Purification and characterization of human muscle pyruvate kinase.
The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.
D000596:Amino Acids / Q000032:analysis; D003460:Crystallization; D006801:Humans; D007118:Immunoassay; D005779:Immunodiffusion; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism
D000596:Amino Acids; D011770:Pyruvate Kinase