Purification and properties of acyl-CoA oxidase from rat liver.
Acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was purified from rat liver. The final preparation was judged to be nearly homogeneous from the results of sedimentation analysis. Ultrogel AcA-34 column chromatography, and phosphocellulose column chromatography. The molecular weight of the enzyme was determined to be 139,000 by the sedimentation equilibrium method and Ultrogel AcA-34 column chromatography. The S020,W of the enzyme was 7.85S. Three protein components, A, B, and C, were found in the enzyme preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights, 75,500, 50,100, and 19,000) and high-pressure liquid chromatrography in the presence of 6 M guanidine . HCl (molecular weights, 71,900, 51,700, and 20,500). It was concluded that components B and C were formed from component A, probably by proteolytic cleavage, based on the result of amino acid analysis of each component. The pI of the enzyme was 9.2. The enzyme contained FAD as a prosthetic group, and exhibited absorption maxima at 278, 378, and 450 nm. The FAD content was 1.22 mol/mol of enzyme. When palmitoyl-CoA was added to the enzyme solution under anaerobic conditions, the bound FAD was reduced. The Km values were lower for C14 to C18 acyl-CoA's than for others tested, whereas Vmax values were roughly the same for C8 to C18 acyl-CoA's. The Km value for O2 was 5 microM. The optimal pH was 8. 3-Ketohexadecanoyl-CoA inhibited the enzyme (Ki=0.47 microM), forming a charge-transfer complex with the enzyme.
D000214:Acyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism; D042962:Acyl-CoA Oxidase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005415:Flavins / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010100:Oxygen; D051381:Rats; D012492:Salts; D013053:Spectrophotometry; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D000596:Amino Acids; D005415:Flavins; D012492:Salts; D005182:Flavin-Adenine Dinucleotide; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; D010100:Oxygen