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Planta

, Volume 174, Issue 3, pp 396–401 | Cite as

Sequence analysis of proteolytic fragments of 124-kilodalton phytochrome from etiolatedAvena sativa L.: Conclusions on the conformation of the native protein

  • Rudolf Grimm
  • Christoph Eckerskorn
  • Friedrich Lottspeich
  • Claudia Zenger
  • Wolfhart Rüdiger
Article

Abstract

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.

Key words

Avena (phytochrome) Endoproteinases Phytochrome (conformation) Trypsin 

Abbreviations

kDa

kilodalton

Pfr

far-red-absorbing form of phytochrome

Pr

red-absorbing form of phytochrome

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Rudolf Grimm
    • 1
  • Christoph Eckerskorn
    • 2
  • Friedrich Lottspeich
  • Claudia Zenger
    • 1
  • Wolfhart Rüdiger
    • 1
  1. 1.Botanisches Institut der Universität MünchenMünchen 19Federal Republic of Germany
  2. 2.Max-Planck-Institut für Biochemie-GenzentrumMartinsriedFederal Republic of Germany

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