Rat hepatic glucokinase: Improved purification and some properties

It is postulated that glucokinase has a distinctly looser quaternary structure than the typical low Km animal hexokinase, and is judged to be 80% pure at this stage.

Abstract

Rat hepatic glucokinase has been purified 2940-fold by means of (NH4)SO4 fractionation, DEAE-Sephadex, DEAE-cellulose, Sephadex G-100, and elution from starch gels. The purified enzyme displays one principal band on polyacrylamide electrophoresis and is judged to be 80% pure at this stage. This is nearly four times the purity achieved previously. The Km for glucose of the purified enzyme is 12 mm. Glucose and various sulfhydryl reagents protect the enzyme against inactivation. If the enzyme is diluted in the absence of glucose of K+ ion there is an inactivation of dilution. Potassium prevents this activity loss. The hexokinases of liver which have low Km values for glucose do not lose activity under these same conditions and do not require potassium. The effect of trypsin and urea on hexokinase activity has also been studied. The molecular weight of the native glucokinase molecule has been estimated to be 68,000 by Sephadex G-100, Bio Gel P-100, and sucrose gradient-density centrifugation. Similar estimations for the low Km hexokinases yield an apparent molecular weight of 96,000. On Sephadex G-100, in the presence of KCl, glucokinase and commercial yeast hexokinase have apparent molecular weights of 48,000. It is postulated that glucokinase has a distinctly looser quaternary structure than the typical low Km animal hexokinases.