Assay type 'Reactomics'

Measurements on Km, Vmax and allosteric activation or inhibition of the heterologously expressed (E. coli) and purifiied main L-lactate dehydrogenase

Artificial transcription elongation compexes are assembled in vitro using synthetic deoxy-oligonucleotides (representing template and non template DNA strands), radiolabelled RNA (representing nascent transcript) and purified RNA polymerase. After high salt wash the incorrect NTP is added and reaction is allowed to proceed for the various amounts of time. Reaction is stopped by addition of formamide-containing loading solution and the products are resolved on high-percentage denaturing polyacryamide
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Concentration of glycolytic intermediates over time

Kinetic characterisation of phosphoglycerate kinase

Kinetic characterisation of glyceraldehyde 3-phosphate dehydrogenase

Kinetic characterisation of triose phosphate isomerase

Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase

TbTryS activity was measured at 37°C in the in vivo-like buffer. All substrate stock solutions were prepared in the in vivo-like buffer and the pH was adjusted to 7.0. The assays were performed in a final volume of 2 ml and contained 0.2 mM NADH, 1 mM phosphoenolpyruvate, 4 units pyruvate kinase, 4 units L-lactate dehydrogenase, 0.17 µM TbTryS, 2.1 mM ATP and varying amounts of glutathione, and spermidine.

Comparison of Kcat values from the model and values from literature.