0-6.0), phosphate (pH 6.0-7.0), Tris–HCl (pH 7.0-9.0), and glycine-NaOH (pH 9.0-10.0) under standard conditions. The pH was adjusted at 50°C. Formation of the transketolase apoform and reconstitution of the holoenzyme Apo-transketolase was obtained by removing the

cofactors THDP and divalent cation through dialysis for 24 hours against Tris–HCl buffer pH 7.5 containing 10 mM EDTA. After removing EDTA NCT-501 price by dialysis, different divalent cations were tested as possible cofactors in the transketolase reaction using Assay I and 1.25 mM X5-P and R5-P, respectively. The effect of metal ions and EDTA, ATP or ADP on TKT activity was measured under standard conditions (Assay I) in the presence of Ca2+, Co2+, Cu2+, Mg2, Mn2+ and Ni2+ at 1 mM final concentration in the reaction mixture. The remaining percentage activities were determined by comparison with no metal ion added. To investigate the effect Trichostatin A cost of EDTA, EDTA salt solution was incubated with TKT for 4 minutes. The measurement was done according to standard assay conditions with 1 mM EDTA final concentration in 1 ml reaction mixture. To study the thermal stability of the TKT proteins, the assay mixture described above was prepared in 1.5 ml reaction tubes and incubated for up to 2 h at 30-80°C. Samples were taken periodically and the residual enzyme activity was measured under standard conditions (Assay

I) in a separate reaction mixture. The TKT activity in the direction of E4-P and X5-P from F6-P + GAP was done by Assay II, a modified version of a previously described assay [31] using the auxiliary enzymes Erythrose-4-phosphate dehydrogenase (E4PDH) from E. coli to detect E4-P from the conversion of F6-P and GAP. The oxidation of NADH was followed setting 1 mmol NADH oxidized equivalent to 1 mmol X5-P consumed. The standard reaction mixture (final volume 1 ml) contained 50 mM Tris–HCl buffer (pH 7.5), 0.25 mM NAD+, 2 mM Mn2Cl, 1 mM dithiothreitol (DTT) 2 U/ml E4PDH Rucaparib manufacturer and purified TKT protein which was preheated for 3 min at 55°C. NAD+ oxidation (ϵ340nm = 6.22 mM–1 cm–1) was followed at 340 nm on a Shimadzu UV1700 spectrophotometer.

The reaction was initiated by the addition of GAP or R5-P respectively (final concentration varied between 0.05 – 10 mM). Hydroxypyruvate (HP) activity (Assay III) was measured by recording the oxidation rate of the α-carbanion intermediate in the presence of IWR-1 mw ferricyanide according to the method of Joshi and coworkers (2008) [32]. The reaction mixture in 1.0 ml contained 50 mM glycyl-glycine buffer (pH 7.6), 2 mM manganese chloride, 0.2 mM THDP, 0.5 mM potassium ferricyanide, 3 mM F6-P/HP and 0.24 mg enzyme protein. The reaction was initiated by the addition of enzyme and the reduction of ferricyanide was monitored at 420 nm using UV-1700 PC spectrophotometer (Shimadzu, Japan). DHAS activity was assayed (Assay IV), depending on the purpose of the experiment, by one of three methods described previously [23, 27], with several modifications.