title: Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: evidence for resolution from a polyphenol oxidase isozyme.
abstract: 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 lower Mr bands. Here we present evidence on highly purified dihydrobenzophenanthridine oxidase from elicited S. canadensis cultures to indicate that this enzyme is the 77-kDa protein and that lower Mr bands include an isozyme(s) of the polyphenol oxidase family that copurifies with it. An antibody raised against the 77-kDa protein and an anti-polyphenol oxidase antibody that recognizes a 70-kDa band were used to monitor chromatographic fractions by immunoblot analysis of the oxidases. Oxidase-containing eluates from DEAE-Sephadex, CM, and HiTrap blue were compared to corresponding flow-through fractions. Bands at 77 and 88 kDa were detected with anti-dihydrobenzophenanthridine oxidase antibody in eluates displaying high dihydrobenzophenanthridine oxidase activity. Polyphenol oxidase specific activity and immunoreactivity partitioned both in flow-through and eluate fractions of the CM and HiTrap columns. Estimation of the dihydrobenzophenanthridine oxidase and polyphenol oxidase specific activities for each step showed increasing enrichment of alkaloidal enzyme accompanied by variable dihydrobenzophenanthridine oxidase/polyphenol oxidase activity ratios. Taken together these observations indicate that the dihydrobenzophenanthridine and polyphenol oxidases have Mr values of 77 and 70 kDa, respectively, and the two enzymes are different entities.
authors.affiliation: E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104-1079, USA.
authors.forename: A
authors.initials: A
authors.lastname: Ignatov
authors.forename: M C
authors.initials: MC
authors.lastname: Neuman
authors.forename: R
authors.initials: R
authors.lastname: Barg
authors.forename: R J
authors.initials: RJ
authors.lastname: Krueger
authors.forename: C J
authors.initials: CJ
authors.lastname: Coscia
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D004156:Catechol Oxidase / Q000302:isolation & purification; D015870:Gene Expression; D015151:Immunoblotting / Q000379:methods; D007527:Isoenzymes / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000276:immunology / Q000302:isolation & purification*; D010946:Plants, Medicinal / Q000201:enzymology*; D017421:Sequence Analysis
chemical_list: D007527:Isoenzymes; D010088:Oxidoreductases; D004156:Catechol Oxidase; C077698:dihydrobenzophenanthridine oxidase