Biomedical subjects

G Grüber

Publications and source records attributed to G Grüber.

22 records · Page 2Linked to original sources

PubMed1996

Differentiation of catalytic sites on Escherichia coli F1ATPase by laser photoactivated labeling with [3H]-2-Azido-ATP using the mutant beta Glu381Cys:epsilonSer108Cys to identify different beta subunits by their interactions with gamma and epsilon subunits.

The ATP binding affinities of the catalytic sites in the three beta subunits of the Escherichia coli F1 ATPase (ECF1) have been explored in relation to the interaction of these subunits with the small subunits gamma and epsilon. ECF1 from the mutant beta E381C:epsilonS108C was reacted with different concentrations of [3H]-2-azido-ATP and covalent insertion of the nucleotide analogue induced by photoactivation of the azide group to a nitrene with single-pulse UV laser excitation. The enzyme showed cooperative binding of [3H]-2-azido-ATP in the presence of Mg2+. The highest affinity site was located at betafree, the one of the three beta subunits in the mutant that does not form disulfide bonds with either the gamma or the epsilon subunit. This beta subunit is, therefore, the site of unisite catalysis in the enzyme. The second mole of [3H]-2-azido-ATP to bind was located in the beta subunit that links to epsilon (betaepsilon), while the lowest affinity binding of the substrate analogue was with the beta subunit that links to gamma (betagamma). In the absence of Mg2+, all three beta subunits bound [3H]-2-azido-ATP with a similar, low affinity. The results show that binding of MgATP is determined by, and/or must determine, the interactions of the different alpha-beta subunit pairs with the single-copy subunits gamma, delta, and epsilon of the enzyme.

Adenosine Triphosphate↗

PubMed1996

Structural changes in the gamma and epsilon subunits of the Escherichia coli F1F0-type ATPase during energy coupling.

Structural changes in the Escherichia coli ATP synthase (ECF1F0) occur as part of catalysis, cooperativity and energy coupling within the complex. The gamma and epsilon subunits, two major components of the stalk that links the F1 and F0 parts, are intimately involved in conformational coupling that links catalytic site events in the F1 part with proton pumping through the membrane embedded F0 section. Movements of the gamma subunit have been observed by electron microscopy, and by cross-linking and fluorescence studies in which reagents are bound to Cys residues introduced at selected sites by mutagenesis. Conformational changes and shifts of the epsilon subunit related to changes in nucleotide occupancy sites have been followed by similar approaches.

Energy Metabolism↗

PubMed1994

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP-synthase. After treatment with 1.5 M LiCl the ATP-synthase dissociated, and subsequently subunit delta (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP-synthase lacking delta by addition of delta showed non-competitive kinetics with a Ki of approximately 5.9 nM. Subunit epsilon from chloroplast F1, which corresponds functionally to the M. luteus F0F1-delta, and chloroplast delta were tested for ATPase inhibitory activity by addition to the partially delta-depleted ATP-synthase from M. luteus. CF1-epsilon inhibited M. luteus ATP-synthase up to 80%, whereas CF1-delta did not show any influence.

Adenosine Triphosphate↗

PubMed1994

ATP synthesis and hydrolysis of the ATP-synthase from Micrococcus luteus regulated by an inhibitor subunit and membrane energization.

After incubation for 70 min in Tris-HCl (pH 8.0), the rate of ATP hydrolysis of free and reconstituted ATP-synthase from Micrococcus luteus multiplied about three times. The apparent increase in activity is due to the reversible dissociation of the delta-subunit. Results of experiments on the temperature dependence of the ATP hydrolysis rate of substrate saturated ATP-synthase exhibited a discontinuity in the Arrhenius plot at 32 +/- 0.5 degrees C for the delta-subunit associated enzyme. Below 32 +/- 0.5 degrees C the activation energy, Ea, was 231.5 +/- 5 kJ mol-1, while above this temperature-level it decreased to 76.4 +/- 3 kJ mol-1. ATP synthesis and hydrolysis of the ATP-synthase, co-reconstituted with monomeric bacteriorhodopsin (Halobacterium halobium), showed a lag of 50 s upon the illumination with green light (505-575 nm). This retardation and the activity depended on the ATP-synthase concentration, being typical of the dissociation of an inhibitor protein. The N-terminal protein sequences of the delta- and epsilon-subunit of the ATP-synthase were identified by automated Edman degradation. Alignment of the amino acid sequence and secondary structure calculations for the delta-subunit did not reveal homology to other known ATP-synthase delta-subunits, but significant equivalence to the epsilon-subunit of E. coli. Sequence analysis of the epsilon-subunit from M. luteus showed homology to equivalent regions in delta-subunits and Oligomycin Sensitivity Conferring Protein (OSCP) of other organisms.

Adenosine Triphosphate↗