Simultaneous preparation of the three biotin-containing mitochondrial carboxylases from rat liver.
Affinity chromatography on avidin-Sepharose column was used to bind the biotin-containing carboxylases from rat liver. With a biotin gradient (0-0.3 mM), peaks of activity of pyruvate, propionly CoA and beta-methylcrotonyl CoA carboxylases co-eluted. Subsequent separation of the three carboxylases was attained using DEAE-Sepharose chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed each of the enzymes to be pure, with pyruvate carboxylase giving a single subunit band (Mr 130 000), propionyl-CoA carboxylase giving two bands (Mr 73 000 and 56 500) and beta-methylcrotonyl-CoA carboxylase giving two bands (Mr 75 000 and 60 000). The specific activity of propionyl-CoA carboxylase (15.8 munits/mg) and beta-methylcrotonyl-CoA carboxylase (24.2 munits/mg) were comparable with reported activities for these purified enzymes, while that of pyruvate carboxylase (1.25 munits/mg) was low. This is a suitable method for the simultaneous preparation of purified carboxylases for the specific purpose of raising antisera to these enzymes.
D000818:Animals; D001710:Biotin; D019735:Carbon-Carbon Ligases; D002262:Carboxy-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D008025:Ligases / Q000302:isolation & purification* / Q000378:metabolism; D043586:Methylmalonyl-CoA Decarboxylase; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011766:Pyruvate Carboxylase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D001710:Biotin; D002262:Carboxy-Lyases; D008025:Ligases; D019735:Carbon-Carbon Ligases; D011766:Pyruvate Carboxylase; C021573:methylcrotonoyl-CoA carboxylase; D043586:Methylmalonyl-CoA Decarboxylase