CLONING AND EXPRESSION OF A XENOPUS LIVER CDNA-ENCODING A FRUCTOSE-PHOSPHATE-INSENSITIVE REGULATORY PROTEIN OF GLUCOKINASE

Xenopus liver contains a protein inhibitor of glucokinase that, in con trast to the mammalian regulatory protein of glucokinase, is insensiti ve to fructose 6-phosphate and fructose l-phosphate [Vandercammen A. a nd Van Schaftingen, E. (1993) Biochem. J. 294, 551-556]. The purpose o f this work was to compare the primary structure and other properties of this Xenopus protein with those of its rat liver counterpart. A Xen opus laevis liver cDNA library was screened using the cDNA encoding th e 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 68 695 Da and 57% identity with the rat liver regulatory protein. This id entity was only about 30% in an internal region (amino acids 349-381) and in the 70 carboxy terminal-residues. The Xenopus cDNA was expresse d in Escherichia coli and the recombinant regulatory protein was purif ied 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 p rotein, both recombinant and native Xenopus regulatory proteins were i nsensitive to fructose B-phosphate, fructose l-phosphate and to physio logical concentrations of P-1, and they inhibited Xenopus glucokinase with greater affinity than rat glucokinase. These results allow one to conclude that the fructose-phosphate-insensitive protein of lower ver tebrates is homologous to the fructose-6-phosphate-sensitive and fruct ose-1-phosphate-sensitive protein found in mammals.