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

Abstract

cDNAs encoding the rat liver regulatory protein of glucokinase were cloned and sequenced. The deduced protein contains 568 amino acids for a molecular mass of 62,867 Da. Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver. No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA. Recombinant regulatory protein expressed in Escherichia~coli was insoluble and inactive, and was presumably contained in inclusion bodies. Western blot analysis showed that the recombinant protein was recognized by antibodies raised against regulatory protein puri8ed from rat liver.

Evolution of glucose utilization: Glucokinase and glucokinase regulator protein

“…Levels of fructose-6-phosphate, which change in parallel with blood glucose levels, regulate the binding of glucokinase with GCKR, allowing glucokinase to be active only when blood glucose levels are elevated (Agius and Peak, 1997;van Schaftingen et al, 1997). GCKR is only abundantly expressed in the liver, and not appreciably in the other sites of glucokinase expression (Detheux et al, 1993;Vandercammen and Van Schaftingen, 1993). The tissue-specific expression of GCKR in the liver therefore permits glucokinase to be post-transcriptionally regulated only in the liver, the one tissue where activity must be regulated to prevent function when blood glucose levels are low.…”

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

“…ml-I heat-denatured herring sperm DNA, 1 mM EDTA and 1OmM sodium phosphate, pH7.1. The probe used for the hybridisation corresponded to nucleotides 1 -1795 of the cDNA encoding rat liver regulatory protein (Detheux et al, 1993). This cDNA was excised from the vector by digestion with EcoRI and PstI, purified and labelled with [ Q -~~P ]~C T P by random priming (Feinberg and Vogelstein, 1983).…”

“…Identical residues are represented by dashes (-) in the rat sequence; the asterisc in the Xenopus sequence (*) represents a one-residue gap. The rat sequence is taken from (Detheux et al, 1993. 100 pM sorbitol 6-phosphate or 200 pM 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).…”

“…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).…”