Abstract
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 dehydrogenasecatalyzed 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 µM, with a Vmax of 51 pmol 3H2O/min/mg protein. The Ki of isovaleryl-CoA dehydrogenase by (methylenecyclopropyl) acetyl-CoA was approximately 2 µM.
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Hyman, D., Tanaka, K. Isovaleryl-CoA Dehydrogenase Activity in Isovaleric Acidemia Fibroblasts Using an Improved Tritium Release Assay. Pediatr Res 20, 59–61 (1986). https://doi.org/10.1203/00006450-198601000-00017
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DOI: https://doi.org/10.1203/00006450-198601000-00017
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