61] Fructose-1,6-bisphosphatase from bumblebee flight muscle

Abstract

This chapter describes the purification and properties of the enzyme fructose-1,6-bisphosphatase (FBPase) from bumblebee flight muscle. The production of fructose 6-phosphate is coupled to the reduction of NADP using phosphoglucoisomerase and glucose-6-phosphate dehydrogenase with the increase in absorbance at 340 nm measured spectrophotometrically. The purification procedure involves preparation of bumblebee flight muscle, homogenization, pretreatment of cellulose phosphate, cellulose phosphate chromatography, removal of fructose 1,6-phosphate from the enzyme preparation, and Sephadex G-200 gel filtration chromatography. The pH optimum of bumblebee FBPase is 8.0, and the molecular weight is 142,000 ± 10,000. During purification of FBPases from other sources, proteolysis can result in a shifting of the pH optimum of the enzyme toward a more alkaline pH. There is no evidence that this occurs with the flight muscle enzyme and the ratio of activity at pH 7.4 compared to pH 9.3 remains constant at 2:1 at all stages of the purification procedure.