Use of ¹²⁵ I-labeled phytochrome to quantitate phytochrome binding to membranes of Avena sativa

Abstract

Purified oat phytochrome was labeled with 125 I without altering the photoreversibility or absorbance properties of the pigment. The radiolabeled phytochrome was used in experiments in vitro to quantitate the binding of the pigment to both crude and purified membrane preparations from oat tissue. After the membranes were allowed to react with 125 I-labeled phytochrome, washed free of unbound material, and pelleted, they were found to have significant levels of radioactivity bound to them. Qualitative identification of phytochrome as the bound radioactive species was confirmed by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the proteins contained in the washed membranes. Data supporting the specificity of the binding are that the binding shows saturation kinetics and that unlabeled phytochrome, but not bovine serum albumin, will competitively inhibit the binding of labeled phytochrome. This technique permits the detection of less than a nanogram of phytochrome and provides a new method for quantifying bound phytochrome that is independent of the spectral detectability of the pigment. It should be useful in elucidating the nature of phytochrome attachment to cellular membranes.

Modulation of a Mitochondrial Function by Oat Phytochrome in Vitro

“…These results do strongly support the postulate that the reactant phytochrome molecules are physically interacting with the mitochondrial membranes; and they encourage a further investigation into the possibility that there are specific binding sites for phytochrome on the mitochondria membrane, as suggested by earlier data (12). Related to this question may be the fact that NADH-dehydrogenase is a flavin mononucleotide-containing flavoprotein, and recent data from W. Smith's laboratory suggest that phytochrome may interact very specifically with flavin mononucleotide in vitro (28).…”

“…Our finding that highly purified phytochrome could bind to mitochondria with a reproducible stoichiometry suggested that there were a limited number of phytochrome binding sites on mitochondria; and competition experiments suggested that these sites were relatively specific for phytochrome (12). Although the intact outer membrane of mitochondria is not freely permeable to molecules larger than 12,000 mol wt (8), phytochrome is probably large enough to span this membrane (29).…”

“…The experiments for d and e were conducted by adding 5 tcg of exogenous phytochrome as Pr or Pfr to the mitochondria prior to the assay. This amount had been determined to be sufficient to saturate the mitochondrial binding sites under the experimental conditions used (12).…”

“…In the present paper, we are reporting our tests of whether Manabe and Furuya's results with pea mitochondria can be obtained with oat mitochondria. We also describe tests of whether exogenously added phytochrome, previously shown to bind to mitochondria with a reproducible stoichiometry (12), could influence the activity of the external NADH-dehydrogenases of mitochondria (22) that we supposed would be accessible to exogenous phytochrome. For these experiments, we have used extensively characterized mitochondrial preparations, and discuss why such characterizations are important for interpretation of the results.…”

Irradiation-enhanced Phytochrome Pelletability

“…The potency of the present batch of colchicine was verified by testing its effect on cell division in a culture of Chiamydomonas reinhardtii. Cell division was completely blocked by 10 m 3 of the drug (data not shown). CONCLUSIONS The present data show that the intracellular dark reaction responsible for enhanced phytochrome pelletability proceeds with a t1/2 of 2 sec at 25 C following Pfr formation in both maize and Avena.…”