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Articles

The Isolation and Partial Characterization of a Membrane Fraction Containing Phytochrome ^1,2

^a The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

If 4-day-old dark-grown zucchini squash seedlings (Cucurbita pepo L. cv. Black Beauty) are exposed briefly to red light, subsequent cell fractionation yields about 40% of the total extractable phytochrome in the far red-absorbing form bound to a particulate fraction. The amount of far red-absorbing phytochrome in the pellet is strongly dependent on the Mg concentration in the extraction medium. The apparent density of the Pfr-containing particles following sedimentation on sucrose gradients corresponds to 15% (w/w) sucrose with 0.1 mM Mg and 40% sucrose with 10 mM Mg. This particulate fraction could be readily separated from mitochondria and other particulate material by taking advantage of these apparent density changes with changes in Mg concentration. Electron microscopy of negatively stained preparations shows that with 1 mM Mg only minute particles are present. These were too small to reveal structural detail with this technique. With 3 mM Mg, separate membranous vesicles between 400 and 600 Ångstroms in diameter appear. At higher Mg concentrations, the vesicles aggregate, causing obvious turbity. The effect of Mg on vesicle formation and aggregation is completely reversible. Above 10 mM Mg, vesicle aggregation persists, but the percentage of bound Pfr decreases.

⁴ Present address: Laboratoire de Photobiologie, C. N. R. S. (L. A. 203). Faculté des Sciences de Rouen, 76130 Mont-Saint-Aignan, France.

⁵ Present address: Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. 94305.

¹ This work was supported by National Science Foundation Grant GB-30964X to W. R. B., by the Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif., by the Deutsche Forschungsgemeinschaft (SFB 46), and by the Délégation Générale à la Recherche Scientifique et Technique. During the work, D.M. held a Max Kade Foundation Fellowship, J. M. M. a National Institutes of Health training grant predoctoral fellowship (Training Grant TOIGM00036), and J.B. a North Atlantic Treaty Organization fellowship. The authors are very grateful for this aid.

² Dedicated to Solon A. Gordon.