Transketolase from yeast, rat liver, and pig liver.

Publisher Summary This chapter describes the assay methods and the purification procedure for transketolase from yeast, rat liver, and pig liver. Transketolase, an enzyme of the pentose pathway of carbohydrate metabolism, catalyzes the cleavage of the C–C bond in keto sugars and the subsequent transfer of a C–C residue to aldo sugars. Transketolase is a thiamine enzyme and belongs to the transferases. It occurs in almost all animal and plant tissues and microorganisms and localizes primarily in a soluble cell fraction. When xylulose 5-phosphate and ribose 5-phosphate are used as substrates, one of the products of the transketolase reaction is glyceraldehyde 3-phosphate and in the presence of glyceraldehyde-phosphate dehydrogenase, the latter is reduced owing to oxidation of glyceraldehyde 3-phosphate. The purification procedure involves extraction, fractionation with acetone, heating, ethanol fractionation, diethylaminoethyl (DEAE)-cellulose chromatography, ammonium sulfate fractionation, and crystallization. Transketolase is sufficiently stable and does not lose activity for several weeks on storage, and has a maximal activity at pH 7.6. Thiamine pyrophosphate and bivalent cations, such as Ca 2+ , Mg 2+ , and some others, are transketolase cofactors.

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