The sialate pyruvate-lyase from pig kidney: purification, properties and genetic relationship.

For further insight into the structural relationship between mammalian and microbial sialate pyruvate-lyases, the enzyme from pig kidney was purified to homogeneity from the tissue homogenate by a heat precipitation step followed by anion exchange and Hydrophobic Interaction Chromatography or native gel electrophoresis, respectively. The pure enzyme preparation exhibited an about 1000-fold increase of specific activity compared to the supernatant after the first centrifugation and revealed a single band at 34-37 kDa after SDS-PAGE, which represents the monomeric form of the protein. While the native enzyme seems to be a trimer according to the molecular weight obtained by gel filtration (108 kDa), crosslinking with dimethylpimelimidate suggests it to be a tetramer. The lyase is optimally active at about 75 degrees C and in the pH range of 7.6 to 8.0 and belongs to the class I-aldolases, due to its non-requirement of metal ions and the presence of lysine as the main functional residue in its catalytic centre. These data are similar to those obtained with bacterial lyases. However, peptide fragments of this enzyme show less similarity to primary lyase structures of microbia than to those derived from expressed sequence tags of mammals.