Further Characterization of the in Vitro Binding of Phytochrome To a Membrane Fraction Enriched for Mitochondria

This study employs (125)I-labeled phytochrome ((125)I-P) from oats to quantitate the binding of phytochrome to a membrane fraction from oats that is highly enriched for mitochondria, and it examines several parameters that influence this attachment. The binding of (125)I-Pfr to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of (125)I-Pr. However, (125)I-Pfr and (125)I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum (125)I-Pfr binding to membranes from light-exposed plants occurs within 30 seconds and is optimized in a reaction buffer containing 5 millimolar MgCl(2) at pH 6.8. Scatchard plots of the binding data for Pfr indicate a single high-affinity site with an affinity constant of 1.79 x 10(11) per molar. When optimal binding conditions are used, over 20% of the (125)I-P added is bound and a stoichiometry of about 100 molecules per mitochondrion is attained. When the specificity of binding is tested using competition experiments with a 15-fold excess of unlabeled phytochrome, (125)I-Pfr shows no specific binding to rat liver mitochondria.