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 Km values of 0.14 and 0.27 mM were obtained, respectively. For ATP, a Km value of 0.080 mM was obtained with either substrate. AMP, ADP, and cAMP inhibited competitively with respect to ATP; Ki 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.
Dills
W L
WL
Department of Chemistry, University of Massachusetts, Dartmouth 02747.
Parsons
P D
PD
Westgate
C L
CL
Komplin
N J
NJ
D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
D017853:Phosphotransferases (Alcohol Group Acceptor); C022550:ribulokinase; C032700:xylulokinase