Article

Photoreversible Conductance Changes Induced by Phytochrome in Model Lipid Membranes

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Abstract

The plant protein phytochrome induces photoreversible conductance changes when added to a black lipid membrane made from oxidized cholesterol. The conductance of the phytochrome-modified membrane is increased by red-light illumination but is decreased by illumination with far-red light. Denatured phytochrome does not affect the conductance of the model membrane.

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... Phytochrome binds to cell membranes (17,19,23) and alters properties of natural (15, 20, 25-27, 29, 30, 33, 34) and model (22) membranes; recent evidence also suggests that changes in membrane properties play a central role in the operation of the biological clock (3,7,12,28,31). Changes in the Pf, level induce phase shifts in endogenous rhythms in Lemna (13) and Phaseolus (1,2) implying that phytochrome interacts with the rhythmic oscillator in these plants. ...
Article
Phytochrome, a membrane-localized biliprotein whose conformation is shifted reversibly by brief red or far-red light treatments, interacts with the rhythmic oscillator to regulate leaflet movement and potassium flux in pulvinal motor cells of Samanea. Darkened pinnae exposed briefly to red light (high Pfr level) have less potassium in motor cells in the extensor region, more potassium in motor cells in the flexor region, and smaller angles than those exposed to far-red light (low Pfr level). Increase in temperature from 24° to 37° increases the differential effect of the light treatments during opening (the energetic phase) but not during closure, implying that phytochrome controls an energetic process. It seems likely that phytochrome interacts with rhythmically controlled potassium pumps in flexor and extensor cells. During nyctinastic closure of white-illuminated pinnae, exposure to far-red light before darkening results in larger angles than does exposure to red. As in rhythmic opening, the angles of all pinnae and the differential effect of the light treatments increases with increasing temperature.
... The assumption that photo-gated channels may involve a direct effect of photoreceptors on membrane permeability is acknowledged in a number of works. For example, early experiments on artificial bilayer phospholipid membranes showed that exogenic phytochrome could change the conductivity of the membranes in relation to its form (Pr or Pfr) [107]. It has been also studied how RL and RL with FRL affect the membrane potential of Characeae cells [108]. ...
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During a plant life, light is necessary not only as a source of energy, but also as a regulatory factor of plant metabolism with information signal function. In this review we consider basic links of primary stages of light signal transduction in higher plants. The transformation circuits and possible pathways of photoreceptor light signal transduction, as well as possible roles of photoreceptor-interacting proteins, secondary messengers and some transcriptional factors are discussed. The review is also focused on examination of rapid signaling events such as activation of ion exchange systems as well as interaction of photoreceptors in signaling pathways.
... Correlation of this association with an in vitro response to Pfr is reported for enhanced peroxidase action in plumular hooks of Cucurbita pepo L. (110). Pfr is known to decrease the resistivity of model mem branes (124). Steps between membrane association of Pfr in a tissue and display undoubtedly follow diverse pathways. ...
... Various photopotentials have been reported, for instance from chloroplasts (Witt and Zickler, 1973;Fowler and Kok, 1974;Bulychev and Vredenberg, 1976) or visual photoreceptors (Brown and Murakami, 1964;Cone and Pak, 1971). There has been extensive work done on model systems, such as planar lipid bilayer membranes, doped either with natural dyes (Tien, 1972;Trissl and Lauger, 1972;Schadt, 1973;Frohlich and Diehn, 1974), synthetic dyes (Verma, 1971;Ullrich and Kuhn, 1972;Duchek and Huebner, 1979), or photosensitive proteins (Roux and Yguerabide, 1973;Montal et al., 1977;Herrmann and Rayfield, 1978;Bamberg et al., 1979;Chen and Berns, 1979;Hong and Montal, 1979). Recently, techniques have been developed to measure interfacial charge displacements with a time resolution on the order of nanoseconds and with a sensitivity of 100l,uV (Huebner, 1979;Trissl, 1980;Trissl and Griaber, 1980). ...
Article
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The concept of chemical capacitance as introduced by Hong and Mauzerall (Proc. Natl. Acad. Sci. U.S.A. 1974. 71:1564) is critically reexamined. This novel capacitance was introduced to explain the time-course of flash-induced photocurrents observed in lipid bilayer membranes containing porphyrins. According to Hong and Mauzerall, the chemical capacitance results from a combination of three fundamental capacitances: the geometric membrane capacitance and the two interfacial double layer capacitances. The concept of chemical capacitance is questioned for the following reasons: (i) The system analysis is insufficiently determinate. (ii) The measured chemical capacitance is approximately 0.16% of that predicted by the theory. (iii) The fact that only 20% of the membrane area is illuminated was not considered in the analysis. The latter point offers an alternative explanation of the capacitance in question: this capacitance may reflect that fraction of the total membrane capacitance that is photochemically active. If so, the concept of chemical capacitance lacks general significance.
... Paleg (1972, 1974) found that gibberellic acid increased glucose permeability of a liposome preparation. Roux (1973) was able to incorporate an impure phytochrome preparation into a black lipid film conferring red/far red light reversible conductivity changes on the film. ...
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In the presence of the plant hormone 2-cis,4-trans abscisic acid, bimolecular lipid membranes exhibited conductance fluctuations of the type associated with channels. Under a voltage clamp of 100 mV, channels were observed of mean conductance 0-4 × 10−10 S and mean lifetime 0·2 msec. Membrane conductance was measured as a function of concentration of abscisic acid and a log-log plot of the data revealed a linear relationship. The slope of the regression line was 0·52 ± 0·08 with Pearson's product moment 0·85. An analysis of current fluctuations is presented. The results are discussed in terms of a process in which multimeric channels are generated from abscisic acid monomers and in which channels are destroyed by collision.
Chapter
Electrical measurements in cells and tissues serve two major purposes. The first, previously reviewed in this series (Findlay and Hope 1976), is in the evaluation of whether the movement of particular ions across membranes is passive or active. The second is in the detection of membrane-localized changes that indicate alterations in the physiological status of plant cells. Since observations of electrical phenomena are made relatively non-destructively in living cells, they can provide a continuous record of cell membrane activity under a variety of physiological conditions. Accordingly, certain electrical measurements have proven to be useful in detailing the initial events of such complex responses as growth, nastic movements, morphogenetic transformations and reaction to stress. Although the electrical phenomena in plants precede conspicuous developmental changes, it is not clear if, or how, the electrical events play a causal role in these responses. This review intends to examine the evidence for light-induced changes in electrical parameters in plant tissue, and evaluate the relationship of such electrical signals to longer-term photomorphogenetic changes.
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Samanea leaflets usually open in white light and fold together when darkened, but also open and dose with a circadian rhythm during prolonged darkness. Leaflet movement results from differential changes in the turgor and shape of motor cells on opposite sides of the pulvinus; extensor cells expand during opening and shrink during closure, while flexor cells shrink during opening and expand during closure but change shape more than size. Potassium in both open and closed pulvini is about 0.4 N. Flame photometric and electron microprobe analyses reveal that rhythmic and light-regulated postassium flux is the basis for pulvinar turgor movements. Rhythmic potassium flux during darkness in motor cells in the extensor region involves alternating predominance of inwardly directed ion pumps and leakage outward through diffusion channels, each lasting ca 12 h. White light affects the system by activating outwardly directed K(+) pumps in motor cells in the flexor region.
