A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN

Abstract
We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol. 92 , 179–185). By incubating pea stem sections in ³²P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°C. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.     

ISOLATION AND CHARACTERIZATION OF THE PRESUMED PHOTORECEPTOR PROTEIN OF Blepharisma japonicum

Abstract— Photosensory responses in the ciliated protozoan Blepharisma japonicum are mediated by a hypericin-like chromophore, blepharismin, localized in granules distributed under the cell membrane. A blepharismin-binding protein, with an apparcnt molecular weight ranging between 35 and 38 kDa, has been isolated by means of column separations and preparative isoelectric focusing and characterized by means of gel electrophoresis, analytic isoelectric focusing as well as absorption and fluorescence spectroscopy.     

PRESUMED PHOTORECEPTOR PROTEIN AND ULTRASTRUCI'URE OF THE PHOTORECEPTOR ORGANELLE IN THE CILIATED PROTOZOAN, Blepharisma

Abstract-The red pigment granule of Belpharisma japonicum is believed to be a photoreceptor organelle mediating photodispersal. Freeze-fracture and thin section electron microscopy revealed that the pigment granules contained a honeycomb-like structure constructed of folded membranes. In the fracture face of the honeycomb-like structure, small membrane particles were observed, which might correspond to pigment—protein complexes. The pigment granules were isolated and detergent-solubilized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the pigment granules mainly contained a 200 kDa membrane protein. The complex of red pigment and 200 kDa protein was isolated by gel-filtration chromatography of the detergent-solubilized components, and the protein was subjected to SDS-PAGE and amino acid analysis. The 200 kDa protein could not be dissociated into subunits by 2-mercaptoethanol, indicating that the protein is composed of a single polypeptide chain. Hydrophobic amino acids contained in the 200 kDa protein were not dominant, suggesting that only partial domains may extend across the membrane of the honeycomb-like structure.     

磷酸化mSA/mCS双极膜的制备与表征

以五氧化二磷、磷酸三乙酯和磷酸为反应剂,制备了磷酸化海藻酸钠(SA),经二茂铁离子改性后作为阳膜层;用乙酰基二茂铁改性壳聚糖(CS)为阴膜溶胶,将阴膜溶胶流延于上述阳膜层上,制备了PSA-二茂铁/CS-乙酰基二茂铁双极膜(mSA/mCSBPM)。测定了双极膜的电荷密度、离子交换容量、交流阻抗、机械性能、热重曲线及离子渗透性。应用红外光谱和扫描电镜对膜成分、形貌与工作性能进行了表征。测试结果表明,mSA/mCS双极膜致密,表面均匀,膜阻抗及工作电压均较小。     

INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

SPECTROSCOPIC AND PHOTOACOUSTIC STUDIES OF HYPERICIN EMBEDDED IN LIPOSOMES AS A PHOTORECEPTOR MODEL*

Abstract— In photoresponsive ciliates, like Blepharisma japonicum and Stentor coeruleus, the photoreceptor pigments responsible for photomotile reactions are hypericin-type chromophores packed in highly osmiophilic subpellicular granules. Liposomes loaded with hypericin can constitute a simple model system, appropriate for understanding the primary light-induced molecular events triggering the sensory chain in these microorganisms. Optical absorption, steady-state and time-resolved fluorescence and pulsed photoacoustic calorimetry have been used to measure spectral distributions, fluorescence lifetimes, radiative and radiationless transition quantum yields of hypericin when assembled into egg L-a-phosphatidylcholine liposomes. With respect to hypericin ethanol solutions, both absorption and fluorescence maxima are 5 nm red shifted when the pigment is inserted into the lipidic microenvironment, regardless of the hypericin local concentration. Increasing by 100 times the hypericin local concentration decreases the relative fluorescence quantum yield by a factor of around 150 and the fraction of thermally released energy, conversely, increases from 0.6 to 0.9. From the analysis of fluorescence lifetimes and their relative amplitudes it appears that a subnanosecond living component is predominant at the highest hypericin local concentrations.     

THE UV PROTEIN FLUORESCENCE OF PURPLE MEMBRANE AND ITS APOMEMBRANE

The ultraviolet fluorescence of the purple membrane of H. halobium and its apomembrane was characterized by measuring emission spectra, polarization, decay lifetimes and the changes induced by pH and temperature. The fluorescence quantum yields of the two membranes are 0.024 × 0.003 and 0.17 × 0.03, respectively. The emission, which shows lifetimes in the 0.4 to 4 ns range, was assigned to heterogeneous populations of emitters, consisting, probably, of two tryptophans in the purple membrane and seven or eight residues in the apomembrane. Acrylamide quenching experiments showed that the accessibility of this neutral quencher to the fluorophors is reduced greatly in both membranes. Fluorimetric methods were also used in an attempt to monitor the purple complex reconstitution process. It was concluded that the fluorescence quantum yields of any monomers, dimers and trimers present in the partially reconstituted membranes should be very similar.
Finally, based on the spectroscopic results and on specific folding patterns of the seven α-helical regions of bacteriorhodopsin (Stoeckenius and Bogomolni, 1982), it is proposed that Trp 137, Trp 138 (and perhaps Trp 10) of the protein molecule are the most plausible fluorophors in the purple membrane. It is also suggested that the protein in the apomembrane takes a more open configuration which is permeable to small ions and molecules.     

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED PHOTOHEMOLYSIS*

Abstract— Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).
Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3ß-hydroxy-5α-hydroperoxy-△⁶-cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bubbling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concentration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.     

PHOTOPHOBIC BEHAVIORAL RESPONSES OF EUGLENA IN A LIGHT INTENSITY GRADIENT AND THE KINETICS OF PHOTORECEPTOR PIGMENT INTERCONVERSIONS*

Abstract— Accumulation of Euglena gracilis in small illuminated regions called light traps is due to a phobic response to the diminished light intensity at the boundary of the region. The rate of such accumulation of cells was measured as functions of both the light intensity within the trap and the change of intensity at the boundary of the trap. The initial rate of accumulation of a population of cells was taken to be a direct measure of the phobic response of a single typical cell. The data indicate that the strength of the behavioral response in a single cell may be described as being proportional, to the rate of change of the amount of photochemically active form of a photoreceptor pigment molecule which can exist in two predominant forms.     

PLASMA POLYMERIZATION OF HYDROPHILIC AND HYDROPHOBIC MONOMERS FOR SURFACE MODIFICATION OF NUCLE-MICROPOROUS MEMBRANE

Surface modification of nucle-microporous membrane by plasma polymerization of HEMA, NVP and D_4 has been studied. The hydrophilicity of membranes was increased with increasing of plasma polymerization time of hydrophilic monomers HEMA and NVP. The flow rate of water through the membrane was increased remarkably after plasma polymerization of HEMA on it.     

EFFECT OF LIGHT QUALITY ON THE in vitro THYLAKOID PROTEIN PHOSPHORYLATION IN THE GREEN ALGA Scenedesmus obliquus

Abstract Thylakoid protein phosphorylation was assayed in vitro with isolated thylakoids of Scenedesmus obliquus by irradiation with monochromatic light of different wavelengths and equal photon fluences. The action spectrum for light-activated protein phosphorylation showed two maxima at 450 and 679 nm. A minimum of activity was reached around 580 nm. At this wavelength, the level of protein phosphorylation barely differed from that of dark-incubated samples. The action spectrum of thylakoid protein phosphorylation resembled the chlorophyll absorption spectrum obtained in vivo. The results show that chlorophyll is the photoreceptor for thylakoid membrane phosphorylation.     

PREPARATION AND CHARACTERIZATION OF SOLUBLE EGGSHELL MEMBRANE PROTEIN/CHITOSAN BLEND FILMS

Biopolymer chitosan was used to modify the mechanical properties of soluble eggshell membrane protein(SEP) films.The SEP/chitosan blend films were prepared by solution casting from 10%aqueous acetic acid.Tensile strength and elongation at break of the blend films increased with increasing amount of chitosan.Microphase separation was observed by field emission scanning electron microscopy,although interaction between the two components was revealed by FTIR.The biocompatibility of SEP/chitosan blend films ...     

THE EFFECT OF INHIBITORS AND UNCOUPLERS OF PHOTOSYNTHETIC PHOSPHORYLATION ON THE DELAYED LIGHT EMISSION OF CHLOROPLASTS*

Abstract— Measurements were made of the 3.7 msec delayed light emission of chloroplasts treated with a variety of agents which affect the rate of electron transport (Hill reaction) or photosynthetic phosphorylation. The presence of the electron acceptors ferricyanide or pyocyanine increased delayed light emission. Inhibitors of electron transport (3-(3,4-dichlorophenyl)-1, -1-dimethylurea or 1,10(ortho)-penanthroline) inhibited delayed light emission. The addition of a phosphate acceptor system inhibited delayed light emission. This inhibition was reversed by inhibitors of the phosphorylation reaction, e.g. Dio-9 or phlorizin. From these results it was concluded that the 3.7 msec delayed light emission probably occurs as a result of back reactions of intermediates in the coupled electron transport and photosynthetic phosphorylation systems.     

FLUORESCENCE OF Euglena gracilis PHOTORECEPTOR PIGMENT: AN in vivo MICROSPECTROFLUOROMETRIC STUDY

Abstract— The spectroscopic characterization of the photoreceptor pigment is one of the main questions in the study of the photosensory transduction chains in photomotile microorganisms. One of the possible techniques that can be used is in vivo microspectrofluorometry. By means of a tunable dye-laser microspectrofiuorometer developed by us, we have investigated some of the spectroscopic properties of the photoreceptor pigment of the green flagellate Euglena gracilis. The in vivo fluorescence excitation spectrum has been determined and the fluorescence quantum yield has been measured. The results show that flavins are indeed present in the paraflagellar body of E. gracilis and that their fluorescence quantum yield is much lower than that of a free flavin. An estimate of the order of magnitude of the rate constants for primary molecular reactions is tentatively given.     

PHOTOREAaIVITY OF CHLORAMPHENICOL in vitro AND in vivo

Abstract The negative side effects of chlorarnphenicol (CAP) mostly involve blood dyscrasias (e.g. irreversible nondose-dependent aplastic anemia), allergic skin reactions and eye damage. To learn the cause of these side effects, most research focuses on metabolically formed nitroso- and hydroxylamino derivatives in the predisposed patient. In previous investigations it was demonstrated that photochemical decomposition of CAP in vitro by UV-A leads to formation of p-nitrobenzaldehyde (pNB), p-nitrobenzoic acid (pNBA) and p-nitrosobenzoic acid (pNOBA); the latter comprises up to 45 mol% of the starting amount of CAP. Incubation of these photoproducts in rat blood showed that pNB and pNOBA rapidly react and that PNBA is stable under these conditions. Reaction products from pNB (half-life 1.7 min) proved to be pNBA and p-nitrobenzyl alcohol (pNBOH) while pNOBA (half-life 3.7 min) was converted into p-aminobenzoic acid (pABA). Exposure of CAP in rat blood to UV-A yielded the same end products: pNBA, PABA and pNBOH. To estimate the amount of oxidative stress generated in vivo by these compounds, the ability to form methemoglobin (MetHb) in erythrocytes was tested; only pNOBA and p-hydroxylaminobenzoic acid (pHABA), a possible intermediate in the decomposition of pNOBA, proved to be reactive. Ultraviolet-A exposure of rats, after intraperitoneal injection of CAP, led to 3.6 times the basic level of MetHb. In addition, covalent binding of ³H-labeled CAP photoproducts to the skin of the back and to the ears was found, which was 9.1 and 3.2 times higher, respectively, than the dark values. Toxicity toward bone marrow cells of all photoproducts was established in vitro. p-Nitrobenzaldehyde, pNOBA andpHABA were 20, 6 and 6 times more toxic than CAP, respectively. These results show that photodecomposition of CAP in vivo does occur. Its reactive photoproducts are able to cause damage that may lead to (systemic) side effects. The latter is supported by the fact that the nature of the reactive products, nitroso- and hydroxylamino derivatives, is the same as the expected metabolites.     

浅谈蛋白质含量量值溯源传递体系的构建

简述了蛋白质含量测定结果溯源到SI单位的重要性,结合中国计量科学研究院开展的工作及当前国际蛋白质计量发展的趋势,阐述了实现蛋白质含量测定结果溯源到SI单位的两种方案,方案中蛋白质标准物质的定值结果可以通过同位素稀释质谱法、滴定法、凝固点下降法等权威计量方法最终与SI单位连接,因此保证了蛋白质标准物质定值结果的准确和可溯源.以蛋白质标准物质作为量值的载体,可实现蛋白质含量量值在应用领域的传递,从而构建起蛋白质含量量值溯源和传递体系.     

PROTEIN COMPOSITION OF SPINACH CHLOROPLASTS AND THEIR PHOTOSYSTEM I AND PHOTOSYSTEM II SUBFRAGMENTS*

Abstract— The proteins of spinach chloroplasts and their subfragments containing photosystem I and photosystem II, obtained by Triton X-100 treatment or French-pressure rupture, were separated by sodium dodecyl sulfate (SDS)-acrylamide electrophoresis at pH 7·0 in phosphate buffer. The individual protein bands were identified where possible by comparing them with known, isolated and characterized proteins from chloroplasts, and their molecular weights were determined. The protein composition of the chloroplast fragments were correlated to the functional properties of these fragments. Distinct patterns were obtained for photosystem I and photosystem II particles. The major protein of photosystem II is expressed in the 23 kilodalton range and photosystem I proteins seem to be clustered mainly in the 50–70 kilodalton range.     

INTERACTION OF THE PEPTIDE HORMONE ADRENOCORTICOTROPIC ACTH(l-24), WITH A MEMBRANE MODEL SYSTEM: A FLUORESCENCE STUDY*

The peptide hormone adrenocorticotropin and a related peptide were studied in solution and in interaction with a model system of membranes (small unilamellar vesicles of dipalmitoylphosphatidylcholine and 17% dimyristoylphosphatidylglycerol) via fluorescence spectroscopy. In aqueous solution, intramolecular distances between the fluorescent residues R(Tyr²-Trp⁹) = 9.2 Å and R(Trp⁹-Tyr²³) 18 Å were obtained, in agreement with molecular models. Interaction of the peptide with the negatively charged membrane is evident from the alteration of the Trp photophysical parameters (quantum yield, fluorescence spectra and anisotropy), with a partition constant between the lipidic and aqueous phase of Kp =1–2 times 10³. The existence of two populations of Trp in the membrane, which are distinctly accessed by acrylamide, was concluded from the tryptophan fluorescence quenching study; the two fractions are located near the membrane interface as inferred from its fluorescence quenching by the 5-doxylstearate and 16-doxylstearate lipophilic quenchers. This result is further supported by energy transfer experiments to the 3-(9-anthroyloxyl)stearic acid and 12-(9-anthroyloxyl)stearic acid probes.     

WAVELENGTH-DEPENDENT EFFECTS OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A in vivo AND in vitro

Abstract Benzoporphyrin derivative monoacid ring A (BPD-MA) is a chlorin-like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P8 15 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon-ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBN2 mice injected with 2 mg/kg of BPD-MA and exposed to light at 125 J/cm², between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630–640 nm and 680–690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680–690 nm caused slightly more damage to normal skin than light delivered at 630–640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 68–95 nm. Antitumor efficacy in tumor-bearing DBN2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor-free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm. When tumor-bearing animals injected with BPD-MA at 2 mdkg and exposed to light 3 h later were treated with either 630 or 690 nm light at various doses, it was observed that 690 nm light was more effective at tumor ablation than was 630 nm light, demonstrating that while similar damage to normal skin may be effected by equivalent doses of light at either wavelength, tumor ablation was greater at 690 nm. Further, our data suggest that alternative light sources with bandwidths greater than those of the argon-ion pumped dye laser (±0.3 nm) may have equivalent efficacy with this photosensitizer.