Chemicals
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).
Activator preparation
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 [14] or from the absorbance at 280 nm.
Determination of enzyme activity
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 [4].
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.
Determination of molecular mass on a Sephadex G-200 column
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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