CHARACTERIZATION AND PARTIAL-PURIFICATION OF INDOLE-3-BUTYRIC ACID SYNTHETASE FROM MAIZE (ZEA-MAYS)

In previous work it has been shown that the route from indoleacetic ac id (IAA) to indolebutyric acid (IBA) is likely to be a two-step proces s with an unknown intermediate designated 'product X'. Our objective w as to characterize and purify enzyme activities that are involved in t hese reactions. Indole-3-butyric acid synthetase was isolated and char acterized from light-grown maize seedlings (Zea mays L.), which were a ble to synthesize IBA from indole-3-acetic acid (IAA) with ATP and ace tyl-CoA as cofactors. The enzyme activity is most likely located on th e membranes of the endoplasmic reticulum, as shown by means of aqueous two-phase partitioning and sucrose density gradient centrifugation, w ith subsequent marker enzyme analysis. It was possible to solubilize t he enzyme from the membranes with a detergent (CHAPS) and high concent rations of NaCl. The molecular mass of solubilized IBA synthetase was ca 31 kDa and its isoelectric point was at pH 4.8. The enzyme forming the reaction intermediate had a molecular mass of only 20 kDa and it s eemed to be located on different membranes. Inhibition experiments wit h reducing agents and sulfhydryl reagents indicated that no sulfhydryl groups or disulfide bridges were present in the active centre of IBA synthetase. KCN inhibited the enzyme activity completely, and sodium a zide by about 50%. Substrate analogs, such as 1-IAA, 2,3-dichloropheno xyacetic acid, phenylacetic acid, and naphthaleneacetic acid, inhibite d IBA formation to a high extent. Experiments with tunicamycin gave ev idence that the enzyme is not a glycoprotein. These findings were conf irmed by affinity chromatography with Concanavalin A, where the enzyme did not bind to the matrix. Further purification of the IBA synthetas e on an ATP-affinity column resulted in a more than 1000-fold purifica tion compared to the microsomal membranes. IBA synthetase activity was also present in other plant families. Our results present further evi dence that IBA is synthesized by a two-step mechanism involving two di fferent enzyme activities.