Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1989 Sep;91(1):79–84. doi: 10.1104/pp.91.1.79

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings 1

Jesus Alvarez Fernandez 1,2,2, Terence G Owen 1,2, Wolfgang G W Kurz 1,2, Vincenzo De Luca 1,2
PMCID: PMC1061955  PMID: 16667047

Abstract

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.

Full text

PDF
79

Images in this article

81Figure 3
on p.81
82Figure 5
on p.82

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. De Luca V., Fernandez J. A., Campbell D., Kurz W. G. Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus. Plant Physiol. 1988 Feb;86(2):447–450. doi: 10.1104/pp.86.2.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. De Luca V., Marineau C., Brisson N. Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2582–2586. doi: 10.1073/pnas.86.8.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eilert U., De Luca V., Constabel F., Kurz W. G. Elicitor-mediated induction of tryptophan decarboxylase and strictosidine synthase activities in cell suspension cultures of Catharanthus roseus. Arch Biochem Biophys. 1987 May 1;254(2):491–497. doi: 10.1016/0003-9861(87)90128-7. [DOI] [PubMed] [Google Scholar]
  5. Engvall E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 1980;70(A):419–439. doi: 10.1016/s0076-6879(80)70067-8. [DOI] [PubMed] [Google Scholar]
  6. Gamborg O. L., Miller R. A., Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968 Apr;50(1):151–158. doi: 10.1016/0014-4827(68)90403-5. [DOI] [PubMed] [Google Scholar]
  7. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES