Purification and properties of γγ-enolase from pig brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as alpha alpha-(pI = 6.5) = 6.5), alpha gamma- (pI = 5.6), and gamma gamma-enolase (pI = 5.2). The pI of purified gamma gamma-enolase was also 5.2. The yy-enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55 degrees C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%-80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, gamma gamma-enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K-m values for 2-PGA, PEP, and Mg2+ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K-m values of other vertebrate enolases. The amino acid composition of pig yy-enolase, as determined by amino acid analysis, shows strong similarity to the compositions of gamma gamma-enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig gamma gamma-enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig gamma gamma-enolase and the other gamma gamma-enolases are almost identical. Li+ proved to be a noncompetitive inhibitor with either 2-PGA or Mg2+ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2(1). An R-symm <5% was obtained for data between 50 and 3.65 Angstrom, but was a disappointing 30% for data between 3.65 and 3.10 Angstrom, indicating crystal disorder.