Isovaleryl-CoA Dehydrogenase Activity in Isovaleric Acidemia Fibroblasts Using an Improved Tritium Release Assay

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 K_m for isovaleryl-CoA of 22 µM, with a V_max of 51 pmol ³H₂O/min/mg protein. The K_i of isovaleryl-CoA dehydrogenase by (methylenecyclopropyl) acetyl-CoA was approximately 2 µM.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account

Continue with Google

Continue with ORCiD