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M Lübben

Publications and source records attributed to M Lübben.

26 records · Page 2Linked to original sources
PubMed1989

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.

Adenosine Triphosphate↗
PubMed1988

Archaebacterial ATPase: studies on subunit composition and quaternary structure of the F1-analogous ATPase from Sulfolobus acidocaldarius.

A modified procedure for the purification of soluble ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius is described. In addition to (alpha) 65 and (beta) 51 kDa polypeptides, further subunits gamma * (20 kDa) and delta * (12 kDa) are demonstrated to be components of the enzyme, exhibiting a total molecular mass of 380 kDa. Molecular electron microscopic images of the native enzyme indicate a quaternary structure probably formed by the gamma *, delta *-complex as a central mass surrounded by a pseudohexagon of the peripherally arranged larger alpha and beta subunits. As can be derived from both molecular mass and electron microscopy data, the archaebacterial Sulfolobus-ATPase emerges to exist as an alpha 3 beta 3-quaternary structure with respect to the larger subunits. This is normally found in typical F1-ATPases of eubacterial and eukaryotic organisms. Therefore it is postulated that F1- and F0F1-ATPases, respectively, can occur ubiquitously in all urkingdoms of organisms as functional units of energy-transducing membranes.

Adenosine Triphosphatases↗
PubMed1987

The plasma membrane ATPase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Purification and immunological relationships to F1-ATPases.

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 (alpha) and 51 kDa (beta) 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 alpha and beta 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 beta 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. coli, chloroplasts and beef heart mitochondria have been tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Archaea↗
PubMed1987

A plasma-membrane associated ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

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 energy-converting 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 mumol 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 vascular ATPase inhibitor nitrate with an [I]50 of 8 mM. Linear Arrhenius plots up to 75 degrees 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 alpha and beta 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.

Adenosine Triphosphatases↗
PubMed1987

Immunoelectron microscopic demonstration of ATPase on the cytoplasmic membrane of the methanogenic bacterium strain Göl.

ATPase was shown to be present on the cytoplasmic membrane of the methanogenic bacterium strain Göl. The enzyme was identified by an immunoelectron microscopic technique by using polyclonal antiserum directed against the beta subunit of Escherichia coli F0F1-ATPase. Negatively stained membrane vesicles exhibited a dense population of stalked particles similar in dimensions and fine structure to typical F0F1-ATPase particles.

Adenosine Triphosphatases↗
PubMed1984

Azidonaphthoyl-ADP: a specific photolabel for the high-affinity nucleotide-binding sites of F1-ATPase.

3'-O-[5-azidonaphthoyl]-ADP has been synthesized as a photoreactive analog to 3'-O-naphthoyl(1)-ADP which is known to bind to the high-affinity nucleotide sites of mitochondrial F1-ATPase, considered to be the catalytic sites. The photolabel in the dark acts as a ligand to F1-ATPase and as a competitive inhibitor with Ki = 11 microM. Binding to the enzyme is accompanied by a quench of endogenous protein fluorescence leveling off at an occupancy of 1 mol/mol F1, whereas the total number of reversible sites accessible to the analog is 3 mol/mol F1 as measured by isotope studies. Covalent insertion by near ultraviolet activation of the probe yields labeling of both alpha and beta polypeptides of F1; it is accompanied by corresponding removal of reversible high-affinity sites for ADP or naphthoyl-ADP and by an inhibition of the enzyme; total inactivation occurs at a covalent occupancy of 2 mol/mol F1. This is the maximum number of sites accessible to covalent modification by the label; one reversible site is still available in the totally inactivated enzyme. This observation is discussed in terms of a stochastic model requiring a minimum of two interacting catalytic domains out of three in order to commence catalysis.

Adenosine Diphosphate↗