Subcellular distribution of hexokinase isoenzymes in pancreatic islet cells exposed to digitonin after incubation at a low or high concentration of D-glucose.
It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in the two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein.
Vanhoutte
C
C
Laboratory of Experimental Medicine, Brussels Free University, Belgium.
Sener
A
A
Malaisse
W J
WJ
D000818:Animals; D002852:Chromatography, Ion Exchange; D004072:Digitonin / Q000494:pharmacology; D005260:Female; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D006593:Hexokinase / Q000378:metabolism*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats
D007527:Isoenzymes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose; D004072:Digitonin