Photosensitive Proteins Research Articles

Wide-band spectral tuning of heat receptors in the pit organ of the copperhead snake (Crotalinae).

Receptors located in the facial pit organ of certain species of snake signal the presence of prey. Infrared radiation is an effective stimulus suggesting that these receptors may be low-threshold temperature receptors. We recorded from the nerve innervating the pit organ of snakes belonging to the family of Crotalinae while stimulating the receptive area with well-defined optical stimuli. The objective was to determine the sensitivity of these receptors to a wide range (0.400-10.6 micro m) of optical stimuli to determine if a temperature-sensitive or photosensitive protein initiated signal transduction. We found that receptors in the pit organ exhibited a unique broad response to a wide range of electromagnetic radiation ranging from the near UV to the infrared. The spectral tuning of these receptors parallels closely the absorption spectra of water and oxyhemoglobin, the predominant chromophore in tissue. Our results support the hypothesis that these are receptors activated by minute temperature changes induced by direct absorption of optical radiation in the thin pit organ membrane.

Mechanism of nonlinear photoinduced anisotropy in bacteriorhodopsin and its derivatives.

Polymer films made with photosensitive chromophore protein bacteriorhodopsin (BR) from the extreme halophile Halobacterium salinarium as well as films made with BR derivatives exhibit a nonlinear photoinduced anisotropy. Two different methods can be used to induce anisotropy in polymer BR films. The first method is based on the anisotropic properties of the initial form of the photocycle, BR570 (B-type anisotropy). Another method is based on the anisotropic properties of the longest-lived photocycle intermediate M412 (M-type anisotropy). CW gas lasers were employed to induce a reversible anisotropy in polymer BR films. Nonlinear photoinduced anisotropy is discussed in the context of a model for the anisotropic photoselection of BR molecules under linearly polarized light. A comparison of the experimental dependencies of nonlinear photoinduced anisotropy on laser intensity with similar calculated dependencies enables one to determine the molecular dichroism of BR and its derivatives not only for the initial form of the photocycle, B but also for the longest-lived intermediate M. Here we present the data showing the correlation between the laser induced nonlinear anisotropic properties and chromophore/protein interactions in BR. The effect of polymer binder on the nonlinear photoanisotropic properties of polymer BR films is also described.

Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

Color vision of ancestral organisms of higher primates.

The color vision of mammals is controlled by photosensitive proteins called opsins. Most mammals have dichromatic color vision, but hominoids and Old World (OW) monkeys enjoy trichromatic vision, having the blue-, green-, and red-sensitive opsin genes. Most New World (NW) monkeys are either dichromatic or trichromatic, depending on the sex and genotype. Trichromacy in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage, but the process of evolutionary change from dichromacy to trichromacy is not well understood. Using the parsimony and the newly developed Bayesian methods, we inferred the amino acid sequences of opsins of ancestral organisms of higher primates. The results suggest that the ancestors of OW and NW monkeys lacked the green gene and that the green gene later evolved from the red gene. The fact that the red/green opsin gene has survived the long nocturnal stage of mammalian evolution and that it is under strong purifying selection in organisms that live in dark environments suggests that this gene has another important function in addition to color vision, probably the control of circadian rhythms.

Langmuir-Blodgett films of photosensitive proteins

The striking properties of monolayers and multilayers of photosensitive proteins obtained by using the Langmuir-Blodgett technique are described. The close packing of the protein molecules, which preserve most of the properties found in solution, seems to be the main cause for their thermal stability, which in some cases reached a temperature of 200°C without the loss of the protein secondary structure. The review is focused on three of the most intensively studied photosensitive proteins, namely photosynthetic reaction centres, bacteriorhodopsin and bovine rhodopsin, and on their possible applications as molecular optical devices.

Design of Intelligent Optical Sensors with Organized Bacteriorhodopsin Films

Oriented purple membrane (PM) films comprised of the photosensitive retinal protein bacteriorhodopsin (bR) was immobilized in an electrochemical photocell by means of bioaffinity bonding with bispecific (BS) anitibodies that recognize bR. Charge displacement photocurrent of significant efficiency was obtained from the homogeneously oriented PM film. Based on this technology, methods are described for the design of intelligent optoelectronic devices, which include image sensors for motion sensing, photoreceptor tips for visual prosthesis of human eyes, and environmental sensors for use in direction sensing and speed sensing.

電極表面修飾とその応用 感光性タンパク質の電極修飾とデバイスへの応用

電極表面修飾とその応用 感光性タンパク質の電極修飾とデバイスへの応用

Photosensitizer targeting in photodynamic therapy I. Conjugates of haematoporphyrin with albumin and transferrin

Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly- l-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer—protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.

