Purification and characterization of human muscle pyruvate kinase

Abstract

The M1 isozyme of pyruvate kinase was purified from human psoas muscle in 7-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, DEAE-cellulose batchwise treatment followed by chromatography on CM-cellulose and Sephadex G-200 gave a product with a specific activity of 383 U[enzyme units]/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 MW of 240,700 and a sedimentation coefficient (S20,W) of 10.04 S. It contains 4 subunits with identical MW 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.