PURIFICATION AND PROPERTIES OF UDPG DEHYDROGENASE FROM BEEF LIVER

UDPG dehydrogenase (UDPglucose:NAD oxidoreductase, EC 1.1.1.22) has been purified from fresh beef liver homogenate by extraction at pH 4.9, ammonium sulfate fractionation between 0.3-0.5 saturation, treatment at 60 °, and pH 4.9 for 1 min, refractionation with ammonium sulfate between 0.35-0.45 saturation, adsorption onto and fractional elution from calcium phosphate gel, chromatography on carboxymethyl cellulose, and finally, chromatography on Sephadex G-200. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis, immunoelectrophoresis, and agar immunodiffusion. Upon sedimentation in a sucrose density gradient there was complete coincidence between protein and enzyme activity. Ultracentrifugal sedimentation velocity analysis at pH 7.0 showed a symmetrical peak with a S20,w of 12.8; at pH 5.5 two peaks corresponding to 13 and 20.4 S20,w were present. The turnover number of the enzyme, assuming a molecular weight of 3 × 105, under optimal conditions at 30 ° was 1050 moles UDP-glucose oxidized per minute per mole enzyme. Dehydrogenase activity against dTDP-glucose and GDP-glucose is due to the same protein as that catalyzing the oxidation of UDP-glucose. No activity could be detected with UDP-galactose, UDP-mannose, or GDP-mannose. © 1969.