PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

A STUDY OF THE FLASH INDUCED 518 NM ABSORBANCE CHANGE KINETICS UNDER ANAEROBIC CONDITIONS

The preillumination induced acceleration of the flash-induced 518 nm absorbance change (ΔA₅₁₈) decay was studied in lettuce leaves and chloroplasts. In leaves, the acceleration was inhibited by DCMU or reversibly by removal of oxygen. In chloroplasts with added ADP and phosphate and/or reconstructed electron transport, the acceleration was also inhibited by DCMU or the lack of O₂.
Anaerobic inhibition of ΔA₅₁₈ decay acceleration was no longer observed when hydroxylamine replaced water as electron donor to PSII. Anaerobiosis was also shown to reversibly inhibit the initial rate of FeCN reduction in chloroplasts. These results suggest the mechanism of anaerobic inhibition of ΔA₅₁₈ decay acceleration to be associated with the O₂ evolving system.     

THE PHOTOCHEMISTRY OF 14 C-5-BROMO-2''-DEOXYURIDYLYL-(3''→5'')-THYMIDINE DETERMINATION OF QUANTUM YIELDS AS A FUNCTION OF pH

Abstract— Quantum yields for the formation of the major photoproduct of 2-¹⁴C-5-bromo-2'-deoxyuridylyl-(3'→5')-thymidine (BrdUpT) have been determined by irradiation of the 2-¹⁴C-BrU-labeled dinucleotide at pH 2.3, 5.9, 7.05, 8.0 and 10.25 with U.V. light at 280nm. At acidic and neutral pH the quantum yield was 0.0063; the value decreased markedly above pH 8.0 to 0.0025 at pH 10.25. Some evidence of the formation of additional photoproducts at high and low pH was found. Some aspects of the mechanism of the reaction are discussed.
Consideration of p K_a values calculated for singlet and triplet excited states indicates that the decrease in the quantum yield of main photoproduct at high pH is due to dissociation of the excited bromodeoxyuridine moiety. It is suggested that the formation of BrdUpT photoproduct and the debromination of bromouracil-labeled DN A occur via different excited states.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

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.     

SPECTRAL CHARACTERIZATION OF LIGHT-REDUCIBLE CYTOCHROME IN A PLASMA MEMBRANE-ENRICHED FRACTION AND IN OTHER MEMBRANES FROM CAULIFLOWER INFLORESCENCES

Abstract— Low temperature spectroscopy has been used to characterize microsomal fractions obtained from cauliflower inflorescences ( Brasska oleracea L.) by differential centrifugation and partition in an aqueous polymer two-phase system. The plasma membrane-enriched fraction (U₃) was found to contain one dominant b -cytochrome, which could be reduced both by blue light and by dithionite. An action spectrum of the blue light-induced absorbance change [LIAC, Δ(A₄₃₀—A₄₁₀)] associated with the reversible reduction of this b -type cytochrome indicated that the primary light-receptor was a flavin-like compound. Another microsomal fraction (L₃) containing membranes from mitochondria, endoplasmic reticulum and other organelles also contained light-reducible cytochrome. One of these could be identified as cytochrome c oxidase, and another may be identical to cytochrome b ₅ of the endoplasmic reticulum.     

THE GECKO OPSIN: RESPONSES TO GEOMETRIC ISOMERS OF RETINAL and 3-DEHYDRORETINAL

Abstract— The opsin of the visual pigment (P521) of the Tokay gecko rapidly regenerates four spectrally different photopigments with the 9-cis and 11-cis isomers of both the vitamin A,- and A₂-aldehydes. The opsin displays the classic stereospecificity for both A₁- and A₂-series of isomers. The two photopigments regenerated with 9-cis- and ll-cw-3-dehydroretinals respond to chloride and nitrate ions as do the comparable pigments formed with 9-cis- and 11-ris-retinal. The result is a family of pigments absorbing with spectral maxima ranging from 464 to 540 nm, a span of some 3000 cm^-1. The photosensitivity of all four pigments was determined and found to be in relative order: 100% (11-cis-A₂), 77% (11- cis -A₂), 36% (9- cis -A,) and 14% (9- cis -A₂).     

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.     

THE PHOTOCHEMICAL INTERACTION BETWEEN THE TRIPLET STATE OF 8-METHOXYPSORALEN AND THE MELANIN PRECURSORL–3, 4 DIHYDROXYPHENYLALANINE

Abstract— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH₂) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH₂ ( k ∼ 2 × 10⁹ dm³ mol^-1 s^-1) to form semireduced 8-MOP and semioxidised dopaH₂.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH₂ at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λ_max and extinction coefficients found were: for 8-MOP at pH 6.5, λ_max= 350 nm (= 9050 dm³ mol^-1 cm^-1); for dopa at pH 6.5, λ_max= 305 nm (ε= 12000 dm³ mol^-1 cm^-1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm³ mol^-1 cm^-1).     

PHOTOBIOLOGIE DU MÉSOCOTYLE D'AVOINE—II. STIMULATION DE L'ÉLONGATION PAR LA LUMIÈRE ROUGE-SOMBRE EN PRÉSENCE DE DIVERSES GIBBÉRELLINES

Abstract— Far-red light has the property of promoting the elongation of Avena mesocotyl sections in the presence of a gibberellin. Part of this effect is not reversible by red light (around 655 mμ ). The wavelengths which are most effective are those in the neignbourhood of 720 mμ , as revealed by the action spectrum. Amongst the 8 gibberellins tested, only A₁, A₃, and A₉, are capable of producing, after far-red irradiation, a greater elongation than the one they cause in the dark.     

Highly Selective Separation of Rhodopsin from Bovine Rod Outer Segment Membranes Using Combination of Divalent Cation and Alkyl(thio)glucoside

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophilic–lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent-to-membrane ratio. This allows remarkable purification of rhodopsin by a single-step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low-speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A₂₈₀/A₅₀₀ value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB-series cations (Zn²⁺ and Cd²⁺) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.     

ULTRAVIOLET IRRADIATION OF POTATO VIRUS X, ITS RNA, AND A HYBRID VIRUS PARTICLE: PHOTOREACTIVATION, KINETIC ISOTOPE EFFECTS, AND QUANTUM YIELD OF INACTIVATION*

Abstract— Studies have been made of the inactivation of potato virus X (PVX), free PVX-RNA, and a phenotypically mixed virus particle composed of PVX-RNA and tobacco mosiac virus (U-1 strain) protein (PVX_RNA: TMV_PRO) by ultraviolet radiation of 254, 280 and 302 nm wavelengths. Rate constants and quantum yields of inactivation have been determined under conditions of photoreactivation and non-photoreactivation in H₂O and D₂O. The ratios of the rate constants in H₂O to those in D₂O obtained for PVX-RNA were greater than unity at the above three wavelengths, as was the case for PVX at 254 and 302 nm. The ratios of the rate constants were, within experimental error, equal to unity for PVX_RNA:TMV_PRO at all three wavelengths and for PVX at 280 nm. It is concluded that, in contrast to the situation observed with TMV and PVX_RNA:TMV_PRO, the behavior of the intact PVX viron upon irradiation closely approximates the behavior of free RNA in solution, on the basis of quantum yields of inactivation, photoreactivated sector, and the ratio of kinetic isotope effects in H₂O and D₂O.     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

EFFECT OF BASE SEQUENCE ON THE ULTRAVIOLET IRRADIATION PRODUCTS OF DOUBLE-STRANDED POLYNUCLEOTIDES CONTAINING BROMOURACIL AND ADENINE

