title: Distribution and degradation of biotin-containing carboxylases in human cell lines.
abstract: Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.
authors.forename: C S
authors.initials: CS
authors.lastname: Chandler
authors.forename: F J
authors.initials: FJ
authors.lastname: Ballard
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D019735:Carbon-Carbon Ligases; D002262:Carboxy-Lyases / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D043586:Methylmalonyl-CoA Decarboxylase; D008928:Mitochondria / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism
chemical_list: D011506:Proteins; D001710:Biotin; D002262:Carboxy-Lyases; D008025:Ligases; D019735:Carbon-Carbon Ligases; D011766:Pyruvate Carboxylase; D000103:Acetyl-CoA Carboxylase; C021573:methylcrotonoyl-CoA carboxylase; D043586:Methylmalonyl-CoA Decarboxylase; D007930:Leucine