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Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg2+, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg2+, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg2+, phytochrome was associated with a "heavy" plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.Since the discovery of phytochrome and the elaboration of its ubiquitous role in the regulation of growth and development of flowering plants, its location within the cell and immediate mode of action have remained unknown. Information concerning its location has been mainly inferential, relating rapid phytochromemediated responses to physiological events at the plasma membrane (10-12) or the nuclear envelope (7). A part of the extracted phytochrome is associated with a particulate fraction (25,26,29); yet, a membrane or membrane fraction which binds phytochrome has not been identified.We examined different fractions obtained from soybean hypocotyls by differential and sucrose gradient centrifugation and observed that phytochrome content in the presence of Mg2+ cor- related with the presence of endoplasmic reticulum with attached ribosomes. In the absence of Mg2+, phytochrome was found in the so-called "heavy" plasma membrane fraction, possibly as a result of contamination of this fraction by fragments of endoplasmic reticulum. MATERIALS AND METHODSPlant Material. Soybean seeds (Glycine max L. cv. Wayne) were soaked in water for 6 hr and grown in moist vermiculite (9). After 4 days in darkness at 29 C, the cotyledons were removed, and the hypocotyl apices (approximately 1.5 cm) were harvested.Membrane Isolation. Isolations of membrane fractions were either in fluorescent room light (Table I) or in dim green light after a 5-min exposure of the tissue to red light (Tables II to IV). By either procedure, the phytochrome was expected to be predominantly in the far red-absorbing form at the time of isolation. Fifty to 100 g of tissue were finely chopped with a razor blade and suspended in cold (0-4 C) homogenizing medium (1 ml/g tissue).For isolations in the absence of Mg2+, the homogenizatio...

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Recent publications have indicated that a KCl-stimulated ATPase from cereal roots is specifically associated with plasmalemma-enriched membrane fractions. However, in previous work we found that relatively high specific activities of this enzyme were also associated with a membrane fraction which did not contain plasmalemma. In an attempt to clarify this discrepancy, we have investigated the effect of density gradient composition upon the association of the enzyme with different membrane fractions isolated from the roots of Zea (10) also found a small peak of KCIstimulated ATPase activity from oat roots at low buoyant densities but regarded it as less significant than the activity associated with the plasmalemma.Because there were obvious discrepancies in the reported distributions of KCl-stimulated ATPase activities in membrane fractions from cereal roots, we felt that the problem merited further investigation. One possible explanation for the anomalies lay in the different centrifugation systems employed. Williamson and Wyn Jones (24) used a Ficoll gradient followed by a sucrose gradient, whereas Hodges and colleagues (6, 10, 12) employed only sucrose gradients. The observation that the composition of density gradients can markedly affect the buoyant density of membrane vesicles (2,15,20)