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Sephadex G-200 was supplied by Amersham Biosciences (Little Chalfont, UK). Diethyl ether and all other solvents (HPLC grade) were from Biosolve (Valkenswaard, The Netherlands). All other reagents were from Sigma-Aldrich (Bornem, Belgium) or Merck KGaA (Darmstadt, Germany). All solutions were prepared with milli-Q water (Millipore S.A./N.V., Brussels, Belgium).
E. coli (strain BL21) were grown overnight (37°C, 250 rpm) in Luria-Bertani (LB) medium (tryptone, 10 g/l; yeast extract, 5 g/l; NaCl, 10 g/l, pH 7.0), collected by centrifugation (10 min, 10 000 × g), and suspended in half the initial volume of M9 minimum medium (Na2HPO4, 6 g/l; KH2PO4, 3 g/l; NaCl, 0.5 g/l; NH4Cl, 1 g/l; CaCl2, 3 mg/l; MgSO4, 1 mM, pH 7.0) containing 10 mM glucose. The culture was incubated for 40 min (37°C, 250 rpm) and centrifuged (10 min, 10 000 × g). The pellet was suspended in 50 mM Tris-HCl buffer (1/33 of the volume of M9 culture), pH 7.4, containing 0.2 mM EDTA, 0.1 M KCl, and frozen at -20°C. Then the cells were thawed, disrupted by sonication (100 kHz, 3 × 1 min) on ice and centrifuged for 30 min at 15 000 × g. The supernatant was boiled for 3 min, put on ice, the precipitate was sedimented (5 min, 15 000 × g), and the final supernatant was used as an activator for enzyme assays. Protein concentrations were measured by the method of Bradford  or from the absorbance at 280 nm.
The standard incubation mixture for AThTP synthesis contained 50 mM sodium maleate, pH 6.5, 1 mM ADP, 0.1 mM ThDP, 10 mM MgSO4, and aliquots of enzyme preparation and 10 μl activator in a final volume of 0.1 ml. Any changes in the protocol are indicated in the legends to the figures. The reaction was carried out at 37°C for 1 h and stopped by addition of 0.5 ml of 12% TCA followed by extraction of the acid with 3 × 1.5 ml of diethyl ether. AThTP was quantified using a HPLC method as previously described [4,15]. Briefly, a 40-μl aliquot of sample was oxidized with 25 μl of 4.3 mM potassium ferricyanide in 15% NaOH and injected into the HPLC system (System 522, Kontron Instruments, Milan, Italy) equipped with a PRP-1 column (Ø 4.1 × 150 mm, Hamilton Co., Reno, NV, USA) protected by a guard column (Hamilton) and a SFM 25 spectrofluorimeter (Kontron Instruments). The separation was performed at a flow rate of 0.5 ml . min-1 in a mobile phase containing 50 mM NaH2PO4, pH 9.5, 25 mM tetra-n-butylammonium hydrogen sulfate and 4% tetrahydrofuran. The peak areas were calculated using the KromaSystem 2000 software (Bio-Tek Kontron Instruments) and compared to the area of a standard solution of chemically synthesized AThTP .
Adenine nucleotides were monitored by UV detection (254 nm) after separation on a 5 μm Chromspher C18 column (150 × 4.6 mm, Varian D.V., Middelburg, The Netherlands). The mobile phase was composed of 25 mM tetra-n-butylammonium hydrogen sulfate, 50 mM NaH2PO4 adjusted to pH 7.0 and 15 % methanol. The flow rate was 1 ml/min.
Data analysis was performed using GraphPad Prism version 4.00 for Macintosh, GraphPad Software, San Diego California USA.
The proteins were separated at 4°C on a Sephadex G-200 column (Ø 2.4 × 65 cm) equilibrated with 20 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl, at a flow rate of 5 cm . hr-1. The column was calibrated with the following standard proteins: apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). The elution volume (Ve) of AThTP-synthesizing enzyme was estimated from its activity and its molecular mass was calculated from the plot of logMr versus logVe/V0.
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