title: Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme.
abstract: Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein. After expression of the cDNA in eukaryotic 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 lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified 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 stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Center for Medical Molecular Biology, Aarhus University Hospital, Skejby Sygehus, Denmark.
authors.forename: P
authors.initials: P
authors.lastname: Bross
authors.forename: T G
authors.initials: TG
authors.lastname: Jensen
authors.forename: B S
authors.initials: BS
authors.lastname: Andresen
authors.forename: M
authors.initials: M
authors.lastname: Kjeldsen
authors.forename: A
authors.initials: A
authors.lastname: Nandy
authors.forename: S
authors.initials: S
authors.lastname: Kølvraa
authors.forename: S
authors.initials: S
authors.lastname: Ghisla
authors.forename: I
authors.initials: I
authors.lastname: Rasched
authors.forename: L
authors.initials: L
authors.lastname: Bolund
authors.forename: N
authors.initials: N
authors.lastname: Gregersen
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000032:analysis / Q000172:deficiency* / Q000235:genetics*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005057:Eukaryotic Cells / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D006579:Heterozygote; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D011499:Protein Processing, Post-Translational
chemical_list: D008715:Methionine; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase