A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay.
The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D010735:Phosphoglycerate Kinase / Q000097:blood* / Q000302:isolation & purification; D010736:Phosphoglycerate Mutase / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism
D004163:Diphosphoglyceric Acids; D000244:Adenosine Diphosphate; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D010735:Phosphoglycerate Kinase; D010751:Phosphopyruvate Hydratase; D010736:Phosphoglycerate Mutase; D008274:Magnesium