Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana

Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 a cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AO alpha and AO beta among the three isoforms, AO alpha, AO beta, and AO gamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AO beta and AO gamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOa, AO beta, and AO gamma were estimated as approximately 290-300 kDa. These results suggest that AO alpha and AO gamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AO beta is a heterodimer of the atAO-1 and atAO-2 products.