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[92] Triosephosphate isomerase from yeast

1975; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(75)41094-1

1557-7988

Wolfgang K.G. Krietsch,

Enzyme Structure and Function

This chapter describes the assay method, purification procedure, and properties of triosephosphate isomerase from yeast. The triosephosphate isomerase is assayed by measuring the rate of formation of dihydroxyacetone phosphate (DAP), which is converted to L-α glycerophosphate (α-GP) by the indicator enzyme L-α-glycerophosphate dehydrogenase. The rate of DAP reduction to α-GP is equivalent to the rate of NADH oxidation, which is measured by the absorbance decrease at 366 nm. The purification procedure includes the preparation of initial extract, heat treatment, ammonium sulfate fractionation, methanol treatment, protamine sulfate precipitation, DEAE-Sephadex chromatography and crystallization, and CM-Sephadex chromatography and recrystallization. The purified triosephosphate isomerase appears to be homogeneous in polyacrylamide gel eleetrophoresis and in the ultracentrifuge. In starch gel electrophoresis, the yeast enzyme is separated into three forms, the maior one of which comprises about 90% of the enzyme protein. The pure enzyme was quite stable in 2.5 M ammonium sulfate at pH 6.5 and has been kept at 0–4° for at least two years without the loss of activity. Other properties are also discussed.