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Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: Evidence for resolution from a polyphenol oxidase isozyme
Year:
1997
Authors :
Barg, Rivka
;
.
Volume :
347
Co-Authors:
Ignatov, A., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
Neuman, M.C., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
Barg, R., Volcani Center, Bet Dagan, 50250, Israel
Krueger, R.J., Department of Pharmacognosy, College of Pharmacy, Ferris State University, Big Rapids, MI 49307, United States
Coscia, C.J., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
Facilitators :
From page:
208
To page:
212
(
Total pages:
5
)
Abstract:
In our initial purification of dihydrobenzophenanthridine oxidase from Sanguinaria cunadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower M(r) 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 M(r) 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 oxiduses. 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 dihydrobensophenanthridine and polyphenol oxidases have M(r) values of 77 and 70 kDa, respectively, and the two enzymes are different entities.
Note:
Related Files :
chromatography
Electrophoresis
gene expression
Oxidoreductases
plant
Sanguinaria canadensis
Show More
Related Content
More details
DOI :
10.1006/abbi.1997.0336
Article number:
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
30158
Last updated date:
02/03/2022 17:27
Creation date:
17/04/2018 00:52
Scientific Publication
Immunoblot analyses of the elicited Sanguinaria canadensis enzyme, dihydrobenzophenanthridine oxidase: Evidence for resolution from a polyphenol oxidase isozyme
347
Ignatov, A., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
Neuman, M.C., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
Barg, R., Volcani Center, Bet Dagan, 50250, Israel
Krueger, R.J., Department of Pharmacognosy, College of Pharmacy, Ferris State University, Big Rapids, MI 49307, United States
Coscia, C.J., E. A. Doisy Dept. of Biochem. and M., St. Louis Univ. School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104-1079, United States
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 cunadensis plant cell cultures, we reported that our most purified preparations contained a major band at 77 kDa and minor lower M(r) 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 M(r) 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 oxiduses. 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 dihydrobensophenanthridine and polyphenol oxidases have M(r) values of 77 and 70 kDa, respectively, and the two enzymes are different entities.
Scientific Publication
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