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.
Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay
- Views Icon Views
- Share Icon Share
F Ahmad, P M Ahmad, A Mendez; Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay. Biochem J 1 June 1986; 236 (2): 527–533. doi: https://doi.org/10.1042/bj2360527
Download citation file: