BINDING OF SORBITOL 6-PHOSPHATE AND OF FRUCTOSE 1-PHOSPHATE TO THE REGULATORY PROTEIN OF LIVER GLUCOKINASE

Using a binding assay in which the ligand protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-phosphate, a negative effector, or of fructose 1-phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3-mu-M for both phosphate esters. Sorbitol 6-phosphate and fructose 1-phosphate competed with each other for the binding. Competition was also observed with psicose 1-phosphate, ribitol 5-phosphate, arabitol 5-phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10 %, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50 % inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of fructose 6-phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory phosphate esters, indicating that it induced conformational changes of the regulatory protein.