OVEREXPRESSION AND CHARACTERIZATION OF THE HUMAN PEROXISOMAL ACYL-COAOXIDASE IN INSECT CELLS

Human liver peroxisomes contain two acyl-CoA oxidases, namely, pahmito yl-CoA oxidase and a branched chain acyl-CoA oxidase, The palmitoyl-Co A 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 ACO X in rat and mouse liver by peroxisome proliferators and the postulate d role of the resulting oxidative stress in hepatocarcinogenesis gener ated interest in characterizing the structure and function of human AC OX. We have constructed a full-length cDNA encoding a 660-amino acid r esidue human ACOX and produced a catalytically active human ACOX prote in at high levels in Spodoptera frugiperda (Sf9) insect cells using th e baculovirus vector, Immunoblot analysis demonstrated that the full-l ength 72-kDa polypeptide (component A) was partially processed into it s constituent 51-kDa (component B) and 21-kDa (component C) products, respectively, Recombinant protein (similar to 20 mg/l x 10(9) cells) w as purified to homogeneity by a single-step procedure on a nickel-nitr ilo-triacetic acid affinity column, Using the purified enzyme, K-m and V-max values for palmitoyl-CoA were found to be 10 mu M and 1.4 units /mg of protein, respectively, The maximal activities for saturated fat ty acids were observed with C-12-18 substrates. The overexpressed huma n ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining, individual expression of either the trunc ated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed la ck of enzyme activity, but co-infection of the insect cells with recom binant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B + C complex which could su bsequently be inactivated by dissociating with detergent.