Purification and Properties of N-Acetylglucosamine Kinase from Rat Liver

Abstract

N-Acetylglucosamine kinase that catalyzes the phosphorylation of N-acetylglucosamine to N-acetylglucosamine-6-phosphate in the presence of ATP and Mg²⁺ has been purified 110-fold from the 105, 000×g supernatant fraction of rat liver by fractionation involving ammonium sulfate precipitation and DEAE-cellulose column chromatography. The purified enzyme is highly specific for N-acetylglucosamine and differs from the enzymes of bacterial origin in that the former does not catalyze the phosphorylation of glucose as does the latter. The activity is greatest at pH 9.4. The K_m for N-acetylglucosamine is 6.0×10^-5M and the K_m for ATP is 4.8×10^-4M. The enzyme is inhibited by ADP, whose effect is partly competitive with respect to ATP and partly noncompetitive. Two rat ascites tumors, namely, Yoshida sarcoma and Yoshida ascites hepatoma (AH 130), possess N-acetylglucosamine kinase at levels comparable to those found in rat liver.