830 MOLECULAR STUDY OF MUTANT MEDIUM CHAIN ACYL-CoA DEHYDROGENASE IN CULTURED FIBROBLASTS FROM PATIENTS WITH MEDIUM CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY

Abstract

We prepared monospecific antiserum in rabbits against medium chain acyl-CoA dehydrogenase (MCAD) purified from rat liver, and studied the biosynthesis of MCAD in 10 cultured skin fibroblast lines from patients with inborn MCAD deficiency. Cells were incubated with [35S]methionine. The labeled MCAD was immunoprecipitated using the anti-MCAD antiserum and Staph A cells and analyzed by SDS-PAGE. We first demonstrated that anti-rat MCAD antibody cross reacted specifically with human MCAD. MCAD is 1-6 kd larger than four other acyl-CoA dehydrogenases such as isovaleryl-CoA- short chain-, long chain-, and 2-methyl-branched chain acyl-CoA dehydrogenases, and can be readily distinguished from other enzymes by its molecular size on slab SDS-PAGE. It is interesting to note that human MCAD is 1 kd larger than rat MCAD, while there were no differences in molecular size between other human acyl-CoA dehydrogenases and the rat counterparts. In all 10 MCAD deficient lines tested, the MCAD activity ranged from 6-13% of the mean of normal control, but the mutant MCAD in all of these cells were indistinguishable from the normal human MCAD on the basis of molecular size, indicating that all the mutant MCAD's are due to point mutation. This is in contrast to the extensive heterogeneity observed in isovaleryl-CoA dehvdrogenase in isovaleric acidemia cells.