Open AccessUse of 125 I-labeled phytochrome to quantitate phytochrome binding to membranes of Avena sativa
Author(s) -
Gradimir Georgevich,
Thomas E. Cedel,
Stanley J. Roux
Publication year - 1977
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.74.10.4439
Subject(s) - phytochrome , avena , biochemistry , sodium dodecyl sulfate , membrane , biology , phytochrome a , biophysics , pigment , absorbance , binding site , chromatography , chemistry , botany , red light , organic chemistry , arabidopsis , gene , mutant
Purified oat phytochrome was labeled with125 I without altering the photoreversibility or absorbance properties of the pigment. The radiolabeled phytochrome was used in experimentsin vitro to quantitate the binding of the pigment to both crude and purified membrane preparations from oat tissue. After the membranes were allowed to react with125 I-labeled phytochrome, washed free of unbound material, and pelleted, they were found to have significant levels of radioactivity bound to them. Qualitative identification of phytochrome as the bound radioactive species was confirmed by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the proteins contained in the washed membranes. Data supporting the specificity of the binding are that the binding shows saturation kinetics and that unlabeled phytochrome, but not bovine serum albumin, will competitively inhibit the binding of labeled phytochrome. This technique permits the detection of less than a nanogram of phytochrome and provides a new method for quantifying bound phytochrome that is independent of the spectral detectability of the pigment. It should be useful in elucidating the nature of phytochrome attachment to cellular membranes.