Robust on-line sampling and analysis during long-term perfusion cultivation of mammalian cells.

In an attempt to support robust automated sampling and analysis of mammalian cell bioreactors, an integrated platform, BaychroMA®, was developed which includes an innovative sterile sampling device, automated sample transport, a sample preparation module, online analyzers, and communication interfaces to process automation systems. The robustness of this platform was verified by applying it to a laboratory-scale perfusion bioreactor that was operated for over 100 days. Both manual and automated samples were collected over the course of the run and a comparison was made for cell density, viability, glucose, and lactate concentrations.

The thermodynamic H+/ATP ratios of the H+-ATPsynthases from chloroplasts and Escherichia coli.

The H(+)/ATP ratio is an important parameter for the energy balance of all cells and for the coupling mechanism between proton transport and ATP synthesis. A straightforward interpretation of rotational catalysis predicts that the H(+)/ATP coincides with the ratio of the c-subunits to beta-subunits, implying that, for the chloroplast and Escherichia coli ATPsynthases, numbers of 4.7 and 3.

Distances between the b-subunits in the tether domain of F(0)F(1)-ATP synthase from E. coli.

The arrangement of the b-subunits in the holo-enzyme F(0)F(1)-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate.

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis.

Binding affinities and protein ligand complex geometries of nucleotides at the F(1) part of the mitochondrial ATP synthase obtained by ligand docking calculations.

F(0)F(1) ATP synthases utilize a transmembrane electrochemical potential difference to synthesize ATP from ADP and phosphate. In this work, the binding modes of ADP, ATP and ATP analogues to the catalytic sites of the F(1) part of the mitochondrial ATP synthase were investigated with ligand docking calculations. Binding geometries of ATP and ADP at the three catalytic sites agree with X-ray crystal data; their binding free energies suggest an assignment to the 'tight', 'open' and 'loose' states.