Separation of Particulate Phytochrome and Plasma Membranes of Maize Coleoptiles by Density Gradient Centrifugation

Abstract

Phytochrome, cross linked in situ to its receptor by glutaraldehyde fixation, and radioactively-labelled membrane material, obtained by lactoperoxidase-catalysed ¹²⁵I-treatment of maize coleoptiles could be separated from each other according to sedimentation velocity and by sucrose density gradient centrifugation. Binding of iodine to membrane material in maize coleoptiles increased several-fold on the addition of reaction-specific enzymes, i.e. lactoperoxidase and glucose oxidase. Mitochondria were considered not labelled because mito-chondrial purification reduced ¹²⁵I incorporation. Membrane material containing incorporated iodine appeared quite heterogenous and sedimented to an equilibrium density position close to, but slightly lighter than that of the mitochondria; participate phytochrome banded at a heavier position. Results obtained therefore suggest that the receptor for phytochrome may not be on the plasma membrane as envisaged in recent hypotheses.