This chapter describes an assay method and the purification procedure for fructose-bisphosphatase (FBPase) from rabbit liver. The enzyme is assayed spectrophotometrically by following the rate of NADPH accumulation at 340 nm in the presence of excess glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase. Neutral FBPase is purified from frozen livers of young, 24-hr-fasted rabbits. For Zn analysis, atomic absorption spectroscopy is employed to measure the concentration of the stock Zn2+ solution and the levels of Zn2+ (and Mn2+) in buffer solutions, water, and FBPase samples. Removal of Zn2+ from FBPase involves various steps; the removal of the tightly bound Zn2+ does not irreversibly alter the FBPase structure. However, experimental studies of other properties of zinc-free FBPase are technically difficult, because the exposure of the enzyme to buffer or other reagents immediately results in binding of the adventitious Zn2+ by FBPase.
All Science Journal Classification (ASJC) codes
- Molecular Biology