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A monoclonal antibody specific for the red-absorbing form of phytochrome

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Abstract

Characterisation of a new monoclonal antibody (mAb), designated LAS 41, directed against 124-kilodalton (kDa) etiolated-oat (Avena sativa L.) phytochrome, indicates that it recognises an epitope unique to the red-light-absorbing form, Pr. In a solid-phase enzyme-linked immunosorbent assay (ELISA), LAS 41 exhibits a seven- to eight-fold higher affinity for Pr than for the far-red-light-absorbing form of phytochrome, Pfr. In addition, in immunoprecipitation assays LAS 41 effectively precipitates 100% of phytochrome presented as Pr but only precipitates a maximum of 24.5% of phytochrome presented as Pfr. These values are indicative of binding exclusively to Pr. Peptide-mapping studies show that LAS 41 recognises and epitope located within a region 6–10 kDa from the aminoterminus of the phytochrome molecule. Since binding of LAS 41 to Pr induces alterations in the spectral properties of Pr, this indicates that at least part of the 4 kDa domain to which the antibody binds is essential for protein-chromophore interaction. Subsequent photoconversion of LAS 41-Pr complexes produces native Pfr spectra, with concomitant production of free antibody and antigen, as shown by a modified ELISA. The specificity of LAS 41 for Pr has facilitated the purification of Pfr which is free of contaminating Pr. This has enabled direct determination of the mole fraction of Pfr established by red light to be 0.874.

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Abbreviations

ELISA:

enzyme-linked immunsorbent assay

kDa:

kilodalton

mAb:

monoclonal antibody

Pfr:

far-red-absorbing form of phytochrome

Pr:

red-absorbing form of phytochrome

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

Δ (ΔA):

difference in absorbance (A Pr665 −A Pr730 )-(A Pfr665 −A Pfr730 )

ΔAr/ΔAfr:

spectral change ratio (SCR)

ϕmax:

mole fraction of Pfr following saturating red light

References

  • Billet, E.E., Gunn, B., Mayer, R.J. (1984) Characterization of two monoclonal antibodies obtained after immunization with human liver mitochondrial membrane preparations. Biochem. J. 221, 765–776

    Google Scholar 

  • Brockmann, J., Rieble, S., Kazarinova-Fukshansky, N., Seyfried, M., Schäfer, E. (1987) Phytochrome behaves as a dimer in vitro. Plant Cell Environ. 10, 105–111

    Google Scholar 

  • Cordonnier, M-M., Greppin, H., Pratt, L.H. (1984) Characterization by enzyme-linked-immunosorbent assay of monoclonal antibodies to Pisum and Avena phytochrome. Plant Physiol. 74, 123–127

    Google Scholar 

  • Cordonnier, M-M., Greppin, H., Pratt, L.H. (1985) Monoclonal antibodies with differing affinities to the red-absorbing and far-red absorbing forms of phytochrome. Biochemistry 24, 3246–3253

    Google Scholar 

  • Daniels, S.M., Quail, P.H. (1984) Monoclonal antibodies to three separate domains on 124 kilodalton phytochrome from Avena. Plant Physiol. 76, 622–626

    Google Scholar 

  • Grimm, R., Lottspeich, F., Schneider, H.A.W., Rudiger, W. (1987) Investigation of the peptide chain of 124 kDa phytochrome: localisation of proteolytic fragments and epitopes for monoclonal antibodies. Z. Naturforsch. 41c, 993–1000

    Google Scholar 

  • Holdsworth, M.L. (1987) Characterisation of phytochrome using monoclonal antibodies. Ph.D. thesis, University of Leicester, UK

    Google Scholar 

  • Hopkins, D.W., Butler, W.L. (1970) Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformations. Plant Physiol. 45, 567–570

    Google Scholar 

  • Jones, A.M., Vierstra, R.D., Daniels, S.M., Quail, P. (1985) The role of separate molecular domains in the structure of phytochrome from etiolated Avena sativa L. Planta 164, 501–506

    Google Scholar 

  • Kelly, J.M., Lagarias, J.C. (1985) Photochemistry of 124-kilodalton Avena phytochrome under constant illumination in vitro. Biochemistry 24, 6003–6010

    Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

    Google Scholar 

  • Lagarias, J.C., Mercurio, F.M. (1985) Structure function studies on phytochrome. Identification of light induced conformational changes in 124-kDa Avena phytochrome in vitro. J. Biol. Chem. 260, 2415–2423

    Google Scholar 

  • Nagatani, A., Yamamoto, K.T., Furuya, M., Fukumoto, T., Yamashita, A. (1983) Production and characterisation of monoclonal antibodies to rye (Secale cereale) phytochrome. Plant Cell Physiol. 24, 1143–1149

    Google Scholar 

  • Nagatani, A., Yamamoto, K.T., Furuya, M., Fukumoto, T., Yamashita, A. (1983) Production and characterisation of monoclonal antibodies which distinguish different surface structures of pea (Pisum sativum cv. Alaska) phytochrome. Plant Cell Physiol. 25, 1059–1068

    Google Scholar 

  • Napier, R.M., Smith, H. (1987) Photoreversible association of phytochrome with membranes II: reciprocity tests and a model for the binding reaction. Plant Cell Environ. 10, 391–396

    Google Scholar 

  • Pratt, L.H. (1982) Phytochrome: the protein moiety. Annu. Rev. Plant Physiol. 33, 557–582

    Google Scholar 

  • Shimazaki, Y., Cordonnier, M-M, Pratt, L.H. (1986) Identification with monoclonal antibodies of a second antigenic domain on Avena phytochrome that changes upon its photoconversion. Plant Physiol. 82, 109–113

    Google Scholar 

  • Shropshire, W., Jr., Mohr, H. (eds.) (1983) Encyclopedia of plant physiology, N.S., vol. 16 A, B: Photomorphogenesis. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Song, P.-S. (1985) The molecular model of phytochrome deduced from optical probes. In: Optical properties and structure of tetrapyrroles, pp. 331–348, Blauer, G., Sund, H. eds. Walter de Gruyter, Berlin New York

    Google Scholar 

  • Thomas, B., Penn, S.E., Butcher, G.W., Galfre, G. (1984) Discrimination between the red and far-red absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies. Planta 160, 382–384

    Google Scholar 

  • Vierstra, R.D., Quail, P.H. (1983a) Purification and initial characterisation of 124 kilodalton phytochrome from Avena. Biochemistry 22, 2498–2505

    Google Scholar 

  • Vierstra, R.D., Quail, P.H. (1983b) Photochemistry of 124 kilodalton Avena phytochrome in vitro. Plant Physiol. 72, 264–267

    Google Scholar 

  • Vierstra, R.D., Quail, P.H. (1986) The protein. In: Photomorphogenesis in plants, pp. 35–60, Kendrick, R.E., Kronenberg, G.H.M., eds. Martinus Nijhoff, Dordrecht Boston Lancaster

    Google Scholar 

  • Whitelam, G.C., Anderson, M.L., Billett, E.E., Smith, H. (1985) Immunoblot analysis of cross-reactivity of monoclonal antibodies against oat phytochrome with phytochrome from several plant species. Photochem. Photobiol. 42, 793–796

    Google Scholar 

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Author notes

  1. Mary L. Holdsworth

    Present address: Molecular Biology Institute, UCLA, 405 Hilgard Avenue, 90024, California, CA, USA

Authors and Affiliations

  1. Department of Botany, University of Leicester, University Road, LE1 7RH, Leicester, UK

    Mary L. Holdsworth & Garry C. Whitelam

Authors
  1. Mary L. Holdsworth
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  2. Garry C. Whitelam
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Holdsworth, M.L., Whitelam, G.C. A monoclonal antibody specific for the red-absorbing form of phytochrome. Planta 172, 539–547 (1987). https://doi.org/10.1007/BF00393872

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  • DOI: https://doi.org/10.1007/BF00393872

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