Characterization and purification of the membrane-bound ATPase of the archaebacterium Methanosarcina barkeri

Abstract

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 HS03-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 (a) and 49,000 (Ii) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the FoF, type ATPase found in many eubacteria.Methanogenic bacteria are strictly anaerobic organisms, and most species are able to grow autotrophically with H2 and CO2 as substrates for methanogenesis. On the basis of studies of the 16S RNA sequence, they are considered to belong to the archaebacteria (1). Other properties of methanogens are also different from those of the eubacteria; the membranes of methanogens contain ether-linked polyisoprenoid glycerol lipids (30) and their cell walls lack peptidoglycan (14). Apart from methanogens, the archaebacteria include extreme halophiles and thermoacidophiles.In the chemiosmotic mechanism of Mitchell (20), a proton motive force established across the cell membrane is a driving force for ATP synthesis. An H+-translocating ATPase (FoF1 ATPase) which catalyzes ATP synthesis has been observed in membranes of both eucaryotes and eubacteria (26). Although membrane-bound ATPase activities were found in membranes of thermoacidophilic archaebacteria, Thermoplasma (28), and Sulfolobus (32) Solubilization and purification of the ATPase. The membrane fraction was suspended in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. After incubation at 8°C for 1 h with gentle stirring, the suspension was centrifuged at 100,000 x g for 90 min. The supernatant (42 ml, 2.4 mg of protein per ml) was applied to a DEAE-Sepharose column (2.6 by 20 cm) equilibrated with 50 mM Tris-maleate (pH 6.9) (column buffer). After the column had been washed with one column volume of the column buffer, a linear gradient from 0 to 0.6 M NaCl in 50 mM Tris-maleate buffer (pH 6.9) was run at 8°C. The fractions containing ATPase activity were pooled (15 ml), and solid ammonium sulfate was added up to reach 65% saturation. The precipitate obtained after centrifugation at 10,000 x g for 20 min was dissolved in a minimal volume of 50 mM Tris-maleate buffer (pH 6.9), and the solution was centrifuged to remove any insoluble material. The supernatant was then applied to a Sephacryl S-300 column (1.6 by 80 cm) equilibrated with the column buffer. The flow rate was 10 ml/min at 8°C, and the fractions with ATPase activity were collec...

Proton translocation coupled to methanogenesis from methanol + hydrogen in Methanosarcina barkeri

“…The presence of this enzyme has been indirectly demonstrated in whole cells of M. barkeri [6,12]. Very recently, Inatomi [13] purified the soluble part of an ATPase from M. barkeri which displays similarities with the F~ part from eubacterial sources: when this Fl-like protein is stripped off the membrane it becomes DCCDinsensitive. However, as long as it is attached to the membrane (probably to the integral F0 part) the ATPase activity is inhibited by DCCD [13].…”

“…Very recently, Inatomi [13] purified the soluble part of an ATPase from M. barkeri which displays similarities with the F~ part from eubacterial sources: when this Fl-like protein is stripped off the membrane it becomes DCCDinsensitive. However, as long as it is attached to the membrane (probably to the integral F0 part) the ATPase activity is inhibited by DCCD [13]. If this enzyme were involved in proton translocation one would expect DCCD to inhibit the acidification usually observed upon methanol addition.…”

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

“…For protein gel blotting, we used antibodies for two members of PSI complexes (PsaA and PsaB), three members of PSII complexes (PsbB, PsbD, and OEC33;Jain et al, 1998;Jeong et al, 2003), the photosynthetic and respiratory ATP synthase (AtpB; Inatomi, 1986), RbcS (Krebbers et al, 1988), and Lhcb3 (Jackowski et al, 2001). Our data showed that PsaB, PsbD, RbcS, and Lhcb3 proteins were undetectable in the ptac14-1 mutant, while the levels of PsaA, PsbB, and OEC33 proteins were significantly decreased in the ptac14-1 mutant compared with the wild-type plant, and the accumulation of AtpB was less affected in ptac14-1.…”

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

“…They are responsible for vacuolar acidification, which plays an important role in a number of cellular processes (1). V o V 1 -ATPases are also found in the plasma membranes of most archea (7)(8)(9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15).…”

“…V o V 1 -ATPases are also found in the plasma membranes of most archea (7-9) and some kinds of eubacteria (10 -12). Several studies indicate that the physiological role of V o V 1 -ATPases of some archea and the thermophilic eubacterium Thermus thermophilus is ATP synthesis coupled to proton flux across the plasma membranes (7,9,(13)(14)(15). Precise understanding of V o V 1 -ATPases would allow the comparison to F o F 1 -ATPases and the elucidation of the common essential mechanism for the coupling of proton translocation across a membrane with ATP formation.…”