INTRACELLULAR BINDING OF GLUCOKINASE IN HEPATOCYTES AND TRANSLOCATIONBY GLUCOSE, FRUCTOSE AND INSULIN

The release of glucokinase from digitonin-permeabilized hepatocytes sh ows different characteristics with respect to ionic strength and [MgCl 2] from the release of other cytoplasmic enzymes. Release of glucokina se is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) an d is inhibited by increasing concentration of KCl [concn. giving half- maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosp hoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydroge nase is independent of ionic strength, but shows a small inhibition by MgCl2 (20 %, versus > 80 % for glucokinase). Lactate dehydrogenase re lease increases with increasing ionic strength [concn. giving half-max imal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of gl ucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl 2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with wh ich the hepatocytes have been preincubated. In hepatocytes pre-culture d with 5 mM glucose the release of glucokinase was much slower than th at of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 muM-1 mM) markedly increased glucok inase release. This suggests that, in cells maintained in 5 mM glucose , glucokinase is present predominantly in a bound state and this bindi ng is dependent on the presence of Mg2+. The enzyme can be released or translocated from its bound state by an increase in [glucose] (A50 15 mM) or by fructose (A50 50 muM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insuli n and counteracted by glucagon. They were inhibited by cyanide, but no t by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-l ike effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D- glucose and 2-deoxyglucose were ineffective. When hepatocytes were inc ubated with [2-H-3,U-C-14]glucose, the incorporation of H-3/C-14 label into glycogen correlated with the extent of glucokinase release. Sinc e 2-H-3 is lost during conversion of glucose 6-phosphate into fructose 6-phosphate, substrate-induced translocation of glucokinase from a Mg 2+-dependent binding site to an alternative site might favour the part itioning of glucose 6-phosphate towards glycogen, as opposed to phosph oglucoisomerase.