M₂ Isozyme of Pyruvate Kinase from Human Kidney as the Product of a Separate Gene: Its Purification and Characterization

The M₂ isozyme of pyruvate kinase has been purified from human kidney. The procedure involved conventional enzyme purification steps plus an affinity chromatography step utilizing the interaction between the dye, Cibracron blue F3GA, and the pyruvate kinase isozyme. During the purification it was observed that the M₂ isozyme is very unstable in the absence of fructose 1,6-bisphosphate. In addition, the electrophoretic mobility of the isozyme in polyacrylamide disc gels at pH 9.3 is greatly affected by the presence or absence of this glycolytic intermediate. The final enzyme product had a specific activity of 127 units/mg of protein and represented a 470-fold purification over the crude extract. The high purity of the enzyme preparation was established by polyacrylamide disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate, by sedimentation velocity and equilibrium analyses, and by NH₂-terminal analysis. Characterization of the purified human M₂ isozyme showed that it is a tetramer of 206 700 daltons with a sedimentation coefficient (s_20,w) of 9.25 S. Sodium dodecyl sulfate gel electrophoresis indicated that the isozyme consists of four subunits of very similar or identical molecular weight. NH₂-terminal analysis suggested that the peptide chains of the enzyme are blocked. The M₂ isozyme cross-reacts with antiserum prepared against the human M₁ isozyme. The amino acid composition of the M₂ isozyme is distinct from that of the M₁ or R isozymes. Based on the amino acid compositions of the purified M₁ and M₂ isozymes we have concluded that they represent the products of separate genes rather than different molecular forms of the same gene product as others have recently proposed.