Abstract
Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.
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Abbreviations
- FPLC:
-
fast protein liquid chromatography
- MBP:
-
myrosinase-binding protein
- MBPRP:
-
myrosinase-bindingprotein-telated protein
- PBS:
-
phosphate-buffered saline
References
Bones, A., Slupphaug, G. (1989) Purification, characterization and partial amino acid sequencing of β-thioglucosidase from Brassica napus L. J. Plant Physiol. 134, 722–729
hadchawan, S., Bishop J., Thangstad, O.P., Bones, A.M., MitchelOlds, T., Bradley, D. (1993) Arabidopsis cDNA sequence encoding myrosinase. Plant Physiol. 103, 671–672
Chew F.S. (1988) Biological effects of glucosinolates. In:Biologically gnactive natural products: Potential use in agriculture (ACS symposium series No. 380), pp. 155–181, Cutler, H.G., ed. American Chemical Society, Washington
Dobberstein, B., Garoff, H., Warren, G. (1979) Cell-free synthesis and membrane insertion of a mouse H-2D histocompatibility antigen and β2-microglobulin. Cell 17, 759–769
Falk, A., Xue, J., Lenman, M., Rask, L. (1992) Sequence of a cDNA clone encoding the enzyme myrosinase and expression of myrosinase in different tissues of Brassica napus. Plant Sci. 83, 181–186
Feick, R.G., Shiozawa, J.A. (1990) A high yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing. Anal. Biochem. 87, 205–211
Fenwick, G.R., Heaney, R.K., Mullin, W.J. (1983) Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18, 123–201
Gallagher, S., Winston, S.E., Fuller, S.A., Hurrell, J.G.R. (1992) In: Current gnprotocols in molecular biology, pp. 10.8.1–10.8.16, Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., eds. Wiley-Interscience, NewYork
Haughn, G.W., Davin, L., Giblin, M., Underhill, E.W. (1991) Biochemical genetics of plant secondary metabolites in Arabidopsis thaliana. Plant Physiol 97, 217–226
Höglund, A.S., Lenman, M., Falk, A., Rask, L. (1991) Distribution of myrosinase in rapeseed tissues. Plant Physiol. 95, 213–221
James, D., Rossiter, J. (1991) Development and characteristics of myrosinase in Brassica napus during early seedling growth. Physiol. Plant. 82, 163–170
Lenman, M., Rödin, J., Josefsson, L.G., Rask, L. (1990) Immunological characterization of rapeseed myrosinase. Eur. J. Biochem. 194, 747–753
Lenman, M., Falk, A., Rödin, J., Höglund, A.S., Ek, B., Rask, L. (1993) Differential expression of myrosinase gene families. Plant Physiol. 103, 703–711
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275
Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497
Sundberg, E., Landgren, M., Glimelius, K. (1987) Fertility and chromosome stability in Brassica napus resynthesized by protoplast fusion. Theor. Appl. Genet. 75, 96–104
Tempst, P., Link, A., Riviere, L.R., Fleming, M., Elicone, C. (1990) Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: Improved methods, applications and gene cloning strategies. Electrophoresis 11, 537–553
Thangstad, O.P., Winge, P., Huseby, H., Bones, A. (1993) The myrosinase (thioglucoside glucohydrolase) gene family in Brassicaceae. Plant Mol. Biol. 23, 511–524
Tookey, H.L. (1973) Crambe thioglucoside glucohydrolase (EC 3.2.3.1): separation of a protein required for epithiobutane formation. Can. J. Biochem. 51, 1654–1660
Towbin, H., Staehelin, T, Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Proc. Natl. Acad. Sci. USA 76, 4350–4354
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Falk, A., Taipalensuu, J., Ek, B. et al. Characterization of rapeseed myrosinase-binding protein. Planta 195, 387–395 (1995). https://doi.org/10.1007/BF00202596
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DOI: https://doi.org/10.1007/BF00202596