A STUDY OF MEMBRANE-ASSOCIATED PHYTOCHROME: HYDROPHOBICITY TEST AND NATIVE SIZE DETERMINATION

We confirmed that after extraction in the absence of added Mg²⁺, a small fraction of phytochrome was associated with pelletable, hydrophobic membranes. When microsomal material of several plant species was subjected to Triton X-114 phase partitioning, a part of phytochrome migrated into the hydrophobic Triton phase in contrast to soluble phytochrome. The amount of bound phytochrome partitioning into the Triton phase varied from 6% for oats to 30% for zucchini and 50% for mustard and maize (0.5–10% of total phytochrome). Membrane-associated phytochrome could be solubilized by the zwitterionic detergent CHAPS and with the nonionic detergent dodecylmaltoside. Subjected to gel filtration on Superose-6/FPLC, oat phytochrome of the CHAPS solubilized sample was eluted in three different molecular weight ranges. There was a main fraction with the molecular weight of purified phytochrome, another fraction (approximately 20%) with a higher molecular weight, and a third small fraction appearing immediately after the void volume. Gel filtration after solubilization by dodecylmaltoside resulted in two distinct fractions: the one eluted at the position of the phytochrome dimer, and the other (approximately 15%) with an apparent molecular weight of 800 kDa. Phytochrome was detected, separated and quantified by SDS-PAGE, and western blotting with the monoclonal antibody Z-3B1. We assume that the distinct, phytochrome positive, high molecular weight fractions contain phytochrome associated with hydrophobic protein.