Cloning and Expression of a Xenopus Liver cDNA Encoding a Fructose‐Phosphate‐Insensitive Regulatory Protein of Glucokinase

Abstract

Xenopus liver contains a protein inhibitor of glucokinase that, in contrast to the mammalian regulatory protein of glucokinase, is insensitive to fructose 6-phosphate and fructose 1 -phosphate [Vandercammen A. & Van Schaftingen, E. (1993) Biochem. J. 294, 551 -5561. The purpose of this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xenopus laevis liver cDNA library was screened using the cDNA encoding the rat liver regulatory protein as a probe. The cloned cDNA was 2534 bp long and encoded a 619-amino-acid protein with a molecular mass of 68695 Da and 57% identity with the rat liver regulatory protein. This identity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expressed in Escherichia coli and the recombinant regulatory protein was purified to near homogeneity and found to have the same size, reactivity to antibodies and effects on the kinetics of glucokinase as the protein purified from Xenopus liver. In contrast to the rat liver regulatory protein, both recombinant and native Xenopus regulatory proteins were insensitive to fructose 6-phosphate, fructose 1-phosphate and to physiological concentrations of Pi, and they inhibited Xenopus glucolunase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower vertebrates is homologous to the fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein found in mammals.The liver of the rat contains a regulatory protein, also called regulator, which inhibits glucokinase competitively with respect to glucose . Fructose 6-phosphate and fructose 1-phosphate are ligands of this regulatory protein and act competitively, fructose 6-phosphate to reinforce the inhibition, and fructose 1-phosphate to antagonize it. In addition, C1-and other monovalent anions reduce the inhibition exerted by the regulatory protein noncompetitively with respect to fructose 6-phosphate. The presence of the regulatory protein in rat liver explains the activating effect of fructose (Van Schaftingen and Vandercammen, 1989;Davies et al., 1990) and of a potassium-rich medium on the phosphorylation of glucose in isolated hepatocytes. The cDNA encoding the rat liver regulatory protein has been recently cloned (Detheux et al., 1993).A fructose-6-phosphate-sensitive and fructose-l-phosphate-sensitive regulatory protein appears to be present in the livers of all mammalian species that express hepatic glucokinase, including man (Vandercammen and Van Schaftingen, 1993). The livers of the amphibians Bufo marinus and Xenopus laevis, and of the turtle Pseudemys scripta elegans con- tain a protein that inhibits glucokinase competitively with respect to glucose but is insensitive to fructose 6-phosphate and fructose 1-phosphate (Vandercammen and Van Schaftingen, 1993). It was of interest to know if this form of regulatory protein of glucokinase was homologous to ...

Identification of Fructose 6-Phosphate- and Fructose 1-Phosphate-binding Residues in the Regulatory Protein of Glucokinase

“…The cultures were incubated in an orbital shaker at 37°C until A 600 ϭ 0.6 and then chilled on ice before addition of the inducer isopropyl-1-thio-␤-D-galactopyranoside (0.4 mM). After a further incubation of 60 h at 18°C in an orbital shaker, the bacteria were isolated by centrifugation, and extracts (50 ml) were prepared (27). For the purification of recombinant GKRPs, polyethylene glycol 6000 was added to a final concentration of 22%.…”

“…The first one is that GKRP found in lower vertebrates, unlike its mammalian counterpart, is insensitive to Fru-6-P and Fru-1-P, inhibiting the enzyme even in the absence of these phosphate esters (26). The cDNA encoding Xenopus GKRP has been cloned and shown to encode a protein with 58% identity to rat liver GKRP (27). A comparison of the sequences of these two proteins may therefore allow identification of residues that potentially interact with Fru-6-P and Fru-1-P.…”

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

“…There was no effect of tagatose on glucose phosphorylation in MIN6 cells (Table 2) or on the rate of glucokinase release during permeabilization with digitonin (data not shown). Another possibility is that ␤-cells contain a protein that shares some similarities with rat liver regulatory protein without being responsive to fructose 1-phosphate, as is the case for Xenopus liver regulatory protein, which lacks a fructose 1-phosphate binding site (30). However, there was no effect of replacement of extracellular Na + by K + (Table 2), which in hepatocytes increases the affinity for glucose by dissociation of glucokinase from the regulatory protein (10).…”

Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase

“…Culture was continued for another 18 h at 18 mC. Cells were harvested by centrifugation (12 000 g, 30 min, 4 mC) and extracted as described in [24]. The recombinant enzyme was purified by chromatography on DEAE-Sepharose and AMP-Sepharose, as described for the native liver enzyme.…”