GEL-FILTRATION OF PARTICLE-BOUND PHYTOCHROME

Pelletable phytochrome from hypocotyl hooks of Cucurbita pepo L. seedlings was separated into 2 fractions by gel filtration on Sepharose CL-2B. One fraction with a Kav [average elution profile] 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 ribonucleoprotein which required Mg (optimal at 10 mM) was overcome by high salt concentrations and prevented by ribonuclease treatment. The 2nd phytochrome fraction was strongly associated with a high molecular weight material with a Kav of < 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 Mg, was unaffected by ribonuclease treatment, and was more resistant to high salt concentrations than was the phytochrome-ribonucleoprotein association. Apparently 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 membrane-associated phytochrome for physiological studies.