Overexpression and characterization of the human peroxisomal acyl-CoA oxidase in insect cells.
Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.
Chu
R
R
Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.
Varanasi
U
U
Chu
S
S
Lin
Y
Y
Usuda
N
N
Rao
M S
MS
Reddy
J K
JK
D042962:Acyl-CoA Oxidase; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005091:Exons; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase