Purification and properties of transketolase from pig liver. I. An attempt to resolve the enzyme into apoenzyme and cofactors.

Transketolase, sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase [EC 2.2.1.1], was extracted from pig liver and purified 96-fold by ammonium sulfate fractionation, followed by column chromatography using DEAE-cellulose and a Sephadex G-200. Transketolase from pig liver was stable at pH 6.0 and above, whereas it was unstable at lower pH values. It could be resolved into apoenzyme and thiamine pyrophosphate in an acidic medium, in contrast to baker's or brewer's yeast transketolase which resolved in an alkaline solution. All the activity of pig liver transketolase was lost upon incubation at pH 5.0 for two hours even at 0 degrees C but about 40% of the original activity could be restored by the addition of excess thiamine pyrophosphate and CaCl2. Restoration of the activity was achieved effectively at pH 7.6-8.0.