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

Richard N. Harkins, John A. Black, Marvin B. Rittenberg

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35 Scopus citations


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 (s20,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.

Original languageEnglish (US)
Pages (from-to)3831-3837
Number of pages7
Issue number17
StatePublished - Aug 1 1977
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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