Purification and characterization of multifunctional enzyme from mouse liver peroxisomes.
A simple and rapid purification procedure for hepatic peroxisomal multifunctional enzyme (delta 3, delta 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase) from clofibrate treated mice is described. The purification is achieved within two days using ion-exchange chromatography and an easily prepared affinity resin. The overall yield is 10% or more after a 100-fold enrichment from the cytosolic fraction of liver tissue. The native enzyme is a monomer with a molecular mass of 75 kDa. The protein is blocked in the N-terminus but internal amino acid sequences was obtained after proteolytic cleavage. Western blot analysis indicated proteolysis of multifunctional enzyme in different subcellular fractions derived from liver tissue. The hydratase activity of the enzyme is heat-labile and highly dependent on the concentration of Tris buffer or potassium chloride present. Optimal activity was found around 37 degrees C and pH 7. The enzyme also shows dehydrogenase and isomerase activity.
Uppsala Genetic Center, Department of Cell Research, Swedish University of Agricultural Sciences.
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Stark
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J
Meijer
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000302:isolation & purification*; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification*; D063994:Peroxisomal Bifunctional Enzyme
D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; C576254:Ehhadh protein, mouse; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases