10] Phosphofructokinase from yeast

Abstract

Publisher Summary This chapter discusses the purification of enzyme phosphofructokinase from yeast and describes some properties of purified enzyme. Phosphofructokinase is an allosteric enzyme that catalyzes the transfer of the γ-phosphoryl group of MgATP to carbon atom one of D(-)-fructofuranose 6-phosphate and produces D(-)-fructofuranose 1,6-bisphosphate and MgADP. Phosphofructokinase is assayed in the coupled optical test using aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase (NAD + ) as auxiliary enzymes. The procedure involves (1) disruption of the yeast cells and extraction of phosphofructokinase, (2) first ammonium sulfate precipitation, (3) chromatography on immobilized cibacron blue F3G-a, (4) second ammonium sulfate precipitation, (5) gel filtration, third ammonium sulfate precipitation, (6) ion-exchange chromatography, and (7) fourth ammonium sulfate precipitation. The specific activity, the electrophoretic mobility of the native enzyme, the subunit pattern, and the behavior in the analytical ultracentrifuge remain unchanged for several months after preparation, when the enzyme is stored at 4 ° C in the buffer mixture.