ON THE NATURE OF THE INTERMEDIATE ELECTRON ACCEPTOR (A1) IN THE PHOTOSYSTEM I REACTION CENTER

Abstract— A sodium dodecyl sulfate-Photosystem I (PSI) complex has been prepared and characterized with respect to its electron acceptors. Component X and iron-sulfur centers A and B are absent from this preparation but the intermediate electron acceptor (A₁) is present. Flash-induced absorbance changes at 25°C show charge separation, followed by a back-reaction with a half-time of 5 µs. The spectrum of the flash-induced change from 350 to 550 nm indicates a contribution from the intermediate electron acceptor, A₁, as well as from P700⁺. EPR studies show that A₁ is associated with a free-radical signal having a g-value of 2.0025 and a linewidth of 12 gauss. A, would appear to be associated with a monomeric form of either Chi α or pheophytin a.     

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.     

Melanocyte-stimulating Properties of Secretory Phospholipase A2

Abstract— Phospholipase A₂ (PLA₂) catalyzes the release of free fatty acids from membrane phospholipids, and its products derived from these fatty acids, such as prostaglandins and leukotrienes, significantly up-regulate the key mela-nogenic enzyme, tyrosinase, in melanocytes. This has led to suggestions that PLA₂ itself triggers melanin synthesis in melanogenesis following UV irradiation or inflammation.
We have examined the effect of secretory PLA₂ (sPLA₂) on melanogenesis in cultured human melanocytes. Secretory PLA₂ stimulated DNA synthesis and melanin synthesis, and these phenomena were completely inhibited by treatment with a phospholipase inhibitor, p- bromophenacyl bromide, demonstrating that the catalytic activity of sPLA₂ is required for melanogenesis. Secretory PLA₂ also stimulated tyrosinase activity, increased the amount of tyrosinase-related protein-1 and up-regulated the expression of both mRNA. These findings suggest that sPLA₂ is an important mediator of UV-induced or postinflammatory pigmentation.     

ELIMINATION OF THE EFFECT OF CONTAMINATING CO2 IN THE 18O-LABELING OF THE CO2 PRODUCED IN BIOLUMINESCENT REACTIONS

Abstract— Although the mechanism of bioluminescent reactions in various species, such as fireflies, ostracod crustaceans ( Cypridina ), sea pansies ( Renilla ), and the deep-sea shrimp Oplophorus , are thought to involve dioxetanone intermediates, studies reported in the past from different laboratories have included widely different experimental results, most likely due to various factors including the effects of contaminating CO₂. With the improved technique employed in the present study, bioluminescent reactions of the firefly and Cypridina in ¹⁸O₂ gas resulted in an incorporation of over 75% of ¹⁸O into one oxygen of the product CO₂. with a reproducibility within a few per cent. When ¹³CO₂. instead of the product CO₂ of the bioluminescent reaction, was studied in an H₂¹⁸O medium, the exchange of one oxygen of ¹³CO₂ with H₂O was 64%. and the effect of contaminant CO₂ amounted to 1418% of the total CO₂. These results suggest that every molecule of CO₂ formed in the bioluminescent reactions of the firefly and Cypridina had intially contained 1 oxygen atom derived from O₂.     

SPECTRAL SENSITIZATION OF SnO2 THIN FILM ELECTRODE BY THE J-AGGREGATE OF CYANINE DYES IN MONOLAYERS

Arachidic acid monolayers containing 5,6,5',6'-dibenzo-I,I-diethyl-2,2'-cyanine chloride were prepared by the spreading method and deposited on the SnO₂ surface using the Langmuir-Blodgett technique. The SnO₂ : dye assembly prepared in this manner was used as a photoelectrode of the conventional electrochemical cell. A distinct J-band was observed in the action spectra of sensitized anodic and cathodic photocurrents. Effects of calcium arachidate barriers inserted between dye layer and either SnO₂ electrode (inside barrier) or electrolyte solution (outside barrier) on the photocurrent were examined. Although the inside barrier was effective in decreasing both anodic and cathodic photocurrents, the outside barrier did not suppress cathodic photocurrent. The following mechanism involving a molecular exciton of the J-aggregate is proposed for the sensitized photocurrent. The anodic photocurrent is caused by hole trapping by some reducing agent and concomitant injection of the electron from molecular exciton to the conduction band of SnO₂. Extraction of conduction-band electron of SnO₂ by molecular exciton and supplying to some oxidizing agent such as dissolved oxygen are responsible for the cathodic photocurrent.     

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.     

SEARCH FOR SINGLET OXYGEN LUMINESCENCE IN THE DISPROPORTIONATION OF HO2/O2

Abstract— During the reaction HO₂+ HO₂ (or O₂^-) = H₂O₂+ O₂ in aqueous solution, no luminescence in the region 620–720 nm, expected if the product O₂ were formed in a singlet state, could be detected. If any singlet O₂ is formed, its yield must be less than 10%. Faint luminescence, sometimes found at shorter wavelengths, was shown to arise from reaction of HO₂ with impurities in the reagents present.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

PHOTOLYSIS OF SUBSTITUTED NAPHTHALENES ON SiO2 AND Al2O3

Abstract— Photolysis of naphthalene on the surface of SiO₂ under an atmosphere of air produces phthalic acid as the only major photoproduct, accounting for 49%o of the consumed naphthalene. Photolysis on Al₂O₃ also produces phthalic acid, in 31% yield. Photolysis of 1 -methylnaphthalene on SiO₂ proceeds under similar conditions to produce 2-acetylbenzoic acid (35%) as the major photoproduct with the production of a small amount of I-naphthaldchyde (6%). 1-Cyanonaphthalene does not photooxidize under similar conditions. The presence of oxygen is necessary for the photodecomposition of naphthalene and 1-methylnaphthalene to proceed. Superoxide formed from the photolysis of naphthalene at the SiO₂/air interface is readily observed by electron paramagnetic resonance spectroscopy. In the absence of naphthalene no superoxide is observed. A mechanism involving electron transfer from the S₁ state of the naphthalene to O₂ is proposed on the basis of these observations and related literature precedent.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.     

ULTRAVIOLET RADIATION-INDUCED PHOSPHOLIPASE A2 ACTIVATION OCCURS IN MAMMALIAN CELL MEMBRANE PREPARATIONS

Ultraviolet erythema in human skin is mediated in part by membrane derivatives of arachidonic acid (AA). UVA (320–400nm) and UVB (290–320nm) have been shown to induce release of AA from intact mammalian cells in culture. In order to investigate the mechanism of this release we examined the effect of UVA and UVB on release of [³H] AA from membrane preparations of murine fibroblasts. C3H 10T1/2 cells were prelabelled for 24 h with [³H] AA. The membrane fractions of the cells were separated after lysis by differential centrifugation. The membranes were irradiated in suspension and the [³H] AA released from the membranes was determined by scintillation spectroscopy of supernatants3–4 h after irradiation. Both UVA and UVB induced release of AA from the membrane preparations. The response to UVB was small but significant, reaching levels approximately 150% of control release at doses of 1,200-4,000 J/m². The response to UVA was larger; doses of 2.5-5.0 J/cm² induced release equal to twice control (200%) levels, while doses of10–20 J/cm² induced maximal release at levels approximately 400% of control. Time course studies with UVB and UVA showed maximal release at 4 h after irradiation. When the membrane preparations were incubated with a polyclonal anti-phospholipase A₂ antibody the UV induced release of [³H] AA was completely inhibited in both UVB (1200 J/m²) and UVA (10 J/cm²) treated cells. These data suggest that activation of phospholipase A₂ is responsible for the UV induced release of AA in mammalian cells and that the mechanism of this activation is due, in part at least, to direct photon-membrane interaction.     

PHOTOOXYGENATION OF BIPHENYL AND ITS DERIVATIVES via PHOTOINDUCED ELECTRON TRANSFER: EFFECT OF Mg(CIO4)2 ON PHOTOOXYGENATION

The 9,10-dicyanoanthracene (DCA)-sensitized photooxygenation of biphenyl derivatives in the presence of Mg(CIO₄)₂ in acetonitrile produces benzoic acid and its derivatives in high yields. In the absence of Mg(CIO₄)₂, the rates for the consumption of biphenyl derivatives decrease by a factor of 0.5-0.8, compared with those in the presence of Mg(CIO₄)₂. In these cases, however, both biphenyls and DCA are oxygenated to give benzoic acids and anthraquinone, respectively, indicating that the addition of Mg(CIO₄)₂ retards the photooxygenation of DCA. With 4-methylbiphenyl, the photooxygenation proceeds efficiently without added Mg(CIO₄)₂, and benzene rings and methyl groups are competitively oxygenated to give benzoic acid, 4-methylbenzoic acid, 4-phenylbenzoic acid, and 4-phenylbenzaldehyde. The addition of Mg(CIO₄)₂ facilitates the oxidation of benzene rings, giving benzoic acid and 4-methylbenzoic acid as major products. These photooxygenations are initiated by a one-electron transfer from biphenyls to the excited singlet DCA and proceed via the radical cations of biphenyls and the radical anion of DCA.     

THE LIGHT DEPENDENT QUENCHING OF CHLOROPLAST FLUORESCENCE BY COFACTORS OF CYCLIC ELECTRON FLOW IN PHOTOSYSTEM I

Abstract—When 3–(3',4'-dichlorophenyl)-1,1-dimethylurea poisoned, intact thylakoids of isolated chloroplasts are illuminated in salt free suspension media, N -methylphenazinium cations (MP⁺) are reversibly taken up. Simultaneously, the chlorophyll fluorescence is reversibly lowered. When inorganic salts in the reaction medium provide membrane permeant charge balancing ions, the extent of the MP⁺ association with the thylakoids is strongly increased, but the fluorescence lowering is hardly affected. lonophoretically active agents inhibit specifically the salt dependent increment of the MP⁺ interaction with the thylakoids, but have only insignificant effects on the fluorescence lowering provided the experimental conditions do not allow the formation of a proton gradient across the thylakoid membrane. On the basis of these results, and of data obtained from comparative studies with other cofactors of cyclic electron transport in PS I, it is suggested that the 'energy dependent' fluorescence lowering is linked to a binding of cationic cofactors to nucleophilic sites in or at the thylakoid membrane. Such sites appear to become exposed in the wake of a light dependent transport of the cofactor, or of protons, into the thylakoid.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).