Liver 3-Phosphoglycerate Kinase. Purification and Some Molecular Properties of the Bovine-Liver Enzyme

3-Phosphoglycerate kinase has been purified for the first time from a gluconeogenetic tissue. A seven-step procedure for the preparation of the bovine-liver enzyme, which results in a specific activity of 130–150 units/mg, is described. The overall purification was 250-fold and 17%, of the original activity was recovered. About 50 mg pure protein can be obtained from 1 kg liver tissue. Optimum conditions for crystallization and stabilization of the enzyme have been elaborated. The existence of at least three different forms of phosphoglycerate kinase in liver extracts has been demonstrated by isoelectric focussing experiments. The main activity peak was purified to homogeneity. Evidence was obtained for a probable tissue specificity of the isoelectric points of the known phosphoglycerate-kinase enzymes (bovine liver pH 6.5, pig liver 6.3; rabbit muscle 7.0; yeast 7.2; mice Ehrlich ascites cells 6.8). Bovine-liver phosphoglycerate kinase was shown to be homogeneous by sedimenting as a regular sharp peak in the analytical ultracentrifuge and by the appearance of a single protein band on both cellulose acetate and polyacrylamide disc gel electrophoresis. The liver and yeast-type enzymes were all distinguishable by their migration distances at pH 4.9, 7.0 and 8.6. Homogeneity of the bovine-liver enzyme was further confirmed by rechromatography on Biogel P-150 and by the determination of the unique NH2- and COOH-terminal amino-acid sequences NH2-His-Lys-Tyr and Thr-Glu-COOH, respectively. The results obtained demonstrate that liver phosphoglycerate kinase, purified by the method described, is a homogeneous protein, distinct from the known muscle and yeast enzymes.