Assays

Cells were grown to mid-exponential phase (OD600nm ~0.2) in GM17 medium at 37°C (with 0.15 mM ZnSO4 where relevant) and 84 ml of culture was mixed by inverting with 8.4 ml of fixing solution (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 30% (v/v) formaldehyde) and incubated at room temperature for 30 min. Cells were disrupted and crosslinked DNA was sheared by sonication. Antibodies coupled to magnetic beads were used to pull down cross-linked complexes. DNA was purified, amplified, ...

Kinetic characterisation of phosphoglycerate kinase

PGK - GAPDH models

Experimental data for the yeast PGK incubations at 30C, with and without recycling of ATP.

A model for the PGK reaction of yeast in presence or absence of the ATP recycling reactions

Changes in metabolite concentrations were either quantified via 31P NMR or enzymatically

PGK 70C model

Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.

L. lactis, S. pyogenes and E. faecalis were grown in C-limited chemostat cultures at various pH's and dilution rates. General flux distribution, yields and other physiological factors were studied.

These files show physiological measurements from the Sheffield Infors chemostat which were made during acetate calibration and also when sampling for the steady-state transcriptional profiles.

The external potassium changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal potassium changes by determining an initial concentration.

Is internal potassium affected by mutations in the Trk1,2 system? Under which conditions? Potassium measurements in wild type and TRK mutants grown and/or incubated under several external conditions.

The potassium fluxes will be estimated from the internal and external concentration changes.

How potassium starvation regulates the parameters of rubidium (potassium) transport. Analysis of transport characteristics during the starvation process. Kinetic characteristics of rubidium transport.

The potassium influx after the addition of a certain amount of KCl to a potassium free medium, followed by the injection of glucose will be measured by using the MIFE and FLISE technique. This reveals a time course of potassium. Also the external potassium concentrations will be measured.

Genes are transcribed in polysictronic messages (pre-mRNA) that are destined for either maturation into mRNAs, or degradation. Since transcription regulation is non-existent with few exceptions, the rate of pre-mRNA processing, together with mRNA decay and translation rates, are believed to control gene expression. In this assay, 2T1 blood form trypanosomes are subject to treatment by ActinomycinD for 5 minutes, inhibiting transcription. The cells are harvested, depleted for ribosomal RNA, and ...

Is protein content of trk1,2 mutants affected? Determination of proteins in wild type and TRK mutants

What are the main proteins identified? Spots sampling and identification by MS

Examples of proteomics templates for gele electrophoresis data that conform to the MIAPE-GE specification

Some examples of proteomics templates for Mass Spectrometry data that conform to the MIAPE specification

The external pH changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal pH changes by determining an initial pH. pH changes will be also determined by using green fluorescent protein dyes. Relating the proton efflux and the change of internal pH allows an estimate of the proton buffering capacity.

Related to the internal pH changes the proton efflux will be estimated from the internal and external concentration changes.

In parallel with the potassium influx the efflux of protons is monitored by measuring the external proton concentration changes with MIFE or FLISE.

Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the ...