Gel filtration of particle-bound phytochrome

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings has been separated into two fractions by gel filtration on Sepharose CL-2B. One fraction with a K av of 0.7 was detected only after red irradiation (in vivo or in vitro). This separated from a ribonucleoprotein fraction during gel filtration. The weak interaction with ribonucleo-protein which required magnesium (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The second phytochrome fraction was strongly associated with a high molecular weight material with a K av of less than 0.1. Low levels of this complex were detected in extracts from dark grown tissue but were increased by red irradiation of excised hooks or crude extracts. The binding of phytochrome to the high molecular weight material did not require magnesium, was unaffected by ribonuclease treatment, and was much more resistant to high salt concentrations than was the phytochrome-ribonucleoprotein association. These results suggest that the association of phytochrome with this membrane-containing fraction is not electrostatic.The separation by agarose-gel filtration offers a useful technique for the preparation of membraneassociated phytochrome for physiological studies.