Purification, properties, and immunocytochemical localization of human liver peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.
A molecular understanding of genetic disease in which peroxisomal functions are impaired depends on analysis of the structure of normal and mutant enzymes of peroxisomes. We report experiments describing the isolation, characterization, and immunocytochemical localization of enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) of the peroxisomal fatty acid beta-oxidation system from normal human liver and compared it with that of rat liver enzyme. The human enzyme, purified approximately equal to 2300-fold by ion-exchange chromatography, is homogeneous as judged by NaDodSO4/PAGE. This PBE is localized exclusively in the matrix of peroxisomes in liver cells by the protein A/gold immunocytochemical method. The human PBE is similar to rat enzyme in size (Mr, approximately equal to 79,000), isoelectric point (pI, 9.8), pH optima, molecular structure as observed by rotary shadowing, and peptide pattern on NaDodSO4/PAGE after proteolytic digestion with Staphylococcus aureus V8 protease. The human and rat enzymes differed in their immunological properties by having partial identity with each other; this is reflected in their slightly dissimilar composition of the amino acids aspartic acid, threonine, glutamic acid, tyrosine, and glycine. COOH-terminal amino acid were similar for both the enzymes: -Gly-Ser-Leu-Ile-COOH. These results suggest that the human and rat liver PBE may be different in their amino acid sequences at their antigenic sites.
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004746:Enoyl-CoA Hydratase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases / Q000302:isolation & purification*; D007106:Immune Sera; D005779:Immunodiffusion; D007535:Isomerases; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D063994:Peroxisomal Bifunctional Enzyme; D051381:Rats
D000596:Amino Acids; D007106:Immune Sera; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D006836:Hydro-Lyases; C576253:EHHADH protein, human; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases