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

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Abstract
This study employs 125I-labeled phytochrome (125I-P) from oats [Avena sativa] 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 125I-Pfr (far-red absorbing form) to the mitochondrial fraction of unirradiated oat seedlings is significantly higher than that of 125I-Pr. 125I-Pfr and 125I-Pr bind in equal quantities to mitochondrial preparations isolated from light-exposed seedlings. Maximum 125I-Pfr binding to membranes from light-exposed plants occurs within 30 s and is optimized in a reaction buffer containing 5 mmol MgCl2 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 .times. 1011 per molar. When optimal binding conditions are used, over 20% of the 125I-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, 125I-Pfr shows no specific binding to rat liver mitochondria.

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