Image sensing and processing by a bacteriorhodopsin-based artificial photoreceptor

Sensing and processing of optical information have been conducted with a unique bioelectronic image sensor that immobilizes bacteriorhodopsin (bR) as a photosensitive retinal protein. A thin film of bR-containing purple membranes was coated on a two-dimensional pixel array of electrodes and was made into a junction with an electrolyte gel layer having a counterelectrode to form an artificial photoreceptor. Photocurrent signals from each pixel showed a differential responsivity to light intensity, intrinsic to this liquid-junction photocell. Images detected and processed by the bR-based artificial photoreceptor were simultaneously displayed on a light-emitting-diode monitor panel through parallel signal-transmission circuitry. The experiment revealed that the photoreceptor is, as a retina model, capable of selectively detecting motion of images in real time and of performing vectorial extraction of their edge components, similar to the visual processing function of biological photoreceptors.

KINETICS OF DYNAMIC HOLOGRAM RECORDING IN POLYMER FILMS WITH IMMOBILIZED BACTERIORHODOPSIN

Abstract— Polymer films with immobilized photosensitive membrane protein, bacteriorhodopsin (BR), were prepared and investigated. A sensitive holographic interferometer with phase modulated optical beams was used for investigation of kinetics of dynamic hologram recording in BR polymer films. It was found that phase hologram formation consisted of two quasi-exponential stages demonstrating existence of a diffusive process in obtained films. It was shown that the kinetics of a phase hologram recording by high intensity beams do not correlate with the exponential law. The applicability of BR polymer films as reversible photorefractive materials for dynamic holography and real-time interferometry was discussed.

Fabricating photoreceptor and image sensor based on the photosensitive protein, bacteriorhodopsin

Fabricating photoreceptor and image sensor based on the photosensitive protein, bacteriorhodopsin

Depalmitylation with hydroxylamine alters the functional properties of rhodopsin.

Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).

The Chemistry of Vitamin A and Vision

AbstractVision is initiated in the retina by the photochemical isomerization of an 11‐cis‐retinal Schiff base to its all‐trans counterpart in the photosensitive protein rhodopsin. All‐trans ‐retinal is released, reduced to form all‐trans ‐retinol (vitamin A), and finally esterified. In order for vision to proceed an 11‐cis ‐retinoid must be regenerated in the eye. Kühne observed in the 1870s that the pigment epithelium must be in place behind the retina in order for rhodopsin to form. And indeed it was in the pigment epithelium that an isomerase system was recently discovered that can produce 11‐cis ‐retinol from added vitamin A. Pauling predicted in the 1930s that certain cis double bonds in carotenoids and retinoids, including 11‐cis ‐retinoids, should be quite unstable with respect to their all‐trans congeners. This prediction is borne out by experiment, but leaves open the crucial question as to where the energy comes from to drive the isomerization process in the eye. This question was answered by the demonstration that the energy is found in the membrane phospholipids. The fact that membranes may serve as an energy source reveals a previously unsuspected biological function of membranes. The chemistry of the novel energy‐linked isomerization process is explored in this review.

The Relationship of the Resonant Recognition Model to Effects of Low-intensity Light on Cell Growth

The resonant recognition model (RRM) is a model of protein-protein and protein-DNA interaction based on a significant correlation between spectra of numerical presentation of the amino acid or nucleotide sequences and their biological activity. Having compared absorption characteristics of photosensitive proteins with their RRM spectral characteristics we obtained a linear correlation between the RRM frequency space and real frequency space, which determines a scaling factor between these two frequency spaces. On applying the RRM model to several groups of peptide growth factors, characteristic RRM frequencies were revealed and the corresponding real characteristic frequencies for these groups of growth factors were calculated using the scaling factor previously obtained. The real frequency characteristics of growth factors obtained in this way correspond with maxima in the action spectra of low-intensity light irradiation effects on cell proliferation.

Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway

Palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter, has been shown to inhibit epidermal growth factor (EGF) binding to both high and low affinity receptors through a protein kinase C-independent pathway. In the present paper, we have investigated the mechanism of palytoxin action in Swiss 3T3 cells. Two lines of evidence indicate that calcium is not required for palytoxin activity. First, palytoxin can induce the loss of EGF binding sites in the absence of external calcium. Second, studies with the photosensitive protein aequorin indicate that palytoxin does not cause the influx of external calcium or the release of calcium from internal stores under the conditions used in these studies. However, palytoxin action does appear to be dependent upon the presence of sodium. When extracellular sodium is replaced by either choline, Tris, or sucrose, palytoxin is unable to decrease EGF binding to either high or low affinity receptors. Studies of sodium influx indicate that palytoxin induces rapid sodium uptake and that the rate of sodium uptake is dose-dependent. Furthermore, there appears to be a direct correspondence between the extent of inhibition of EGF binding by palytoxin and the rate of sodium uptake. Finally, the palytoxin-induced inhibition of EGF binding can be mimicked by monensin, a sodium ionophore. The specificity of this sodium dependence was tested by substituting lithium, potassium, or cesium for sodium. Although lithium is an effective substitute for sodium, palytoxin can no longer inhibit EGF binding when sodium is replaced by either potassium or cesium. Marked inhibition of palytoxin action is also obtained when 5.4 mM potassium or 5.4 mM cesium are added to the sodium-containing medium. These studies suggest that palytoxin is able to down-modulate the EGF receptor through a novel mechanism involving the activation or formation of a sodium pump or channel.

Comparison of the Effects of the Vasoconstrictive Dihydropyridines BAY K 8644 and CGP 28392 on Isolated Rat Aorta

The effect of BAY K 8644 was compared to that of CGP 28392 in isolated rat thoracic aorta. In low concentration both dihydropyridines had a direct concentration dependent contractile effect that could be eliminated in Ca-free medium and by the Ca-antagonist nifedipine. Readdition of Ca to the Ca-free medium restored the contractile response to both agents. In high concentrations (greater than or equal to 10(-5) M), both agents depressed active tone. In physiological salt solution containing 15.8 mM, potassium BAY K 8644 was far more potent in contracting rat aorta than CGP 28392. The responses to BAY K 8644 and CGP 28392 were not affected by alpha- and beta-adrenoceptorblockade. Both compounds shifted the noradrenaline- and potassium-concentration response curve to the left and increased the maximal response. Ultraviolet radiation (UVR) had a reversible and marked effect on BAY K 8644 and no effect on CGP 28392 contractions. The UVR-mediated relaxation of BAY K 8644-induced contractions could partly be eliminated by raising extracellular Ca. The results showed that differences according to potency and mode of action exist between BAY K 8644 and CGP 28392 in rat aorta. To explain the effect of UVR on BAY K 8644-induced contractions, a modification of a Ca channel model is proposed that implies the existence of "photosensitive proteins" located to "Ca antagonist" binding sites. According to this hypothesis, UVR may induce a reversible binding of BAY K 8644 to "Ca antagonist receptors."

Mechanism of colour discrimination by a bacterial sensory rhodopsin.

A photosensitive protein resembling the visual pigments of invertebrates enables phototactic archaebacteria to distinguish colour. This protein exists in two spectrally-distinct forms, one of which is a transient photoproduct of the other and each of which undergoes photochemical reactions controlling the cell's swimming behaviour. Activation of a single pigment molecule in the cell is sufficient to signal the flagellar motor. This signal-transduction mechanism makes evident a colour-sensing capability inherent in the retinal/protein chromophore.

KINETICS OF DYNAMIC HOLOGRAM RECORDING IN POLYMER FILMS WITH IMMOBILIZED BACTERIORHODOPSIN

AbstractPolymer films with immobilized photosensitive membrane protein, bacteriorhodopsin (BR), were prepared and investigated. A sensitive holographic interferometer with phase modulated optical beams was used for investigation of kinetics of dynamic hologram recording in BR polymer films. It was found that phase hologram formation consisted of two quasi‐exponential stages demonstrating existence of a diffusive process in obtained films. It was shown that the kinetics of a phase hologram recording by high intensity beams do not correlate with the exponential law. The applicability of BR polymer films as reversible photorefractive materials for dynamic holography and real‐time interferometry was discussed.

Isolation of water-soluble chlorophyll protein from the leaves of Chenopodium album

1. 1. A water-soluble, photosensitive chlorophyll protein was obtained from the leaves of Chenopodium album. 2. 2. The substance, purified by ammonium sulfate fractionation and column chromatography, showed a single-peaked sedimentation pattern on ultracentrifugal analysis. 3. 3. The native chlorophyll protein (prepared in the dark) showed absorption bands at 277, 429 and 667 mμ. Irradiation caused a marked change of the spectrum. New peaks appeared at 362, 399, 567 and 743 mμ. 4. 4. The chlorophyll protein contained chlorophylls a and b. The irradiated protein had three additional pigments which were not found in the native one.