Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Miki Nakazawa, Junko Kakihana, Toru Hisabori, Katsushi Manabe

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

Procedures for the purification of native phytochrome from etiolated pea seedlings without the use of immuno-purification techniques are described. Phytochrome (in the PFR form) was purified by polyethyleneglycol fractionation, adsorption to pentyl agarose and batch elution, chromatography on DEAE-Sepharose, adsorption to phenyl Toyopearl and batch elution, and chromatography on Red Toyopearl. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280 of PR) that ranged from 0.55 to 0.6. The subsequent chromatography on Sephacryl S-300 yielded very pure phytochrome with a SAR of 0.98. PR and PFR peaks in the difference spectrum of the phytochrome were centered at 665 and 730 nm, respectively. The spectral change ratio (ΔAr/ΔAfr) of the difference spectrum was unchanged after the chromatography on phenyl Toyopearl, and the value was 1.05-1.08, indicating that the spectral properties of this preparation were intact. The absorption spectra indicated that the peak absorbance of PFR was at 728-730 nm and that of PR was at 666-667 nm. These peak positions were essentially same as those obtained with the undegraded oat phytochrome. Incubation of the sample purified on Sephacryl S-300 at 25°C for 5 h in either the PR or PFR form did not result in degradation of the molecule. The rate of dark reversion of PFR observed with the purified pea phytochrome was similar to that observed in vivo. The addition of dithionite had no effect on the reversion rate.

Original languageEnglish
Pages (from-to)789-796
Number of pages8
JournalPlant and Cell Physiology
Volume31
Issue number6
Publication statusPublished - 1990
Externally publishedYes

Keywords

  • PFR dark reversion
  • Phytochrome
  • Pisum sativum
  • Purification

Field of Science

  • 1.6 Biological sciences

Publication Type

  • 1.1. Scientific article indexed in Web of Science and/or Scopus database

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