The purification of properties of rat liver fructose 1,6-bisphosphatase

Abstract

Fructose 1,6-bisphosphatase has been isolated in homogeneous form from rat liver by a simple and convenient procedure, including adsorption on carboxymethyl cellulose and substrate elution. It is a tetrameric protein, with molecular weight of approximately 140,000. The amino acid composition shows some similarity to that of the rabbit liver enzyme, with a blocked NH 2-terminus and leucine, rather than alanine, as the COOH-terminal residue. The purified enzyme contains no tryptophan. It requires Mg 2+ or Mn 2+ and is activated by NH 4 +, which also decreases the affinity for both substrate and divalent cations. In the absence of chelating agents, the pH optimum is 8.0, but this is shifted to 7.0, 7.2, and 7.5 on addition of EDTA, histidine, and citrate, respectively. Ca 2+ is a potent inhibitor and appears to compete for the Mg 2+-binding site. The rat liver enzyme is inhibited by AMP in a cooperative manner, with a Hill coefficient of about 2. Saturation is reached when approximately 2 mol of AMP are bound per mole of enzyme, but no AMP binding is observed in the absence of the substrate. Antibody to the purified enzyme, produced in the rabbit, reacts with fructose 1,6-bisphosphatase in rat liver and rat kidney with equal affinity. It also reacts with rat muscle fructose 1,6-bisphosphatase, with 1/100th the affinity.