NADP+-dependent D-xylose dehydrogenase from pig liver. Purification and properties

Abstract

An NADP+-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An M(r) value of 62000 was obtained by gel filtration. PAGE in the presence of SDS gave an M(r) value of 32000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio k(cat.)/K(m(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+;NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. K(m) values for D-xylose and NADP+ were 8.8 mM and 0.09 mM res