A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.
