K. Uehara, S. Hosomi
1982
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Journal
Methods in Enzymology
Abstract
Publisher Summary This chapter describes the assay method of D-erythrulose reductase isolated from the beef liver. D-erythrulose reductase activity is measured by following the decrease in the absorption of nicotinamide adenine dinucleotide phosphate dehydrogenase [NAD(P)H] at 340 nm, accompanying the reduction of D-erythrulose. The reaction is initiated at 28° by the addition of enzyme. A blank without D-erythrulose must be run to correct for any nonspecific oxidation of NAD(P)H. The steps involved in the purification procedure are (1) homogenization, (2) acetone fractionation, (3) diethylaminoethyl (DEAE)-cellulose chromatography I and II, (4) hydroxyapatite chromatography I and II, and (5) crystallization. D-erythrulose reductase contains 2-3 mol of bound NADP+ per mole of enzyme, so that the extinction coefficient of the enzyme at 280 nm is not constant. Thus, the concentration of pure enzyme protein is calculated from absorbance measurement at 290 nm; at this wavelength, the contribution of NADP+ to the absorbance is negligible. The molecular weight of the enzyme is estimated by sedimentation equilibrium analysis, Sephadex Go200 gel filtration, and sucrose density gradient centrifugation. The subunit molecular weight is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.