ASSAY, PURIFICATION, AND PROPERTIES OF BOVINE LIVER D-XYLULOKINASE

Convenient specific assay methods using commercially available materials were developed for the measurement of D-xylulokinase and D-ribulokinase activities. Using these assays, D-xylulokinase from bovine liver has been purified to homogeneity. Purification involved column chromatography on DEAE-cellulose, Sephadex G-100, Dyematrex Red A, and Superose 12. The enzyme preparation was obtained with final yields of 15-30% and had activity toward both D-xylulose and D-ribulose. The final specific activities ranged from 2.5 to 7.5 and 1.9 to 5.9 mol/min for the two substrates and K(m) values of 0.14 and 0.27 mm were obtained, respectively. For ATP, a K(m) value of 0.080 mm was obtained with either substrate. AMP, ADP, and cAMP inhibited competitively with respect to ATP; K(i) values were 0.34, 0.35, and 1.0 mm, respectively. Xylulokinase thus prepared was a monomeric protein of 51,000 Da and had a pH optimum between 7.4 and 8.2. The kinetics of the enzyme do not support any significant regulation of flux through the enzyme by metabolite level changes. (c) 1994 Academic Press, Inc.