Rat liver pyruvate carboxylase. Purification, detection and quantification of apo and holo forms by immuno-blotting and by an enzyme-linked immunosorbent assay.
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.
D000273:Adipose Tissue / Q000201:enzymology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification*; D001059:Apoproteins / Q000302:isolation & purification*; D002262:Carboxy-Lyases / Q000302:isolation & purification; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007122:Immunoelectrophoresis; D008099:Liver / Q000201:enzymology*; D043586:Methylmalonyl-CoA Decarboxylase; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology; D011766:Pyruvate Carboxylase / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D001051:Apoenzymes; D001059:Apoproteins; D002262:Carboxy-Lyases; D011766:Pyruvate Carboxylase; D043586:Methylmalonyl-CoA Decarboxylase