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. Fractionally 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 10.04 S. It contains four subunits with identical molecular weights of 61 000. 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.