Transketolase from Human Erythrocytes Purification and Properties

Human erythrocyte transketolase (sedoheptulose‐7‐phosphate: D‐glyceraldehyde‐3‐phosphate glycolaldehyde‐transferase) was purified 8200‐fold by adsorption onto hydroxylapatite, DEAE‐cellulose treatment, acetone fractionation, and chromatography on Sephadex G‐100. The purified transketolase could not be separated from glyceraldehyde‐3‐phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde‐3‐phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G‐100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde‐3‐phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose‐7‐phosphate from xylulose‐5‐phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.