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.     

Energy metabolism of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of the archaebacterium and mechanisms of acidophily, membrane bound ATPase, cytochromes and NADH dehydrogenase of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Typea cytochrome was found in the membrane. The organism was sensitive to cyanide and azide, and though cytochromec is lacking in this organism, these respiratory poisons inhibited a terminal oxidase, when assayed with cytochromec from other sources. NADH dehydrogenase was highly purified from the crude extract of the cells. The enzyme was able to transfer electrons from NADH to caldariellaquinone, a unique benzothiophenequinone in the genusSulfolobus. Thus, the enzyme is a possible member of the respiratory chain. Membrane fraction contained two types of ATPase, one was active at neutral pH and slightly activated by sulfate; the other was an acid apyrase and inhibited by sulfate. Typical characteristics of F₀F₁ATPase could not be found in these enzymes. These results suggest that (1) the thermoacidophilic archaebacteria are phylogenically distant from both eubacteria and eukaryotes, (2) the archaebacterial thermoacidophiles can be classified in a different subgroup from methanogens and extreme halophiles, and (3) in spite of the aerobic nature of the organism, the energy yielding mechanisms appear quite unique, when compared to those of other aerobes and mitochondria.     

Purification and properties of a thermophilic and thermostable DNA polymerase from the archaebacterium Sulfolobus solfataricus

A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation. It requires Mg++ for its activity and has a pH optimum of 7.7. The activity is sharply dependent on the ionic strength. The enzyme is thermostable; its properties and activity requirements were characterized. The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.     

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

Abstract The bioenergetic properties of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserved energy by oxidative phosphorylation and how the involved catalysts are related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system are identified and a novel type of heme- aa ₃ containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an F ₁-analogous ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfolobus are discussed as likely components of an F ₀ F ₁-analogous ATP-synthase. The structural and functional properties of this and other archaebacterial ATPases are compared to each other and with respect to evolutionary relations.     

Isolation and properties of the SuaI restriction endonuclease from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A new restriction endonuclease SuaI was isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspR1; it recognizes tetranucleotide GGCC and cleaves DNA in the center of this sequence. SuaI requires Mg2+, the optimal concentration being 6 mM. KCl at concentrations above 25 mM significantly inhibits the enzyme activity. The pH optimum lies within the range of 6--7 at 70 degrees C, the temperature optimum is at 70--75 degrees C. The enzyme is highly stable at temperatures up to 80 degrees C. DNA of S. acidocaldarius is not cleaved by the enzyme.     

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.     

A membrane-bound ATPase from Halobacterium halobium: purification and characterization

An ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1 M or over. The molecular weight of the native enzyme was around 320,000; it is most likely composed of two pairs (alpha 2 beta 2) of 86,000 (alpha) and 64,000 (beta) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4 M at any pH. The Km value for ATP was 1.4 mM and the K1 value for ADP (competitive to ATP) was 0.08 mM. Neither azide (a specific inhibitor for F0F1-and F1-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCl concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobacterial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).     

Chemiosmotic energy conversion of the archaebacterial thermoacidophile Sulfolobus acidocaldarius: oxidative phosphorylation and the presence of an F0-related N,N'-dicyclohexylcarbodiimide-binding proteolipid

The energy-transducing mechanism of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius DSM 639 has been studied, addressing the question whether chemiosmotic proton gradients serve as an intermediate energy store driving an F0F1-analogous ATP synthase. At pH 3.5, respiring S. acidocaldarius cells developed an electrochemical potential of H+ ions, consisting mainly of a proton gradient and a small inside-negative membrane potential. The steady-state proton motive force of 140 to 160 mV was collapsed by protonophores, while N,N'-dicyclohexylcarbodiimide (DCCD) caused a hyperpolarization of the membrane, as expected for a reagent commonly used to inhibit the flux through proton channels of F0F1-type ATP synthases. Cellular ATP content was strongly related to the proton motive force generated by respiration and declined rapidly, either by uncoupling or by action of DCCD, which in turn induced a marked respiratory control effect. This observation strongly supports the operation of chemiosmotic ATP synthesis with H+ as the coupling ion. The inhibition of ATP synthesis by [14C]DCCD was correlated with covalent reactions with membrane proteins. The extraction of labeled membranes with organic solvents specifically yielded a readily aggregating proteolipid of 6 to 7 kilodaltons apparent molecular mass. Its amino acid composition revealed significant similarity to the proteolipid found in eubacteria, such as Escherichia coli, as an extremely hydrophobic constituent of the F0 proton channel. Moreover, the N-terminal amino acid sequence of the Sulfolobus proteolipid displays a high degree of homology to eubacterial sequences, as well as to one derived from nucleic acid sequencing of another Sulfolobus strain (K. Denda, J. Konishi, T. Oshima, T. Date, and M. Yoshida, J. Biol. Chem. 264:7119-7121, 1989). Despite certain structural similarities between eucaryotic vacuolar ATPases and the F1-analogous ATPase from Sulfolobus sp. described earlier, the results reported here promote the view that the archaebacterial ATP-synthesizing complex functionally belongs to the F0F1 class of ATPases. These may be considered as phylogenetically conserved catalysts of energy transduction present in all kingdoms of organisms.     