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Plants growing in the natural environment are exposed daily to prolonged periods of high intensity irradiation. Many plant photomorphogenic responses are fully expressed only under prolonged exposures to high irradiances of light. The intensive study of these responses, the “High Irradiance Responses” (HIR) of plant photomorphogenesis, which started about 20 years ago, has been essentially directed—so far—toward the identification of the HIR photoreceptor, or photoreceptors, a problem that has not been satisfactorily and definitively solved, as yet. There is a great deal of evidence in support of the hypothesis that phytochrome, the pigment mediating the redfar red reversible plant photo-responses to low fluences of light, is involved in the photocontrol of the HIR. It seems likely that phytochrome may be the only photomorphogenic receptor responsible for the photocontrol of HIR responses brought about by irradiation at wavelengths longer than 600 nm. Phytochrome is probably also involved in the photocontrol of the HIR effects brought about by irradiation in the 350 to 500 nm region of the spectrum, but it cannot be excluded that other photochemical systems may also be involved. From a theoretical point of view, it does not seem unreasonable that the final expression of an HIR response may involve an interaction between phytochrome and other photochemical systems, with phytochrome probably playing the primary role and being responsible for the control of the activity of the other systems. Numerous “phytochrome only” interpretations (models) of the HIR have been proposed. Some of them have been developed to a fairly high degree of elaboration and have allowed the prediction of at least some of the features of the HIR. These “models,” although not rigorously and completely tested yet, seem to provide a reasonable interpretation for the HIR effects displayed under prolonged far red irradiation and for those HIR responses for which far red is the most effective spectral region. However, they do not provide a satisfactory explanation for the HIR responses for which blue is the most or the only effective spectral region, nor for the high effectiveness of white light. But, in spite of these problems, the “phytochrome only” interpretations of the HIR can be considered more satisfactory than those based on an interaction between phytochrome and other photochemical systems, especially in relation to the fact that the identity of these other photochemical systems has not been defined yet.
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Phytochrome (P), a chromoprotein of 120,000 MW, occurs at low concentrations in all higher plants. The chromophore is an open tetrapyrrole. The pigment exists in two light-absorbing forms: Pr, which absorbs at 660 nm, and Pfr, which absorbs at 730 nm. These forms are interconvertible by light. Pr, the physiologically inactive form, exists in dark-grown plants; Pfr, the active form, appears after irradiation with red light, P-mediated responses, of which about 80 are known, range from short-time effects (sec) such as bioelectric potentials, to long-time effects (hr) such as increases in enzymatic activity. Measurements of phototransformation in vivo with polarized light suggest that P is localized in the plasma membrane. Particulate cell fractions contain about 70% of total extractable P if Pfr is present and only 4% if Pr is present. Evidence indicates that the fraction containing Pfr may be the plasma membrane. One can isolate a partially solubilized membrane system, which can be reversibly reconstituted by adding Mg. The reformed vesicles bind Pfr in vitro. Pfr binding increases with decreasing pH and decreases with increasing monovalent cation concentration. Pfr is released from the membrane by far red light (Pr is formed) and by Triton X-100. We suggest that Pfr binding to a membrane induces conformational changes; the functional properties of this membrane are altered, which might lead to the observed phytochrome-mediated responses.
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Maize (Zea mays L. cultivar WF-9XM-14) and pumpkin (Cucurbita pepo L. cultivar Black Beauty) were grown in the dark at 25° C. Collection and all subsequent manipulations were performed in green safelight. The upper 1 to 2 cm of 5-d-old corn coleoptiles and the hypocotyl hook of 5-d-old pumpkin seedlings were used as starting material.
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The absorption of light energy not only nourishes the plant through photosynthetic phosphorylation and concomitant carbon dioxide fixation, but it also determines the nature and the direction of plant growth. This review will summarize the major advances in the physiology of both of the latter processes over the last half-century. Because of severe limitations of space, only a few key concepts can be mentioned, let alone emphasized, and some fields, like algal phototaxis, must be omitted entirely. Although there was certainly provocative earlier work by Klebs and others (100), our present understanding of the control of form by light, or photomorphogenesis, began with the clear enunciation of the principles of photoperiodism by Garner and Allard (55, 56) a little more than 50 years ago. Photomorphogenetic transformations are mediated mainly by the longer wavelengths of the visible spectrum (105), and are these days attributed mainly or exclusively to absorption by phytochrome. The development of the phytochrome concept by
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Moderate doses of red (660 nanometer) irradiation cause a rapid increase in the translocation of fluorescein in dark-grown mung bean hypocotyl (Vigna radiata L.) segments. The increase fails to appear following large doses of red (660 nanometers) irradiation. The red induced increase is prevented by a subsequent far red (730 nanometer) irradiation. Reversibility suggests the participation of phytochrome in the process. The increase in translocation is attributed to the generation of a positive electrostatic charge in the plasma membrane by some action of phytochrome on membrane molecules.
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A red light-induced, far red reversible stimulation of proton efflux from apical segments of etiolated Avena sativa L. cv. Victory coleoptiles was observed. The acidification responses to red light and also to auxin were not the consequence of respired CO(2). The response to red light was strongly inhibited by cycloheximide and carbonyl cyanide, m-chlorophenyl hydrazone, but mannitol had a stimulatory effect. Red light and auxin applied together yielded a greater than additive response, in comparison to the effects of the two stimuli applied separately.
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Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light-induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site.
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Abstract— Photobiological processes such as photosynthesis, photomorphogenesis, photomovement, and photoreception are all associated with the membranous portions of cells. The unique properties of membrane surfaces are apparently required to achieve biologically relevant energy transduction and photocontrol phenomena and consequently the use of model membrane systems is suggested as an advantageous approach to elucidation of the important physical and chemical processes involved. Black lipid membrane (BLM) and liposome techniques are critically reviewed as preferred techniques for constructing and manipulating lipid bilayers. The lipid bilayer is considered to be the basic foundation for biological membrane models, and specific physical phenomena observed with the bilayers and their biological ramifications are analyzed. Light-stimulated polarization of the membrane and electron transfer across the bilayer are viewed as appropriate analogs of vision and photosynthesis, respectively. Bilayer-adsorbed dye experiments are the simplest systems explored that exhibit polarization and charge transfer across the membrane. Chloroplast extract BLM experiments are cited as an example of the light-stimulated transfer of electrons across the membrane under the influence of a preexisting redox gradient. Biliprotein (phycocyanin or phycoerythrin) on one side of the chloroplast extract membrane permits the direction of electron flow across the membrane so that a redox gradient is created in a manner truly analogous to photosynthesis. The potential for solar energy conversion from such membranes is explicitly considered utilizing a schematic photoelectrochemical cell. Model membranes containing bacterial rhodopsin and phytochrome represent examples of ionic gradients that result in biological energy transduction. Studies of membranes that exhibit transient photoeffects are considered potentially relevant for the elucidation of phototaxis. The analysis of many properties of photosensitive membranes is greatly aided by the use of appropriate theoretical models. It is apparent that there is a great potential for the application of photosensitive model membranes in many research areas involving complex photobiological phenomena and novel methods for solar energy conversion.
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Abstract—The dose response curve for light (phytochrome)-induced anthocyanin synthesis was determined in the mustard seedling. The curve gives the amount of anthocyanin (A) synthesized within 24 h as a function of the amount of Pfr* produced by a brief light pulse. The [Pfr] response curve is composed of two linear parts with very different slopes (a1,2) connected by a relatively narrow transient range (curved segment). The [Pfr] response curve extrapolates precisely through zero [Pfr]. The reciprocity law is valid over the whole range investigated (up to 320 s of irradiation). It is concluded that the initial (or primary) reaction of Pfr (Pfr+ X → PfrX) does not involve any significant cooperativity in the case of phytochrome-mediated anthocyanin synthesis. It is speculated that the linear parts of the [Pfr] response curve truly reflect the mode of phytochrome action (A=a1,2 [Pfr]; X does not come into play since it is not rate limiting) whereas the curved segment represents a transition of the reaction matrix of Pfr. The large difference between a1 and a2 seems to indicate that the physiological effectiveness of a given amount of Pfr (or PfrX) is determined by [Pfr] through a Pfr-induced change in the reaction matrix.
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Highly purified samples of phytochrome from etiolated oat (Arena satira L.) seedlings were analyzed for sugar content. Phytochrome was found to be a glycoprotein, with about 4% of its weight as carbohydrate. In addition, modifications in the procedure for purifying phytochrome are proposed here and certain details about the protocol are discussed that improve the yield and reproducibility of previously published methods.
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The effect of light on the size of intact protoplasts isolated from the primary leaves of etiolated Triticum aestivum was studied. A 2-min red-light irradiation in the presence of 1 mM KCl was sufficient to cause a swelling of protoplasts compared with those maintained in darkness. The effect was photoreversible by far-red light over two light cycles, indicating the involvement of phytochrome. At 4°C, escape from reversibility occurred between 2 and 5 min after the exposure to red light. In exposure-response experiments, 20 s red light at 27 μmol m(-2)s(-1) was sufficient to saturate the response. Exogenous gibberellic acid added in darkness in the presence of KCl also induced protoplast swelling. Gibberellins may act as an intermediate in the phytochrome-induced swelling of protoplasts.
Chapter
The kinetics of phytochrome-mediated responses can be a powerful analytical tool in the quest to elucidate this photoreceptor’s molecular mechanism of action. Clearly those responses most rapidly detectable upon photoconversion are the ones most likely to be closest in sequence to the primary action of the pigment. The observation that several rapid phytochrome-mediated responses appeared to involve changes in membrane properties led Hendricks and Borthwick (1967) to propose the “membrane hypothesis” of phytochrome action. In its most explicit formulation, this hypothesis proposes that phytochrome modifies the functional properties of one or more cellular membranes as its primary action upon photoconversion, and that this modification results from the direct physical interaction of the pigment with the components of those membranes. All other observed alterations in cellular and molecular function are then postulated to ensue in cascade fashion from this single molecular mechanism of action.
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During a plant life, light is necessary not only as a source of energy, but also as a regulatory factor of plant metabolism with information signal function. In this review we consider basic links of primary stages of a light signal transduction in a plant cell. The transformation circuits and possible pathways of transfer of a light signal by major photoreceptors, phytochrome and cryptochrome, as well as possible roles of effectory proteins and secondary messengers are discussed.
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Phytochrome is a chromoprotein that serves as the photoreceptor for a wide range of photomorphogenic responses in plants. This chromoprotein exists in two photointerconvertible forms at physiological temperatures. One form absorbs maximally near 665 nm (Pr) and is considered inactive, while the other absorbs maximally near 730 nm (Pfr) and is morphogenically active. Since the discovery of this pigment some 25 years ago (Borthwick, 1972), two different, although not necessarily mutually exclusive, approaches to an understanding of its mode of action have been taken. One approach has been to investigate phytochrome-mediated responses in attempts to deduce the nature of the primary events that lead to these responses. A second approach has been to investigate the biophysical and biochemical properties of the pigment so that a direct path to understanding its molecular mechanism of action might become available.
Chapter
Publisher Summary In higher plants, the process of photosynthesis occurs within specific membrane bounded organelles called “chloroplasts.” All the chloroplasts exhibit three major structural regions— namely, highly organized internal sac-like flat compressed vesicles called “thylakoids,” an amorphous background rich in soluble proteins called “stroma,” and a pair of outer membranes known as the “envelope.” The envelope essentially renders functional and structural integrity to the chloroplast. This chapter discusses the structure, isolation, chemical composition, and origin of the higher plant chloroplast envelope. The chapter examines the multiple functions of this important membranous system involved in the regulation of the inflow of raw materials for photosynthesis and the outflow of photosynthetic products. The chloroplast envelope of higher plants is a permanent structure and consists of two morphologically and topologically distinct membranes separated by a region about 10–20 nm thick, which appears electron-translucent. The structure of both envelope membranes is consistent with the lipid-globular protein mosaic model of membrane structure as proposed by Singer and Nicolson.
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The association between phytochrome and multilamellar soybean lecithin dicetylphosphate-stigmasterol vesicles, was examined by means of red light-enhanced pelletability. The presence of stigmasterol increased the liposome :phytochrome interaction. Far-red light partially reversed the binding and this reversibility was increased after 2 h in the dark. Partial phytochrome purification by high speed centrifugation or addition of protamine did not reduce binding. The amount of bound Pfr was increased by preincubation with liposomes in the dark before illumination. This procedure, was applied to a wheat phytochrome extract that did not show in vitro red light enhanced pelletability. More than half the phytochrome pelleted after a dark preincubation with liposomes at 25°C followed by a red light irradiation at 25°C, suggesting that pelletability is dependent on temperature and on the physicochemical properties of the phytochrome extract (possible presence of endogenous binding inhibitors). Phytochrome binding on liposome could be a useful model for the study of phytochrome-membrane interactions and related membrane properties.
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The hypothesis was tested whether phytochrome interacts with black lipid membranes (BLM) as indicated eventually by red/far red reversible changes of the electrical membrane resistance. Such changes have not been found with unmodified BLM made from phosphatidylcholine, with membranes modified in their surface charge by octadecylamine or by TMHA salts and with membranes from oxidized cholesterol. The experiments do not exclude more specific interactions between cellular membranes and phytochrome.
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PHYTOCHROME is a chromoprotein that has been shown to initiate and control major developmental responses of plants to light1. It has two spectrally different forms that can be reversibly interconverted by light, as indicated:The spectral properties of phytochrome are sensitive to changes in the microenvironment of the covalently bound chromophore(s). Relatively mild denaturing treatments, such as incubation at 25 °C for over an hour2 and dehydration3, result in a significant loss of photoreversibility. In view of this susceptibility to spectral denaturation, it is remarkable that phytochrome can be proteolytically degraded to a molecular weight (MW) of 60,000, or half its native subunit size, without exhibiting any loss of photoreversibility or even any blue shift in its peak absorbance as Pr4. The additional fact that further proteolysis of phytochrome to 40,000 MW drastically reduces photoreversibility5 suggests that the 60,000 MW fragment has a sequence and configuration that are both sufficient and necessary to preserve photoreversibility. It is not clear whether this fragment is the product of one or more centrally located cleavages of the native subunit to yield two pieces of MW near 60,000 per native subunit or whether it is the product of extensive proteolysis which yields a single 60,000 MW fragment per native subunit. We have compared the amino acid composition and the peptide map of the proteolytically-derived 60,000 MW fragment with those of the native 120,000 MW subunit. Our results strongly support the conclusion that the native 120,000 MW subunit is composed of two halves with nearly identical sequences.
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Abstract— This paper describes a method for rapidly monitoring early changes in electrolyte permeability induced by phytochrome from salt-loaded liposomes. The method allows for the continuous monitoring of low-level ion efflux from liposomes by measuring the conductivity of a liposome suspension medium which has osmotic and chemical potentials that promote a slow. passive efflux of the compartmented electrolytes. The addition of the far-red absorbing form of phytochrome (Pfr) to this system at 20°C immediately produces efflux rates which are 2–3 times greater than if the red-absorbing form (Pr) is added. This differential effect is not evident at 4°C and varies with the lipid composition of the liposomes. Under conditions in which Pfr induces a 2-fold greater change in the electrolyte permeability of liposomes than Pr. only about 18% more 125I-labeled Pfr than 125I-labeled Pr binds to the liposomes. At equimolar concentrations. the photochromic small peptide of phytochrome (60 000 dalton monomer) and the more native‘large’phytochrome (120000 dalton subunits) induced equivalent changes in the electrolyte permeability of liposomes. No differential leakage of ATP, glucose, or trvpsin from liposomes was observed after Pr and Pfr reacted with vesicles enclosing these substances. The Pfr form of phytochrome promoted greater turbidity in liposome suspensions and a greater degree of aggregation and/or vesiclc fusion than Pr. The kinetics of these changes suggested that they were not the hasis of the differential permeability effects of Pr and Pfr.
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The electric potential difference changes observed on etiolated oat coleoptiles in response to phytochrome transformation have been further studied using contacts on the coleoptile surface. Results are given, at 0.4 second resolution, for the first 1.5 minutes after saturating flashes of light each lasting 1 second.Responses to initial red (662 nanometers), to far red (about 700 nanometers and above) 10 minutes later, and to second red 10 minutes later still, all have time courses that are approximately Gaussian sigmoid in shape. The response to far red is of opposite sign to the response to red. Approximate magnitudes of the three changes, 1 minute after the light flash, are +6 millivolts for red, -10 millivolts for far red, +3.5 millivolts for the second red. It is argued that the observations reflect a hyperpolarization of the plasmalemma of coleoptile cells following red light and depolarization following far red. The response to red is not produced by a change in membrane permeability to K(+). The mechanism could include a change to Na(+) or Cl(-) permeability or a modulation of an electrogenic pump: enhanced H(+) extrusion, Ca(2+) extrusion, or Cl(-) uptake. The response to far red could be produced by the reverse of one of those changes.The Gaussian curve is fitted to the data to determine the time at which the responses begin. Each response begins 4.5 seconds after the start of the flash of light. These delays are not related to the time course of phytochrome pelletability or redistribution in the cell. The delays may be due to some interaction of the transformed phytochrome with the plasma-lemma. Alternatively, the transformed phytochrome may interact quickly with some other structure which initiates a signal that takes 4.5 seconds to reach the plasmalemma.
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The polarities of a large number of soluble and membrane proteins have been calculated by summing the mole fractions of polar amino acids. It was found that 85% of the 205 soluble proteins considered in this study had polarities of 47 +/- 6%. Only 2% of the soluble proteins had polarities below 40%, whereas 47% of the 19 membrane proteins had polarities below 40%. The membrane proteins with polarities below 40% could be separated from their respective membranes only by detergents or organic solvents, indicating the importance of hydrophobic forces in their interaction with other membrane components. It is concluded that the majority of "intrinsic" membrane proteins have low polarity, and that the polarity index is therefore a useful parameter for characterization of membrane proteins.
Article
Action potentials are constructed step by step in bimolecular lipid membranes by adjusting the membrane composition, ionic gradients, pH, temperature and the concentration of two proteinaceous adsorbates: an excitability inducing material (EIM) of mol. wt less than 105 and protamine sulfate. They show most bioelectric kinetic phenomena and generally conform to the Hodgkin and Huxley theory for action potentials in nerve. The evidence indicates that the system consists of two ion selective channel types. One, produced by EIM, develops a cationic e.m.f.; the other, resulting from a complex between EIM and protamine, develops an anionic e.m.f. Both contain a double gating mechanism showing two negative resistances which are controlled by the voltage and by chemical factors including membrane lipid composition, ionic strength, pH, some alkaloids, acridine and phenothiazine derivatives and divalent ions. The action potentials result from the interplay of e.m.f.'s and resistances of the two channel populations each acting as a parallel battery and a voltage dependent variable resistive load on the other coupled via the membrane potential. Some possible molecular mechanisms responsible for the conductance changes are discussed.
Article
Phytochrome was extracted from etiolated oat seedlings and purified 750-fold by a four-step procedure involving chromatography on calcium phosphate, chromatography on Sephadex G-200, continuous electrophoresis on a free-flowing film, and finally gel filtration on Bio-Gel P-150. The product obtained was homogeneous to electrophoresis on cellulose polyacetate or acrylamide gel. Exclusion chromatography indicated that phytochrome had a molecular weight of about 60,000. The molecular extinction coefficients for the red-absorbing form maxima were ε280 82,000, ε382 26,000, and ε664 76,000.
Article
Phytochrome is consistently observed in pellets centrifuged from homogenates of etiolated, 5-day-old oat seedlings. The majority of pigment associated with the pellet cannot be removed by buffer washes, nor can appreciable quantities of additional phytochrome be adsorbed onto the sedimented material. Over 70% of phytochrome in the pellet is released by 1% Triton X-100.Storage at 0 degrees , irradiation by white light, and Triton treatment all cause much greater loss of photoreversibility in pelleted phytochrome than in supernatant phytochrome. We conclude that the phytochrome in the 1500 to 40,000g (30 min) pellet is distinct from the soluble phytochrome in the supernatant.
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