The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58 |
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Authors: | W Russell McLauchlan Raymond A McKee David M Evans |
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Institution: | (1) Molecular Biology Group, Department of Genetics and Microbiology, AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich Research Park, NR4 7UA Norwich, UK |
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Abstract: | The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K
m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO
N-methylputrescine oxidase
- PVDF
polyvinylidene difluoride
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper. |
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Keywords: | Alkaloid biosynthesis Enzyme purification Nicotiana N-Methylputrescine oxidase |
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