Yeast Pyruvate Kinase

Abstract

Abstract A scheme for the preparation of pyruvate kinase (EC 2.7.1.40) in gram amounts from yeast is described which utilizes two (NH4)2SO4 fractionations followed by successive chromatography on DEAE-cellulose and cellulose phosphate columns in 50% aqueous glycerol. The resulting enzyme had a specific activity of 219 µmoles per min per mg at 30°. Under certain conditions it exhibited a cold lability, but means are described to maintain a stable preparation. The protein sedimented as a single symmetrical peak in the Beckman model E ultracentrifuge with schlieren optics. Over 95% migrated as a single band on polyacrylamide gel electrophoresis. At 2.0 mg per ml, s20w, = 8.52 and D20w, = 4.98 x 10-7 cm2 sec-1, giving an s/D molecular weight of 166,000. From this molecular weight and a dry weight analysis, the molar extinction coefficient was 1.08 x 105 m-1 cm-1 at 280 nm. An amino acid analysis of the enzyme is presented.