TIME-GATED FLUORESCENCE OF BLEPHARISMIN, THE PHOTORECEPTOR PIGMENT FOR PHOTOMOVEMENT OF Blepharisma

Abstract— A computer-controlled apparatus for time-resolved laser fluorescence spectroscopy has been used to measure fluorescence lifetimes, time-integrated and time-gated spectra of crude extracts of blepharismin, the photoreceptor pigment of the ciliated photoresponsive protozoan Blepharisma japonicum , in ethanol, aqueous solutions and detergent micelles. The effect of hydroxyl concentration has been investigated in both alcohol and water solutions. A short-living (0.2-0.4 ns) molecular species, emitting at 600 nm, is predominant in aqueous solutions at pH < 11.7, whereas in pure ethanol solutions an intermediate-living species (about 1 ns), still fluorescing at 600 nm, prevails. Upon increasing OFF concentration, a third, long-living (about4–6 ns) molecular species, emitting at 660 nm, is formed in all the examined media. This species has been tentatively identified as the negatively charged form of the photoreceptor pigment, whereas the short-living and the intermediate-living fluorescence emissions have been attributed respectively to the phenolic and the quinonic neutral forms of blepharismin. The phenolic form in its ground state is suggested to be the molecular species from which proton release occurs.     

ADDITIONAL EVIDENCE FOR BLEPHARISMIN PHOTORECEPTOR PIGMENT MEDIATING STEP-UP PHOTOPHOBIC RESPONSE OF UNICELLULAR ORGANISM, Blepharisma

Abstract— In the ciliated protozoan, Blepharisma japonicum, the pink-colored pigment (blepharismin) contained in the pigment granules is believed to be the photoreceptor pigment responsible for the step-up photophobic response. When the cells partially bleached by extrusion of the pigment granules caused by cold shocks were subsequently cultured under illuminated conditions, the pigment-less granules regenerated and the cells were further bleached (pigment content below 0.5%). The photosensitivity of such colorless cells disappeared completely. In contrast, the blepharismin pigment regenerated gradually when such colorless cells were transferred to darkness. The photosensitivity of the cells also recovered with regeneration of the pigment. We found that blepharismin pigment was not photobleached in the absence of O₂. The step-up photophobic response was also completely repressed in the absence of O₂. These results strongly confirm that blepharismin is a photoreceptor pigment mediating photobehavior of Blepharisma and that O₂ is required for the early step in the phototransduction of the light-excited pigment.     

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.     

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: PIGMENT RELEASE BY Mg2+-GRADIENT ELUTION

Abstract— Gradient elution is used to facilitate controlled withdrawal of Mg²⁺ from phytochrome-rich particulate fractions from irradiated Avena sativa L. shoots. The bound pigment from red-irradiated tissue is released in a discrete band when the Mg²⁺ falls to just below 1 mM. This phytochrome has an apparent molecular weight of ?300 kilodaltons upon gel filtration, indistinguishable from that of the unbound pigment in the same extract and from that in the 50,000 × g supernatant from non-irradiated Avena. This indicates that the bound phytochrome is released as a soluble molecule at a Mg²⁺ concentration above that which permits release of the particulate binding partner from other particulate components. These findings appear to preclude the possibility that the phytochrome-binding partner association can be selectively preserved at a Mg²⁺ level that would permit separation and analysis of phytochrome-bearing particles without the complication of Mg²⁺-induced membrane and RNP aggregation. “Cycled” P_fr (that from tissue irradiated with a red-far red sequence prior to homogenization) is released at 0.1 to 0.2 mM Mg²⁺. This indicates that “cycled P_r is more tenaciously bound by the particulate fraction than is P_fr. This effect is photoreversible both by further in vivo and subsequent in vitro irradiations, suggesting that the state of the pigment, rather than of the binding partner, directly controls the tenacity of the interaction. Increasing concentrations of KCl release the pigment from the particulate fraction in the presence of 10 mM Mg²⁺; increasing Triton X100 concentrations do not. This confirms the ionic nature of the pigment-particulate fraction interaction and indicates strongly that the phytochrome is located external to any membrane vesicles present (although not necessarily that it is bound directly to such vesicles). The data further suggest that phospholipid polar head groups are not primarily responsible for the binding.     

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.     

月季花红色素的提取及稳定性的研究

一．引言月季，别名月月红，月季花（ＲｏＳａＣｈｉｎｅｎｓｉｓＪａｃｑ），属蔷薇科，蔷薇属，从每年的３月到１０月蓓蕾续放，开花不绝，现广泛栽培，主要分布于河北、陕西、山西等地。月季花主要成分为萜醇类化合物［１］，花、根及叶均可入药，内服可治妇女病，外用...     

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.     

红藻条斑紫菜R-藻红蛋白中的能量传递过程

本文通过对条斑紫菜R-PE(藻红蛋白)及其α-β-γ亚基的吸收光谱和荧光光谱进行计算机解叠,研究了R-PE内发色团之间的能量传递过程,并对R-PE及亚基内的各发色团进行了“s”和“f”型的指认。发现在亚基中为“f”型的发色团在R-pE(αβ)₆γ中起着“s”型发色团的作用,且将能量传递给最后的“f”型发色团。荧光激发偏振光谱进一步证明了R-PE内的能量转移过程与计算机解叠的结果一致。     

EXTRACTION AND SEPARATION OF RADISH RED PIGMENT FROM A WASTE WATER OF SALTING RADISH BY D61 RESIN

Extraction and separation of Radish red pigment from a waste water of salting Radish was studied on D61 resin.The exchange capacity of adsorption the pigment was equal to 60.91mg&#183;g^-1 wet resin,and the equilibium time only 40mins.All Radish red pigment adsorbed on D61 resin was eluted using a eluent in which concentraction of HCl or alcohol was 0.1mol&#183;L^-1 or 80% at 50℃ when the flow rate was at 2BV&#183;hm^-1.Stability of D61 resin was very well,and while the resin was recycled fifteen times, the exchange capacity was only decreased 11.9%,and the exchange capacity didn′t changed.Because of negative ion of mustard oil,it could′t be adsorbed on the resin.Finally, a paste product with yield of 1.96mg&#183;100ml^-1 waste water was obtained after alcohol evaporation and vacuum drying.     

ON THE PHOTORECEPTOR PIGMENT FOR PHOTOTROPISM AND PHOTOTAXIS: IS A CAROTENOID THE MOST LIKELY CANDIDATE?*

Abstract— The absorption spectrum of lycopene can be altered to show significant absorption in the 350–360 nm region in an ethanol-water mixture, thus resembling the phototropic action spectrum of Avena coleoptiles. The hypochromicity (bleaching) of the main absorption band and appearance of the new band at 350–360 nm can be attributed to exciton interactions between two stacked lycopene molecules. β-Carotene does not show anomalous bleaching under identical conditions. Thus, the apparent modification of the absorption spectra of carotenoids in ethanol-water mixtures cannot be used as an argument to resolve the action spectrum in terms of carotenoids. In addition, we have critically reviewed the spectroscopic characteristics of carotenoids. Short lifetimes of the excited singlet states and inefficient intersystem crossing of carotenoids are not compatible with the suggestion that carotenoids are the most likely candidate for the photoreceptor pigment in phototropism.     

减压柱层析—分光光度法分离和测定辣椒色素中的辣椒素

减压柱层析──分光光度法分离和测定辣椒色素中的辣椒素刘敬兰陈连文（河北师范大学实验中心０５００１６）（河北经贸学院轻工系０５００６１）关键词：减压柱层析，分离，辣椒素辣椒的辣味主要来源于辣椒素。辣椒素的化学名称为Ｎ－（３－甲氧基－４－羟苄基）－８－甲...     

ABSORPTION CHANGES IN BACTERIAL CHROMATOPHORES-II. A NEW CHLOROPHYLL-LIKE PIGMENT FROM THE OXIDATION OF CHROMATQPHORES FROM RHODOSPIRILLUM RUBRUM

Abstract— Evidence is presented which points to (at least) two bound forms of bacteriochlor-ophyll present in chromatophores of Rhodospirillum rubrum , both of them readily converted to unbound bacteriochlorophyll (abs. max 770 mμ) when the chromatophores are extracted with acetone or ethanol. Controlled oxidation of the chromatophores with Ir(IV) or with Zn (II) and ferricyanide preferentially destroys the more strongly absorbing pigment (abs. max 880 mμ) but brings about only a slight decrease in the magnitude of the photoinduced absorption changes at 810 and 792 mμ. Such oxidations yield a new pigment, absorbing at 715 mμ in the aqueous preparation and, more strongly, at 680–684 mu when the pigment is extracted into organic solvents. This pigment is formed irreversibly and is therefore different from the material formed by photooxidation of chromatophores. Its visible spectrum and the spectrum of the material formed from it by acidification suggest that it is a chlorophyll-like substance, possibly derived from bacteriochlorophyll by (two-electron) oxidation of one of the dihydropyrrole rings to a pyrrole ring. Directions are given for separation of this pigment from other colored compounds present in the oxidation mixtures.     

ORIGIN OF THE RED SHIFTED ABSORPTION IN PHYCOCYANIN 632 FROM Mastigocladus laminosus

A phycocyanin (PC) with γ_max= 632 nm (PC632) was isolated from extracts of the cyanobacterium Mastigocladus laminosus. The complex contained three polypeptides migrating in SDS-PAGE around 22 kDa. The N-termini of the three polypeptides are identical. They are homologous to rod-core linker polypeptides from this and another cyanobacterium and are identical to one of them. Reconstitution of PC (γ_max=614–620 nm) with the 22 kDa polypeptide produced again the red-shifted PC632. Phycobilisomes isolated at pH 6 or 7 contain, if any, only traces of 22 kDa polypeptides. Origin and functions of the polypeptides are discussed, they are most likely proteolytic fragments of allophycocyanin-PC rod-core linkers that contain the PC-binding domain.     

RED CHEMILUMINESCENCE FROM RAM SEMINAL VESICLE MICROSOMES: PITFALLS IN THE USE OF SPECTRALLY RESOLVED RED CHEMILUMINESCENCE AS A TEST FOR SINGLET OXYGEN IN BIOLOGICAL SYSTEMS

Abstract— The assignment of the red chemiluminescence bands in the ram seminal microsome system to singlet oxygen as previously reported by Cadenas and Sies (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 519-528 was re-evaluated. Measurements of 1268 nm emission demonstrated that ram seminal vesicle microsomes produced very small quantities of singlet oxygen (0.41 2 0.05 p.M) when they metabolized high concentrations of 15-hydroperoxyarachidonic acid (1 mM). The red chemilumi- nescence, however, was not due to singlet oxygen, since it failed to increase in deuterium oxide and it was five orders of magnitude larger than the predicted emission from singlet oxygen produced in this system. Quantitative measurements of the time integral of the square root of the red chemiluminescence intensity may be a useful test to confirm the assignment of red emission to singlet oxygen in other biochemical systems.     

ISOLATION AND CHARACTERIZATION OF PROTEINS FROM THE PUTATIVE PHOTORECEPTOR FOR POSITIVE PHOTOTAXIS IN THE DINOFLAGELLATE, Peridinium gatunense NYGAARD

Abstract –A chromoprotein fraction was isolated from the freshwater dinoflagellate, Peridinium gatunense, which is assumed to contain the photoreceptor chromoproteins for phototaxis, as its absorption and fluorescence spectra correspond to the action spectrum for phototaxis. The chromoproteins of this fraction were separated by anion-exchange column chromatography and further by sodium dodecylsulfate gel electrophoresis.     

RELEASE OF VOLATILES FROM CUBIC PHASES: MONITORING BY GAS SENSORS

Structured fluids such as emulsions and liquid crystalline mesophases can be used to control aroma release

This study shows that the use of a gas sensor array coupled with pattern recognition analysis can be particularly useful in studying volatile profiles

A mixture of 8 carefully selected volatile, hydrophilic and hydrophobic compounds was entrapped in 4 different matrices: water, lipids (sunflower oil, unsaturated monoglycerides), emulsion (20% wt water) and cubic phases (20 and 3% wt water)

A clear discrimination between the release pattern of the different matrices has been measured by the sensor array system. It has been shown that the cubic phase release patterns is not only controlled by its composition but also depended strongly on ihe lipid/water interfacial area

The combined use of structured fluids (structure-release relationship) and sensor arrays allows to control and to distinguish the release patterns of volatile.     

PHOTODYNAMIC RELEASE OF PROTOPORPHYRIN FROM INTACT ERYTHROCYTES IN ERYTHROPOIETIC PROTOPORPHYRIA: THE EFFECT OF SMALL REPETITIVE LIGHT DOSES

Abstract— Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to (hemo)globin. Irradiation of these cells causes a shift in fluorescence emission maximum and a decreased fluorescence intensity which is consistent with transfer of protoporphyrin from (hemo)globin to the cell membrane. When the erythrocytes were irradiated intermittently, nearly 70% of the protoporphyrin was released and the hemolysis was less than 3%. Giving the total light dose as a single pulse, resulted in 84% protoporphyrin release and 16% hemolysis.
In vivo the erythrocytes obtain small, repetitive light doses when circulating in the dermal capillaries. We suggest the possibility that in patients with erythropoietic protoporphyria these small light pulses could be sufficient to photodamage the binding place of protoporphyrin on (hemo)globin. In the dark, protoporphyrin can then move from (hemo)globin through the cell membrane and bind to albumin in the serum. Our findings indicate that if protoporphyrin is not present in the cell membrane during irradiation, no photohemolysis will occur. This may explain why patients with erythropoietic protoporphyria have no abnormal hemolysis. The effect of intermittent light pulses may also contribute to the understanding of the protoporphyrin release from erythrocytes in patients with erythropoietic protoporphyria.     

RUTHENIUM RED INHIBITION OF OXYGEN EVOLUTION AND SPECIFIC RELEASE OF THE EXTRINSIC 16 kDa POLYPEPTIDE IN A PHOTOSYSTEM II PREPARATION

The inhibitory effect of the dye ruthenium red was studied in photosystem II-enriched submembrane fractions. A number of distinct types of interaction were found, which differed in their concentration range and required incubation time. Ruthenium red instantaneously quenches the initial chlorophyll a fluorescence level (F₀) and the maximum fluorescence level (F_m) by enhancing radiationless deactivation in the chlorophyll light harvesting complex. Associated with this quenching of fluorescence is an instantaneous decrease in the quantum yield of oxygen evolution. Ruthenium red also inhibited the light saturated rate of oxygen evolution and the variable fluorescence, monitored 80 µs after a saturating excitation-flash. These inhibitions increased with incubation time and became greater than 50% within 5 min. Although ruthenium red was known to affect Ca²⁺ or Cl^? sites specifically, the inhibitory action was more pronounced than simple Ca²⁺ or Cl^? depletion. Incubation with ruthenium red for 5 min blocks the Z P680⁺ → Z⁺ P680 charge transfer reaction. Upon mixing with the photosystem II preparation, ruthenium red induced specific release of the extrinsic 16 kDa polypeptide associated with water-splitting without release of Mn. It is proposed that the inhibitor produces an ionic imbalance which alters the configuration of the donor side of photosystem II.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

PHOTORECEPTOR INTERACTION IN PLANT PHOTOMORPHOGENESIS: THE LIMITS OF EXPERIMENTAL TECHNIQUES AND THEIR INTERPRETATIONS

Abstract— Problems concerning the interpretation of interactions of higher plant photomorphogenetic receptors are discussed. The theory that action of a blue light photoreceptor serves only to maintain responsiveness to phytochrome (Responsiveness Theory) is demonstrated to be unable to be properly tested with present techniques. This theory is also unable to explain experimental results any better than an alternative theory that a blue light photoreceptor may require the presence of the active form of phytochrome to express its activity (Presence Theory). This tatter theory is also incapable of being fully tested. There does not appear to be an adequate current theory to explain photoreceptor interactions. Other issues discussed include the use of displacement transducers in growth studies, the induction of phytochrome-type responses by blue light, and the relative importance of the photoreceptors. New data are introduced on the effect of blue light in the end-of-day growth response to phytochrome of the light-grown Cucumis sativus L. hypocotyl, and on the light equivalence principle in the same species.