Purification and regulatory properties of pigeon erythrocyte pyruvate kinase

1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.