The preparation of transketolase free from D-ribulose-5-phosphate 3-epimerase.
A procedure for the purification from Candida utilis of transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) free from D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was developed using acetone precipitation, elution from DEAE-cellulose, adsorption of epimerase by thiopropyl-Sepharose, and chromatography on D-ribose 5-phosphate-Sepharose and DEAE--Sephadex. The final product had a specific activity of 43 units/mg, a transketolase/epimerase activity ratio greater than 53 000 to 1, an apparent Km for D-xylulose 5-phosphate and D-ribose 5-phosphate of 77 and 430 microM, respectively, and ran as a single band using electrophoresis on polyacrylamide gel. It was inhibited by D-arabinose 5-phosphate and D-glucose 6-phosphate. During the purification by column chromatography, multiple forms of the enzyme were detected by gel electrophoresis but these gradually disappeared as the enzyme was further purified.
D000096:Acetone; D002175:Candida / Q000201:enzymology*; D002238:Carbohydrate Epimerases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D007700:Kinetics; D010428:Pentosephosphates / Q000494:pharmacology; D012267:Ribosemonophosphates; D012274:Ribulosephosphates / Q000302:isolation & purification; D014174:Transketolase / Q000302:isolation & purification*
D005958:Glucosephosphates; D010428:Pentosephosphates; D012267:Ribosemonophosphates; D012274:Ribulosephosphates; C028055:arabinose 5-phosphate; D000096:Acetone; C031626:ribose-5-phosphate; D019298:Glucose-6-Phosphate; C031625:xylulose-5-phosphate; D014174:Transketolase; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase