title: Isovaleryl-CoA dehydrogenase activity in isovaleric acidemia fibroblasts using an improved tritium release assay.
abstract: Isovaleric acidemia is a disorder of leucine metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase. At least two clinical subgroups of patients exist: a severe form, in which symptoms occur within the 1st wk of life, and a milder variant in which manifestations develop later in life. We developed a modified version of the tritium release assay to accurately measure residual isovaleryl-CoA dehydrogenase activity in fibroblasts from patients with both forms of isovaleric acidemia. In the modified assay, specific isovaleryl-CoA dehydrogenase-catalyzed tritium release from [2,3-3H]isovaleryl-CoA was determined by including an inhibitor of isovaleryl-CoA dehydrogenase, (methylenecyclopropyl)acetyl-CoA, in one of the tubes in paired assays, to determine the nonspecifically released 3H2O. Residual activities of the nine isovaleric acidemia lines tested ranged from 0 to 0.67 pmol 3H2O/min/mg protein (controls 19.4 +/- 8.0). The three lines from mildly affected individuals all had no detectable activity, whereas the severe cases had a mean of 0.41 pmol 3H2O/min/mg protein. Normal human fibroblast isovaleryl-CoA dehydrogenase had a Km for isovaleryl-CoA of 22 microM, with a Vmax of 51 pmol 3H2O/min/mg protein. The Ki of isovaleryl-CoA dehydrogenase by (methylenecyclopropyl)acetyl-CoA was approximately 2 microM.
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000201:enzymology*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D045782:Hemiterpenes; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D010088:Oxidoreductases / Q000172:deficiency / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010421:Pentanoic Acids / Q000097:blood*; D012867:Skin / Q000201:enzymology; D014316:Tritium; D014631:Valerates / Q000097:blood*
chemical_list: D045782:Hemiterpenes; D010421:Pentanoic Acids; D014631:Valerates; D014316:Tritium; C008216:isovaleric acid; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase; D007930:Leucine