Intracellular binding of glucokinase in hepatocytes and translocation by glucose, fructose and insulin.
The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocytes have been preincubated. In hepatocytes pre-cultured with 5 mM glucose the release of glucokinase was much slower than that of other cytoplasmic enzymes measured. However, preincubation with glucose (10-30 mM) or fructose (50 microM-1 mM) markedly increased glucokinase release. This suggests that, in cells maintained in 5 mM glucose, glucokinase is present predominantly in a bound state and this binding 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 microM). The effects of glucose and fructose were rapid (t1/2 5 min) and reversible, and were potentiated by insulin and counteracted by glucagon. They were inhibited by cyanide, but not by cytochalasin D, phalloidin or colchicine. Mannose had a glucose-like effect (A50 approximately 15 mM), whereas galactose, 3-O-methyl-D-glucose and 2-deoxyglucose were ineffective. When hepatocytes were incubated with [2-3H, U-14C]glucose, the incorporation of 3H/14C label into glycogen correlated with the extent of glucokinase release. Since 2-3H 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 partitioning of glucose 6-phosphate towards glycogen, as opposed to phosphoglucoisomerase.
Agius
L
L
Department of Medicine, University of Newcastle upon Tyne, U.K.
Peak
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology*; D004795:Enzyme Stability; D005632:Fructose / Q000494:pharmacology*; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D010590:Phalloidine / Q000494:pharmacology; D010733:Phosphoglucomutase / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D000199:Actins; D007328:Insulin; D010590:Phalloidine; D015638:Cytochalasin D; D005632:Fructose; D011189:Potassium Chloride; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005941:Glucokinase; D010733:Phosphoglucomutase; D008274:Magnesium; D005947:Glucose; D004072:Digitonin