IMMUNOBLOT ANALYSES OF THE ELICITED SANGUINARIA-CANADENSIS ENZYME, DIHYDROBENZOPHENANTHRIDINE OXIDASE - EVIDENCE FOR RESOLUTION FROM A POLYPHENOL OXIDASE ISOZYME

In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria canadensis plant cell cultures, We reported that our most purified preparations contained a major band at 77 kDa and minor lowe r M-r bands. Here we present evidence on highly purified dihydrobenzop henanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower M-r bands inclu de an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphe nol oxidase antibody that recognizes a 70-kDa band were used to monito r chromatographic fractions by immunoblot analysis of the oxidases. Ox idase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions, Bands at 77 and SS k Da were detected with antidihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity . Polyphenol oxidase specific activity and immunoreactivity partitione d both in flow-through and eluate fractions of the CRI and HiTrap colu mns. Estimation of the dihydrobenzophenanthridine oxidase and polyphen ol oxidase specific activities for each step showed increasing enrichm ent of alkaloidal enzyme accompanied by variable dihydrobenzophenanthr idine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphe nol oxidase have M-r values of 77 and 70 kDa, respectively, and the tw o enzymes are different entities. (C) 1997 Academic Press.