Abstract
Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- DTT:
-
dithiothreitol
- HIC:
-
hydrophobic interaction chromatography
- RA:
-
rosmarinic acid
- RAS:
-
rosmarinic acid synthase
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Bauw, G., Holm Rasmussen, H., Van den Bulcke, M., Van Damme, J., Puype, M., Gesser, B., Celis, J.E., Vandekerckhove, J. (1990) Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: A direct link between proteins and genes. Electrophoresis 11, 528–536
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
De-Eknamkul, W., Ellis, B.E. (1987a) Tyrosine aminotransferase: the entrypoint enzyme of the tyrosine-derived pathway in rosmarinic acid biosynthesis. Phytochemistry 26, 1941–1946
De-Eknamkul, W., Ellis, B.E. (1987b) Purification and characterization of tyrosine aminotransferase activities from Anchusa officinalis cell cultures. Arch. Biochem. Biophys. 257, 430–4438
Ellis, B.E., Towers, G.H.N. (1970) Biogenesis of rosmarinic acid in Mentha. Biochem. J. 118, 291–297
Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158
Häusler, E., Petersen, M., Alfermann, A.W. (1991) Hydroxyphenylpyruvate reductase from cell suspension cultures of Coleus blumei Benth. Z. Naturforsch. 46c, 371–376
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Mizukami, H., Ellis, B.E. (1991) Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures. Plant Cell Rep. 10, 321–324
Oakley, I.M., Kirsch, D.R., Morris, N.R. (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361–363
Petersen, M.S. (1991) Characterization of rosmarinic acid synthase from cell cultures of Coleus blumei. Phytochemistry 30, 2877–2881
Petersen, M., Alfermann, A.W. (1988) Two new enzymes of rosmarinic acid biosynthesis from cell cultures of Coleus blumei: hydroxyphenylpyruvate reductase and rosmarinic acid synthase. Z. Naturforsch. 43c, 501–504
Petersen, M., Häusler, E., Karwatzki, B., Meinhard, J. (1993) Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth. Planta 189, 10–14
Razzaque, A., Ellis, B.E. (1977) Rosmarinic acid production in Coleus cell cultures. Planta 137, 287–291
Author information
Authors and Affiliations
Additional information
The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Two-dimensional gel electrophoresis was done with the help of Dr. Guy Bauw, University of Gent, Belgium.
Rights and permissions
About this article
Cite this article
Petersen, M. Purification of rosmarinic acid synthase from cell cultures of Coleus blumei Benth. Planta 191, 18–22 (1993). https://doi.org/10.1007/BF00240891
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00240891