CHARACTERIZATION OF WILD-TYPE HUMAN MEDIUM-CHAIN ACYL-COA DEHYDROGENASE (MCAD) AND MUTANT ENZYMES PRESENT IN MCAD-DEFICIENT PATIENTS BY 2-DIMENSIONAL GEL-ELECTROPHORESIS - EVIDENCE FOR POSTTRANSLATIONAL MODIFICATION OF THE ENZYME

Two-dimensional gel electrophoresis was used to study and compare wild type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and miss ense mutant enzyme found in patients with MCAD deficiency. By comparin g the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with po int mutations changing the net charge of the protein can be readily re solved from the wildtype protein. After expression of the cDNA in euka ryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to low er pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. T he observed pI shift would be compatible with phosphorylation of one a spartic acid residue per monomer. Comparison of pulse labeling and ste ady-state amounts of MCAD protein in overexpressing COS 7 cells confir ms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild type protein, but is degraded much more r eadily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modifie d form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively st ronger both in pulse labeling and in steady state, indicating that eit her the efficiency of modification or the stability of the modified fo rm is affected by the K304E mutation. Detection of both wild-type and K304E mutant MCAD was achieved in lymphoblastoid cells from patients a nd carriers of the mutation. Both spots for the wild-type but only the nonmodified spot for the K304E mutant could be detected. In lymphobla stoid cells from carriers, the intensity of the spot representing the mutant protein is much weaker than the two spots representing wild-typ e MCAD, emphasizing that the K304E mutant protein is more susceptible to degradation than wild-type MCAD. The absence of detectable amounts of mod ified K304E mutant MCAD protein in these cells suggest that the conclusion drawn from COS-7 cell expression is also valid in patient cells. (C) 1994 Academic Press, Inc.