Glucokinase and Hexokinase in Liver in Relation to Glycogen Synthesis

Abstract

The first step in the pathway from glucose to glycogen has not yet been satisfactorily accounted for. The enzyme responsible for the phosphorylation of glucose in liver has not been clearly identified. Qualitatively, Slein, Cori, and Cori (1) postulated a “glucokinase,” but Crane and Sols (2) reported the identification of a hexokinase that apparently had a Michaelis constant (K,,,) for glucose not as low as those of the hexokinases of other tissues. Quantitatively, the finding by Long (3), in a survey of glucose phosphorylation rates by homogenates of rat organs, that liver was the least active of all has been a long standing puzzle. An important advance was made recently by DiPietro, Sharma, and Weinhouse (4, 5) when they found that at a high glucose concentration, the glucose phosphorylation rate of liver extracts approaches that required to account for glycogen synthesis from glucose in normal fed rats. Nevertheless, a major difficulty for the involvement of hexokinase as the first step in the pathway of glucose to glycogen in liver stems from the finding by Leloir et al. (6) that the liver UDP-glucose-glycogen glucosyltransferase (glycogen synthetase) is strongly dependent for activity on a rather high concentration of glucose-&P, a strong inhibitor of animal tissue hexokinases (2). Because of these opposing effects of glucose-6-P, hexokinase and glycogen synthetase would not be able to work efficiently in sequence. Figueroa, Pfeifer, and Niemeyer (7) have recently raised doubts as to whether glucose6-P is a necessary step in the synthesis of glycogen from glucose in liver. We report here the identification in rat liver of two enzymes that can phosphorylate glucose. One of them seems to fulfill the requirements for the first and limiting step in the synthesis of glycogen from glucose in liver. Fresh liver homogenates from normal white rats fed ad Zibitum and killed early in the morning were prepared and assayed as described in the legend to Table I. The effect of the concentration of glucose on its rate of phosphorylation, when the glucose present in the homogenate (measured with glucose oxidase) was taken into account, suggested the occurrence of one enzyme with high affinity for glucose (as reported by Lange and Kohn (8)) and another with low affinity (as found by DiPietro, Sharma, and Weinhouse (5)). 1,5-Anhydroglucitol-B-P, a nonmetabolizable analogue of glucose-6-P which, like the latter, strongly inhibits animal tissue hexokinases noncompetitively with glucose (9) but competitively with ATP-Mg (lO),l could fully inhibit