Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites.
Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.
Agius
L
L
Department of Diabetes and Metabolism, The Medical School, The University of Newcastle upon Tyne, Framlingham Place, Newcastle upon Tyne, NE2 4HH, UK. loranne.agius@nc.l.ac.uk
Stubbs
M
M
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002478:Cells, Cultured; D005941:Glucokinase / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D005941:Glucokinase; D005947:Glucose