A coding-length clone of rat liver fructose-1,6-bisphosphatase (EC 3.1.3.11) was isolated by immunological screening of a cDNA library in Agtll. Its identity was verified by comparing the deduced amino acid sequence with that obtained by direct sequencing of a complete set of CNBr and proteolytic peptides from the purified protein. The enzyme subunit is composed of 362 amino acids and has N-acetylvaline as the amino-terminal residue. The cDNA, 1255 base pairs (bp) long, consisted of 1086 bp of coding region, 15 bp of 5' untranslated sequence, and 154 bp at the 3' untranslated end. The 3' untranslated sequence contained a polyadenylylation signal (AATAAA) followed after 30 bp by a stretch of 7 adenines at the end of the clone. The deduced amino acid sequence was identical to the primary sequence of the protein and confirmed the alignment of five nonoverlapping peptides. It also confirmed the 27-residue extension, unique to the rat liver subunit, ending with a carboxyl-terminal phenylalanine. RNA blot analyses using the radiolabeled liver cDNA as a probe revealed a single band of fructose-1,6-bisphosphatase mRNA, 1.4 kilobases long, in liver and kidney but not in nongluconeogenic tissues. Fructose-1,6-bisphosphatase mRNA was increased 10-fold in livers from diabetic rats and was reduced to control levels after 24 hr of insulin treatment, suggesting that the changes in enzyme activity observed in diabetes and after insulin treatment are due to alterations in mRNA abundance.The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate, catalyzed by fructose-1,6-bisphosphatase (Fru-1,6-P2ase; EC 3.1.3.11), is an essential step in the gluconeogenic pathway (1) and is under both acute and long-term hormonal control. Acute hormonal regulation occurs primarily by modulation of the potent inhibitor fructose 2,6-bisphosphate (Fru-2,6-P2) (2, 3), which has been postulated to bind either to the active site (2, 4) or to a separate allosteric site (3, 5). Most of the studies on allosteric regulation of Fru-1,6-P2ase by Fru-2,6-P2 have been carried out on the rat liver enzyme, but identification ofthe Fru -2,6-P2 binding site has been hampered both by lack of primary sequence and x-ray crystallographic information and by lack of a cDNA clone with which to do site-directed mutagenesis studies. Yeast (6) and rat liver (7) Fru-1,6-P2ase have also been reported to be regulated by cAMP-dependent phosphorylation. Phosphorylation of the rat liver enzyme at a site(s) located on a carboxyl-terminal extension unique to this form (8, 9)t has been reported to increase enzyme activity (7, 10), but the changes have not been universally observed (8). Phosphorylation of the yeast enzyme, on the other hand, at an amino-terminal extension (11) results in a marked inhibition of activity (6). Liver Fru-1,6-P2ase activity has been reported to increase during diabetes and starvation (12, 13), suggesting that the enzyme is subject to long-term regulation at the levels of transcription and/or translation.Since the DNA encoding mammalian ...
