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1996 (1)
1995 (1)
1Author    Susanne Bickel-Sandkötter, Martina UferRequires cookie*
 Title    Properties of a Dissimilatory Nitrate Reductase from the Halophilic Archaeon Haloferax volcanii  
 Abstract    Grown under anaerobic conditions in presence of nitrate, Haloferax volcanii shows nitrate reduction and accumulation of nitrite in the culture medium. We found a membrane-asso-ciated nitrate reductase, which could easily be solubilized by gently stirring of isolated mem­ branes. Surprisingly, this nitrate reductase requires no NaCl for its activity. A medium pH of 7.5 and high temperatures up to 80 °C are necessary for optimum activity. Kinetic studies showed that the apparent K M was 0.36 mmol/1 for nitrate and 80 ^mol/1 for dithionite-reduced methyl viologen. The respiratory chain inhibitor cyanide effects nitrate reduction noncompe-titively with respect to nitrate with a K \ of 0.3 mmol/1. Azide was a strong inhibitor: The concentration required for half maximal inhibition was 60 |amol/l, whereas thiocyanate and chlorate were much weaker inhibitors. The isolated enzyme was partially purified by frac­ tionated precipitation using polyethylene glycol. SDS gel electrophoresis resulted in three putative subunits of the nitrate reductase of molecular masses of about 100, 61, and 31 kDa. 
  Reference    Z. Naturforsch. 50c, 365—372 (1995); received February 6/February 27 1995 
  Published    1995 
  Keywords    Archaeon, Haloferax volcanii, Dissimilatory Nitrate Reductase, Nitrate Reduction, Inhibition 
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 TEI-XML for    default:Reihe_C/50/ZNC-1995-50c-0365.pdf 
 Identifier    ZNC-1995-50c-0365 
 Volume    50 
2Author    K. Erstin Steinert, SusanneB. Ickel-S, AndkötterRequires cookie*
 Title    Isolation, Characterization, and Substrate Specificity of the Plasma Membrane ATPase of the Halophilic Archaeon H aloferax volcanii  
 Abstract    Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg2+ or Mn2+, high concentra­ tions of NaCl, a pH value of 9, and high temperatures with an optimum at 60 °C. We have not found any phosphatase activity in the preparations. We developed a purification method for the isolated enzyme with an enrichment factor o f 90. SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa. N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cysteines, inhibited the enzyme slightly. Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides. Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the Aj part o f the halophilic ATPase. The described genes are organized in an operon in the order D. C, E, B, A , named alphabetically according to their decreasing size. The deduced products o f 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification o f the ATPase. Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn2+ higher rates of ATP hydrolysis could be observed than in presence of Mg2+, but free manganese ions inhibited the enzyme activity of the ATPase. Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of M ichaelis-Menten constants for ATP in the hydrolysis direction of 1 m M in presence of MgCl2 and 0.24 m M in presence of MnCl2. Sodium chloride concentrations in the molar range induce changes in K M by a factor of about 10. The enzyme is specific for ATP; other nucleotides including GTP and A D P are competitive inhibitors o f ATP hy­ drolysis. 
  Reference    Z. Naturforsch. 51c, 29 (1996); received June 26/Novem ber 11 1995 
  Published    1996 
  Keywords    Archaeon, Haloferax volcanii, Plasma Membrane ATPase, Subunits, ATP-Hydrolysis 
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 TEI-XML for    default:Reihe_C/51/ZNC-1996-51c-0029.pdf 
 Identifier    ZNC-1996-51c-0029 
 Volume    51