Quantitative determination of triosephosphates during enzymatic reaction by high performance liquid chromatography: effect of isomerase on aldolase activity.
Fructose-1,6-bisphosphate and triosephosphates have been separated by high performance liquid chromatography utilizing a SynChropack AX anion exchange column with 50-200 mM KH2PO4, pH 2.5-4.6 as mobile phase. The best resolution for each compound was reached in a system of 150 mM KH2PO4, pH 2.5. If radioactive fructose-1,6-bisphosphate as initial substrate was enzymatically converted in triosephosphates, the recoveries of metabolites after the precipitation and chromatographic procedures were higher than 95%. The concentration of radioactive 3-phosphoglycerate measured by liquid scintillation shows a good correlation (correlation coefficient: 0.997) with the spectrophotometrically determined concentration of NADH, which is formed from [U-14C]fructose-1,6-bisphosphate in equimolar concentration with 3-phosphoglycerate in aldolase and glyceraldehyde-3-phosphate dehydrogenase system. The method developed was applied to detect the inhibitory effect of triosephosphate isomerase on aldolase activity which takes place due to the heterologous complex formation.
D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000032:analysis; D014305:Triose-Phosphate Isomerase / Q000378:metabolism*
D009243:NAD; D005634:Fructose-Bisphosphate Aldolase; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase