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.     

PROTON, Mg2+ AND PROTEIN AS COMPETING LIGANDS FOR THE FLUORESCENT PROBE, MAG-INDO-1: A FIRST STEP TO THE QUANTIFICATION OF INTRACELLULAR Mg2+ CONCENTRATION

Abstract Increasing evidence of the role of magnesium in various cellular mechanisms has led to the need to develop an accurate method for the evaluation of magnesium concentration in cells. 1H-indole-6-carboxylic acid, 2–(4- bis- [carboxymethyl]amino-3–[carboxy]ethoxy) (mag-indo-1) is used as a fluorescent indicator for ionized magnesium concentration. A physicochemical study of this probe has pointed out (1) that at concentrations higher than 10 μ M , the presence of dimers can alter the different equilibria and (2) at concentrations, avoiding the dimer (≤ 10 μ M ), three fluorescent forms are in equilibrium with the deprotonated form of mag-indo-1 (L), which are the protonated form LH, the magnesium-bound form LM and the protein-bound form LP. A model is proposed that takes into account the equilibria between the four species. In a solution containing magnesium and protein, a complex fluorescence spectrum can be resolved by a combination of the three fluorescence spectra (L, LM, LP). However, under these conditions, the LH fluorescence spectrum is not taken into account for the spectral resolution. Finally, from the contribution of characteristic fluorescence spectra in the experimental fluorescence spectrum, the magnesium concentration can be estimated with accuracy. Such a method should be further applied to magnesium determination in different cell lines.     

THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

OPTICAL PROPERTIES OF PHYTOCHROME IN THE BLUE- SPECTRAL REGION AS MEASURED IN VIVO AND IN VITRO

In the blue spectral region, the phototransformation difference spectrum of oat phytochrome extracted as P_fr differs from that of phytochrome extracted as P_r. The difference absorbance maximum for phytochrome extracted as P_fr is at 420 nm, while that extracted as P_r is at 412 nm. The phototransformation difference spectrum measured in the blue in oat coleoptile tips without inner leaves, corresponds very well with that of phytochrome as extracted in its P_fr form. There is, however, a slight apparent attenuation of the blue difference band relative to those in the red-far-red. In coleoptile tissue containing inner leaves, the blue difference band is relatively even more highly attenuated. A similar attenuation is observed in the blue, in the protochlorophyllide to chlorophyllide phototransformation difference spectrum. In the spectrum measured with excised coleoptile without inner leaves, there is a small attenuation, while in coleptile tissue with inner leaves the attentuation is nearly 9-fold. These data suggest that the observed attenuation is probably artifactual. Neither instrumental non-linearity nor fluorescence induced by the measuring beam could explain the observed attenuation. It is suggested that the observed attenuation is probably mainly the result of wavelength dependent scatter amplification, the amplification in the blue being attenuated by the high background absorption of other pigments in this region.     

RADIATION ACTIVATION OF CARCINOGENS AND THE ROLE OF OH AND O2-

Abstract— Radiation-induced covalent binding of labelled carcinogens to DNA has been investigated under a variety of conditions using ultrafiltration or millipore filtration of TCA precipitable complexes. High yields of carcinogen binding at high DNA concentrations are also observed for a variety of small molecules and are not carcinogen-specific. At high carcinogen concentrations, radiation-induced unstable electrophilic carcinogenic species are produced, and undergo free-radical reactions which simulate cellular redox reactions involved in metabolic carcinogen activation, leading to the formation of covalently bound carcinogen adducts to DNA as a potential target macromolecule. The yields of carcinogen-DNA adducts increase linearly with dose and depend upon carcinogen concentration. The results of scavenger studies indicate that the oxidising species O₂^- and OH are the principal activating species. Rate constants for the selective radiation-induced oxidation reactions of various chemical carcinogens with superoxide have been measured by a competition kinetic method using pulse radiolysis. The relatively long-lived superoxide radical reacts with carcinogens at a rate which is two orders of magnitude slower than the diffusion-controlled rate for the hydroxyl radical, thus allowing a measure of O₂^- specificity in the presence of competing reactants within the cell.     

LOCATION OF THE CAROTENOID 2Ag-STATE AND ITS ROLE IN PHOTOSYNTHESIS

The location of the carotenoid 2A_g-state (S₁) was studied by fluorescence spectroscopy in two series of carotenoids. One consisted of natural polyenes like phytoene, phytofluene, β-carotene, and neurosporene, and the other of minicarotenes, i.e., compounds similar to β-carotene, but with a smaller number of double bonds (n=3–9). A decrease of the S₁-S₀ energy gap with n was observed in both series, and extrapolation to n= 11 gave the energies 14 500 and 13 200 cm^?1 for β-carotene and lycopene, respectively. The neurosporene S₁ state was located at ca 16 000 cm^?1. A good relationship between the nonradiative relaxation rate and the number of conjugation was observed. The rate increased by a factor of 2.5 per double bond. The possible role of the S₁ state in reversible carotenoid ← chlorophyll electron-exchange energy transfer is discussed.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES

Abstract
In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M N and N bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES*

Abstract— In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M → N and N → bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M → N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

THE CONTRIBUTION OF RHODOSPZRZLLUM RUBRUM FERREDOXIN II TO IN VIVO EPR SIGNALS AND ITS ROLE IN ELECTRON TRANSPORT

Evidence is presented that Fd II, an iron-sulfur protein containing 4 irons and 4 acid-labile sulfurs, is responsible for a number of signals previously reported detectable in cells of R. rubrum. When oxidized, Fd II exhibits a g = 2.012 EPR signal which is readily detected in R. rubrum cells. In our hands, Fd II is photochemically reduced to an EPR-silent product contrary to the results of other investigators. However. in the presence of reducing agents. the reduced form is apparently denatured upon freezing. The denatured form exhibits EPR signals similar to some also previously observed in whole cells. Fd II catalyzes the ascorbate-DCPIP linked photoreduction of NAD with R. rubrum chromatophores even in the presence of an inhibitor which suppresses the formation of pH and emf gradients. This result raises the possibility of a role for Fd II in non-cyclic electron transport in R. rubrum.     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

REGENERATION OF NAD+ AND NADP+ COFACTORS BY PHOTOSENSITIZED ELECTRON TRANSFER

Abstract The irradiation with visible light of photosensitizer dyes like methylene blue or N-methyl phenazonium methyl sulfate leads to the oxidation of reduced coenzymes such as pyridine nucleotides (NADH or NADPH). This photoredox reaction can be used to regenerate the oxidized form of these coenzymes in enzymatic reactions and total consumption of a substrate with catalytic amounts of enzyme, coenzyme and photosensitizer can be performed. The process has been studied on two common enzymatic reactions: ethanol oxidation by alcohol-NAD ⁺ -oxidoreductase and gluconate-6-phosphate oxidation by 6-phospho-D-gluconate-NADP⁺-2-oxidoreductase. In the first case, a turnover number of 1125 has been obtained for the photoregeneration of NAD ⁺ from NADH.     

Na+/K+ TRANSPORT AND PHOTOSENSITIVITY OF THE COLORLESS FLAGELLATE Peranema trichophorum (Euglenida)

Abstract— Perimycin, ouabain and elevation of extracellular K⁺ concentrations cause an increase in the fluence rate thresholds (white light) for the step-up photophobic response in Peranema trichophorum . Elevation of extracellular Na⁺ concentration decreases the thresholds for this response in comparison to the control level. The fluence rate threshold of perimycin-treated cells increases before the side effect of an antibiotic action appears. Removal of K⁺ ions from the medium of K⁺-treated cells to a concentration of 1 mM depresses the threshold for the step-up response to the control level. By addition of K⁺ or Na ⁺ ions to perimycin- or ouabain-treated cells the threshold returns to the control value. It is suggested that the flagellar and cell membrane are responsible for changes of P. trichophorum photosensitivity.     

PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2

The photophysics of the complex forming reaction between Quin-2 and Ca²⁺ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca²⁺ buffer: k₀₁= 8.6 times 10⁸ s^?1, k₂₁= 1 times 10¹¹M^?1 s^?1, k₀₂= 8.8 times 10⁷ s^?1, k₁₂= 4 times 10⁴ s^?1. k₀₁ and k₀₂ denote the respective deactivation rate constants of the Ca²⁺ free and bound forms of Quin-2 in the excited state. The constant k₂₁ represents the second-order rate constant of binding of Ca²⁺ and Quin-2 in the excited state while k₁₂ is the first-order rate constant of dissociation of the excited Ca²⁺:Quin-2 complex. From the estimated values of k₁₂ and k₂₁ the dissociation constant K_d* in the excited state was calculated. It was found that pK_d* (6.4) is slightly smaller than pK_d (7.2). There was no interference of the excited-state complex forming reaction with the determination of K_d. Intracellular Ca²⁺ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca²⁺ indicator.     

在Y型分子筛中Co2+、Ni2+配合物的合成及其应用研究

用气相法和液相法分别在Y型分子筛的超笼中合成了Co²⁺、Ni²⁺的乙二胺配合物,并用红外(FT-IR)、顺磁共振(ESR)等方法对配合物的组成和稳定性进行了研究。CO²⁺的单核配合物在室温下可与O₂分子形成加合物,这种优先并可逆地吸附氧气的性能在氧气富集、对空气进行氧氮分离和食品保鲜等方面有广泛的用途。     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

THE REACTIVITY OF SUPEROXIDE (O2-) and ITS ABILITY TO INDUCE CHEMILUMINESCENCE WITH LUMINOL

Abstract— The quantum yield and the kinetics of O -induced luminol chemiluminescence was investigated in a broad pH interval at varying luminol and concentrations. It is suggested that the weak chemiluminescence observed is mediated via a luminol-superoxide-adduct proposed to be an a-hydroxyperoxyl radical. At pH 7 the maximum quantum yield of chemiluminescence per initial percent was determined to be 4 times 10^-8. The degree of involvement in phagocytosis and related processes should be viewed against this maximum limit.     

DETERMINATION OF THE BINDING CONSTANT OF H14 CO−3 TO THE PHOTOSYSTEM II COMPLEX IN MAIZE CHLOROPLASTS: EFFECTS OF INHIBITORS AND LIGHT

The binding (dissociation) constant for HCO^?₃ to the photosystem II complex in maize chloroplasts is approximately 80 μM. One HCO^?₃ binds per 500–600 chlorophyll molecules. In the dark, formate is a competitive inhibitor of HCO^?₃ binding, while 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea (DCMU) inhibits HCO^?₃ binding non-competitively. Light decreases HCO^?₃ binding in the presence of formate. Light increases the binding of HCO^?₃ in the presence of DCMU. The high binding constant for HCO, discriminates strongly among the various hypotheses attempting to explain the “bicarbonate-effect” on photosystem II. The proposal by Stemler and Jursinic (Arch. Biochem. Biophys. 221, 227–237 1983), that HCO^?₃ is one of a class of monovalent anionic inhibitors of photosystem II, is favored. These anions compete for a specific binding site on the photosystem II complex.     

PHOTOCHEMICALLY INDUCED BINDING OF Rh(phen)2Cl2+ TO DNA†

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.     

EFFECTS OF EXTRACELLULAR Ca2+ CONCENTRATION AND PAPAVERINE ON THE VISUAL CELL FUNCTION IN THE ISOLATED FROG RETINA

Abstract— Effects of extracellular Ca²⁺ concentration and papaverine on the PIII response of the electroretinogram and the dark adaptation process of the visual cells were studied in the isolated, aspartate-treated bullfrog retina. The amplitudes of both the fast and slow PIII responses are increased in 0.01 m M Ca²⁺ solution, but decreased in Ca²⁺-free solution containing 1 m M EDTA. The application of 0.1 m M papaverine in the presence of 1 m M Ca²⁺ led to the enhancement of the slow PIII response at lower stimulus intensity and the prolongation of the slow PIII response, but these effects of papaverine on the response were lost when Ca²⁺ was removed from the bathing fluid. The half-time of recovery of the fast PIII response amplitude after switching off the adapting light was a linear function of the amount of bleached rhodopsin. Papaverine in the absence of Ca²⁺ produced about 2-fold increase in the half-time of recovery of the response. These findings suggest that chemical reactions which are sensitive to papaverine in the absence of Ca²⁺ are implicated in the dark adaptation process of the visual cells.     

CYTOPROTECTION AGAINST MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF THE Na+,K+-ADENOSINE TRIPHOSPHATASE IN LEUKEMIA CELLS: GLUTATHIONE AND SELENOPEROXIDASE INVOLVEMENT

When irradiated with broad-band visible light in the presence of merocyanine 540 (MC540), murine leukemia L1210 cells grown under selenium-deficient conditions (Se(-) cells) accumulated lipid hydroperoxides and lost viability more rapidly than selenium-satisfied controls (Se(+) cells). These findings suggest that cytoprotection against photoperoxidation and photokilling is mediated at least in part by selenoperoxidase (SePX) action. Similar protection against photoinactivation of an intrinsic membrane enzyme, the Na⁺,K⁺-ATPase, has been observed. Thus, irradiation of MC540-sensitized Se(-) cells resulted in an immediate and progressive inactivation of ouabain-sensitive Na⁺, K⁺-ATPase; by contrast, activity loss in Se(+) cells was preceded by a prominent lag. Enzyme photoinactivation in Se(-) cells was inhibited by ebselen, an SePX mimetic, confirming that SePX(s) is (are) involved in natural protection. Desferrioxamine treatment (iron sequestration/inactivation) resulted in higher hydroperoxide levels and slower Na⁺,K⁺-ATPase inactivation during MC540/light exposure, whereas ferric-8-hydroxyquinoline treatment (iron supplementation) had the opposite effect. Thus, iron appears to play an important role in both of these processes. In contrast, photoinactivation of another intrinsic enzyme in L1210 cells, acetylcholinesterase (AChE), was unaffected by selenium or iron manipulation. On the basis of these findings, we propose that lipid peroxidation plays an important role in the photoinactivation of Na⁺,K⁺-ATPase, but not AChE. This is consistent with the fact that Na⁺, K⁺-ATPase's active site lies within the membrane bilayer, whereas AChE's active site lies outside the bilayer.