[59] Fructose-1,6-bisphosphatase from rat liver

This chapter describes the purification and properties of the enzyme fructose-l,6-bisphosphatase obtained from rat liver, which requires a divalent cation for activity and shows maximum activity at neutral pH.

Abstract

This chapter describes the purification and properties of the enzyme fructose-l,6-bisphosphatase obtained from rat liver. Fructose-l,6-bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi. The enzyme activity can be determined spectrophotometrically by following the rate of formation of NADPH at 340 nm in the presence of excess glucosephosphate isomerase and glucose-6-phosphate dehydrogenase. The purification of the enzyme involves extraction, heat treatment, phosphocellulose AffiGel blue chromatography, ammonium sulfate fractionation, and AffiGel blue chromatography. Rat liver fructose-1,6-bisphosphatase exhibits a single protein band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis with a subunit molecular weight of 42,000. Purified rat liver fructose-1,6-bisphosphatase and the enzyme present in crude extracts shows maximum activity at neutral pH. Fructose-1,6-bisphosphatase requires a divalent cation for activity and this requirement can be fulfilled either by Mg2+ or Mn2+. Zn2+, Fe2+, and Fe3+ are strongly inhibitory to the enzyme.