Purification and properties of an ATPase from Sulfolobus solfataricus.

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzofurazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-Cl was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuriphenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethylmaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.     

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.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

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.     

Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Functional characterization of an extremely thermophilic ATPase in membranes of the crenarchaeon Acidianus ambivalens.

A plasma membrane-bound adenosine triphosphatase with specific activities up to 0.2 micromol min(-1) (mg protein)(-1) at 80 degrees C was detected in the thermoacidophilic crenarchaeon Acidianus ambivalens (DSM 3772). The enzymatic activity exhibited a broad pH-optimum in the neutral range with two suboptima at pH 5.5 and 7.0, respectively. Sulfite activation resulted in only one pH optimum at 6.25. In the presence of the divalent cations Mg2+ and Mn2+ the ATPase activity was maximal. Remarkably, the hydrolytic rates of GTP and ITP were substantially higher than for ATP. ADP and pyrophosphate were only hydrolyzed with small rates, whereas AMP was not hydrolyzed at all. Both activities could be weakly inhibited by the classical F-type ATPase inhibitor N,N'-dicyclohexylcarbodiimide, whereas azide had no influence at all. The classical inhibitor of V-type ATPases, nitrate, also exerted a small inhibitory effect. The strongly specific V-type ATPase inhibitor concanamycin A, however, showed no effect at all. The P-type ATPase inhibitor vanadate had no inhibitory effect on the ATPase activity at pH 7.0, whereas a remarkable inhibition at high concentrations could be observed for the activity at pH 5.5. Arrhenius plots for both membrane bound ATPase activities were linear up to 95 degrees C, reflecting the enormous thermostability of the enzyme.     

Intrinsic DNA-dependent ATPase activity of reverse gyrase

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.     

Purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius

A succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. It consists of four subunits: a, 66 kDa; b, 31 kDa; c, 28 kDa and d, 12.8 kDa. In the 141-kDa native protein, the four subunits are present in an equimolar stoichiometry. The complex contains acid-non-extractable flavin, iron and acid-labile sulphide. Maximal succinate dehydrogenase activities were recorded at pH 6.5, which coincides with the internal pH of Sulfolobus cells. The temperature optimum of 81 degrees C defines the Sulfolobus succinate dehydrogenase as a thermophilic enzyme complex. The Km for succinate was found to be 1.42 mM (55 degrees C). Similar to the mitochondrial soluble succinate dehydrogenase, this enzyme is capable of transferring electrons to artificial electron acceptors, for instance phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine and ferricyanide. In contrast to the mitochondrial succinate dehydrogenase, the archaebacterial enzyme reduces 1,4-dichloroindophenol also in the absence of phenazine methosulfate. Caldariella quinone, the physiological electron mediator in the Sulfolobus respiratory chain, was only slowly reduced under adjusted conditions. The succinate--phenazine methosulfate-(1,4-dichloroindophenol) oxidoreductase of the isolated complex was strongly inhibited by tetrachlorobenzoquinone. In plasma membranes the complex reduces molecular oxygen in a cyanide-sensitive reaction. Polyclonal Sulfolobus anti-a antibodies crossreacted with 66-67-kDa polypeptides from membranes of Thermoplasma acidophilium, Sulfolobus solfataricus and beef heart submitochondrial particles.     

Oxalacetate decarboxylase and pyruvate carboxylase activities, and effect of sulfhydryl reagents in malic enzyme from Sulfolobus solfataricus

Malic enzyme (S)-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decarboxylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decarboxylation of L-malate. The oxaloacetate decarboxylase activity was stimulated about 50% by NADP but only in the presence of MgCl2, and was strongly inhibited by L-malate and NADPH which abolished the NADP activation. In the presence of MnCl2 and in the absence of NADP, the Michaelis constant and Vm for oxaloacetate were 1.7 mM and 2.3 mumol.min-1.mg-1, respectively. When MgCl2 replaced MnCl2, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the Km and Vm values for L-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5'-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decarboxylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both L-malate and MnCl2, and strongly enhanced by the carboxylic acid substrates; NADP + malate + MnCl2 afforded total protection. The inactivation of the oxaloacetate decarboxylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decarboxylase activity.     

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.     

Characterization and purification of a membrane-bound archaebacterial pyrophosphatase from Sulfolobus acidocaldarius.

Plasma membranes of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate-hydrolyzing activity [M. Lübben & G. Sch?fer (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 degrees 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.     

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.