The Preparation and Characterization of Pure Rat Liver Glucokinase

Glucokinase from rat liver has been purified over 10,000-fold and appears homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. The enzymatically active protein represents 0.007 % of the total liver protein and possesses a specific activity of 80 units per mg for purified enzyme. The use of potassium phosphate buffer gradients greatly enhances enzyme resolution during ion-exchange chromatographic procedures. In addition, use of substrate and sulfhydryl protecting reagents in all buffers prevents significant losses of activity. To our knowledge this is the highest degree of purity ever achieved for a mammalian hepatic ATP:d-hexose-6-phosphotransferase. Rat liver glucokinase has been characterized with respect to stability, Km, Vmax, and phosphorylation coefficient toward several substrates as well as behavior in the presence of several inhibitors. Spectral analysis fails to reveal the presence of a prosthetic group. The molecular weight determined by sodium dodecyl sulfate acrylamide gel electrophoresis and Sephadex G-100 gel filtration is 53,000 and 57,000, respectively. The turnover number is calculated to be 4,346 moles of d-glucose phosphorylated by ATP per min per mole of enzyme at 25°.