [ABSTRACT FROM AUTHOR]

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotein complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies. A. ambivalens Ffh encodes a 50.4-kDa protein that is structured by three distinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-binding domain (G) and a C-terminal located C-domain (C). The A. ambivalens Ffh sequence shares 44–46% sequence similarity with Ffh of methanogenic archaea, 34–36% similarity with eukaryal SRP54 and 30–34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A. ambivalens. Recombinant Ffh has a melting point of tm = 89 °C. Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts. Highest rates of GTP hydrolysis have been achieved at 81 °C in presence of 0.1–1 mm Mg2+. GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents. The Km for GTP is 13.7 µm at 70 °C and GTP hydrolysis is strongly inhibited by GDP (Ki = 8 µm). A. ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus. Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic... [ABSTRACT FROM AUTHOR]

The terminal quinol oxidase of the cytochrome aa type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldorius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50°C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (ΔΦ, interior negative), and transmembrane pH gradient (ΔpH, interior alkaline) at temperatures up to 70°C. The successful functional reconstitution of the cytochrome aa-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon. [ABSTRACT FROM AUTHOR]

The plasma-membrane-associated ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius characterized in a previous work [M. Lübben & G. Schäfer (1987) Eur. J. Biochem. 164, 533 - 540] has been solubilized. It can be easily removed from the membrane by mild treatment with zwitterionic detergents, therefore it appears to be a peripheral membrane protein analogous to the soluble F1-ATPase of eubacteria and eukaryotes. Further purification has been achieved by subsequent gel permeation and ion-exchange chromatography. The final purity is greater than 70% as judged by staining intensities after SDS/polyacrylamide gel electrophoresis. The ATPase consists of two major polypeptides of 65 kDa (α) and 51 kDa (β) in comparable quantities; a minor band (20 kDa) is assumed to be a contaminant or a constitutive part of the enzyme, possibly copurified in substoichiometric amount. The native molecular mass of the solubilized ATPase determined by gel permeation is 430 kDa. Considering the precision of these methods, it remains open whether a 3:3 stoichiometry reflects the contribution of a and β subunits to the quaternary structure, in analogy to known F1-ATPases. The catalytic properties resemble those of the membrane-bound state. There are two pH optima at 5.3 and 8.0 in the absence and only one optimum at 6.5 in the presence of the activating anion sulfite. Activity is strictly dependent on the divalent cations Mg2 + or Mn2 + ATP and dATP are hydrolyzed with highest rates; also other purine and pyrimidine nucleotides are cleaved significantly, but not ADP, pyrophosphate and p-nitrophenyl phosphate. The ATPase is insensitive to azide or vanadate but is inhibited by relatively low concentrations of nitrate. Polyclonal antisera have been raised against the β subunit of the Sulfolobus ATPase. Cross-reactivities with cellular or membrane extracts of a number of archaebacteria, eubacteria and chloroplasts have been analyzed by means of Western blotting and immunodecoration. A strong cross-reactivity with other genera of the Sulfolobales is observed, also with Methanobacterium, Methanosarcina, Methanolobus and Halobacterium. Even membranes of the eubacterium Escherichia coli and of eukaryotic chloroplast react with the antibodies. With one exception, in all cases the molecular mass of the cross-reacting polypeptide falls in the range of 51 -56 kDa. Only in Halobacterium halobium, bands at 66 and 68 kDa have been detected. In order to identify the cross-reacting polypeptides, the purified F1-ATPases of E. coil, chloroplasts and beef heart mitochondria have been tested. Unequivocally the /3 subunits of these coupling factors are labeled by the Sulfolobus anti-β antibody. Our data support the idea that the Sulfolobus ATPase is at least closely related or even affiliated to the group of eubacterial and eukaryotic F0F1-ATPases. In addition, the ATPase could be localized as a constituent of the plasma membrane by immuno-electron microscopy. [ABSTRACT FROM AUTHOR]