Studies of the regulation of rat liver fructose 1,6-bisphosphatase

Fructose 1,6-bisphosphatase has been isolated from rat liver by a newly developed procedure which includes absorption on a column of Procion Red HE-3B immobilised to cross-linked Sepharaose-6B, then specific elution using a step of fructose 1,6-bisphosphate and AMP. A number of criteria indicated that the enzyme was not subjected to any significant degree of proteolytic modification during the purification. Purified fructose 1,6-bisphosphatase was homogeneous as judged by polyacrylamide gel electrophoresis and was composed of identical subunits in the molecular weight range of 39000-42000. Molecular weight values of 158000 and 148000, obtained by gel filtration chromatography and sedimentation equilibrium analysis respectively, indicated that the native enzyme was tetrameric. The amino acid composition of the enzyme was similar to that reported by Tejwani et al (1976); the enzyme contained no tryptophane. The catalytic subunit of cyclic AMP-dependent protein kinase catalysed incorporation of [32]P from [gamma-32P] ATP into homogeneous rat liver fructose 1,6-bisphosphatase in vitro. Approximately 4 moles of phosphate were incorporated per mole of the tetrameric enzyme, but this phosphorylation was not associated with an increase in enzyme activity using standard assay conditions. Fructose 1,6-bisphosphatases from rabbit muscle and from ox liver could not be phosphorylated by cyclic AMP-dependent protein kinase. The dephosphorylation of phosphorylated rat liver fructose 1,6-bisphos-phatase in vitro was catalysed by phosphoprotein phosphatases 1, 2A and 2C from rat liver. Phosphatase 2A was the most active of these three against fructose 1,6-bisphosphatase and this finding is in agreement with the postulate that phosphatase 2A is the major phosphoprotein phosphatase involved in the control of gluconeo- genesis (Cohen, 1982). The phosphorylation of fructose 1,6-bisphosphatase in vivo was also examined. Isolated rat hepatocytes were incubated with 32P[i] and subsequently treated with either glucagon (10