The mechanism by which rat liver glucokinase is inhibited by the regulatory protein

Abstract

The fructose-6-phosphate-sensitive and fructose-I-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J . Bioclzern. 179, 179-1841 was purified close to homogeneity by a procedure involving poly(ethyleneglyco1) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer.The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecularmash inhibitor.Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1 -phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.Our laboratory has recently reported the occurrence in the liver of a regulatory protein which, in the presence of Fru6P, inhibits glucokinase competitively with respect to glucose [I]. This inhibition is antagonised by FrulP, which acts competitively with Fru6P. The properties of this protein allow one to account for the fact that fructose stimulates the phosphorylation of glucose in isolated rat hepatocytes [2].Several mechanisms could conceivably account for the action of the regulatory protein on glucokinase. The regulator could cause a covalent modification of the enzyme. It could also catalyse the formation of a low-molecular-mass inhibitor, possibly from Fru6P, since this compound greatly reinforces the inhibition exerted by the regulatory protein [I]. Finally the protein could inhibit glucokinase by forming a co...

Stimulation of Hepatocyte Glucose Metabolism by Novel Small Molecule Glucokinase Activators

“…We confirmed that fructose 1-phosphate causes a concentration-dependent dissociation of the complex (Fig. 3A), as previously reported by Vandercammen and Van Schaftingen (33). However, GKA1 (10 mol/l) did not cause dissociation of the complex (results not shown).…”

Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase

“…Rat liver regulatory protein purified to near-homogeneity [2] was submitted to two-dimensional gel electrophoresis [6]. The proteins Correspondence address: E. Van Schaftingen, UCL 75.39, Avenue Hippocrate 75, B-1200 Brussels, Belgium.…”

“…The effect of this protein is greatly reinforced by fructose 6-phosphate and antagonized by fructose l-phosphate. The regulatory protein has been purified to nearhomogeneity and identified as a 62 kDa polypeptide [2]. It inhibits glucokinase by forming an inactive complex with this enzyme (reviewed in [3]).…”

Glucokinase and candidate genes for Type 2 (non-insulin-dependent) diabetes mellitus

“…The mechanism of the fructose effect has been investigated by Dr. E. Van Schaftingen and his colleagues. In an outstanding series of papers [54][55][56][57][58][59] they show that hepatic GK is inhibited by fructose 6-phosphate (Ki 40 gmol/1 [59]), that the effect of fructose 6-phosphate is reversed by fructose 1-phosphate (K0.5 4.4 gmol/1), and that the effects of fructose phosphates require a specific regulatory protein [55]. In liver as in other tissues fructose 6-phosphate and glucose 6-phosphate are held in equilibrium by phosphoglucoisomerase.…”

“…The mechanism of action of the regulatory protein has been published [55,56], and a comprehensive list of inhibitory and stimulatory analogues acting through the regulatory protein has also been published [59].…”