Human erythrocyte pyruvate kinase. Its purification and some properties

Abstract

1. 1. Human erythrocyte pyruvate kinase (EC 2.7.1.40) was purified 30 000-fold by successive (NH 4) 2SO 4 precipitation and column chromatography with blue dextran 2000. The resulting enzyme preparation had a specific activity of 150 μmoles NADH · min−1 · mg−1 at 25°. 2. 2. The molecular weight as determined by gel filtration with Sephadex G-200 was 205 000 ± 5000. 3. 3. With starch gel electrophoresis only one band was observed after-detection of the enzyme activity with the fluorescent technique. 4. 4. The variation of activity of pyruvate kinase at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. 5. 5. The stimulatory effect of Fru-1,6-P 2 as well as the inhibitory effect of ATP on the activity of pyruvate kinase were pH dependent. 6. 6. Phosphorylated hexoses ( Fru-1,6-P 2 and Glc-6-P ), P 1 and the substrate PEP overcame the inhibition of the activity of pyruvate kinase by ATP at physiological pH. 7. 7. Phosphorylated hexoses and P 1 stimulated the activity of pyruvate kinase.