Abstract —The effects of ultraviolet irradiation of double-stranded synthetic polynucleotides containing BrU and A have been investigated. Homopolymer pairs and alternating copolymers composed of either ribo- or deoxyribo-nucleotides were prepared and were irradiated with either 313 nm or ˜ 285 nm light. Strand separation and a modest amount of strand breakage followed irradiation of the homopolymer pairs. Changes in the ultraviolet absorption spectra of the polymers during irradiation reflected the sum of hyperchromic increases caused by progressive strand separation and loss of absorbance caused by photoproduct formation. Extensive debromination occurred. An RNase digest of irradiated poly(rA)–poly¹⁴C(rBrU), analysed by column chromatography, showed components similar to those found previously upon irradiation of single-stranded poly(rBrU). Little photoproduct was released by RNase digestion as mononucleotides. The major photoproduct was in the dinucleotide fraction, and may be 5,5'-diuracil. Base sequence had a profound effect on the sensitivity of the polynucleotides. Irradiation of alternating copolymers with doses of light comparable to those that produced major photochemical changes in the homopolymer pairs brought about little if any change in the copolymers of alternating base sequence.     

PHOTOBIOLOGIE DU MÉSOCOTYLE D''AVOINE-III. DOUBLE EFFET DE LA LUMIÈRE BLEUE

Abstract— Using excise sections of oat first-internodes, a dual effect of blue light can be demonstrated on elongation when the sections are first irradiated in distilled H₂O, then incubated with gibberellic acid (GA). At low light energies (230 ergs/cm² per sec, for 2 min), a pretreatment with blue light enhances the GA effect above the elongation it can produce in the dark. At high energies (650 ergs/cm²/sec for 45 min), the same wavelengths cause an inhibition of the GA-induced elongation. An action spectrum for the two effects show a maximum near 435 mμ in both cases. Neither light effect is visible when indoyl-3-acetic acid is used instead of GA. Several physiological effects distinguish the two blue effects. The promotive effect is most marked in the young regions of the mesocotyl, whereas the maximum inhibitive effect is located in slightly older tissues. Time-course experiments showed that the promotive effect is partly due to an extension of the duration of elongation. The inhibitory effect is only temporary and vanishes about 30 hr after the beginning of the experiment. The promotive effect of blue light resembles the effect of far-red light, but the former can be observed with gibberellins A₂, A₄, A₅, A₆ and A₇ which are practically inactive after an irradiation with far-red light. The inhibitory effect of blue light is different from the red-light effect as shown by the time-course experiments.     

PHOTOLYSIS OF AQUEOUS SOLUTIONS OF POTASSIUM CIS-DIAQUOBIS (OXALATO) CHROMATE (III)

Abstract— The photolysis of aqueous solutions of cis -[Cr(C₂O₄)₂(H₂O)₂]- at 254 nm and pH 4 produced CO₂ and H₂ in nearly equal yields. The quantum yield of hydrogen, φ², increased by 9% and the yield of carbon dioxide, φ, by 65% as the pH was lowered from 4 to I. The total gas yield, φ_gas, decreased in the presence of added oxalate or chromium (II) ions and when the light intensity was lowered. The gas yield in D₂O was appreciably higher than in H₂O. The variation of φ_gas with pH (D) and with added oxalate ion was roughly parallel in the two liquid media. The gas yield increased in the series:
A tentative mechanism is suggested to explain the results.     

Cell Cycle Kinetics Following UVA Irradiation in Comparison to UVB and UVC Irradiation

Abstract— There is limited information about the carcinogenic effect of longwave ultraviolet radiation (UVA: 315-400 nm). In particular very little is known about the relevant genotoxic damage caused by physiological doses of UVA radiation. A general response of cells to DNA damage is a delay or arrest of the cell cycle. Conversely, such cellular responses after UVA irradiation would indicate significant genotoxic damage. The aim of this study is to compare cell cycle kinetics of human fibroblasts after UVC (190-280 nm radiation), UVB (280-315 nm radiation) and UVA irradiation. Changes in the cell cycle kinetics were assessed by bivariate flow cytometric analysis of DNA synthesis and of DNA content. After UVC, UVB or UVA irradiation of human fibroblasts a suppression was seen of bromodeoxyuridine (BrdU) incorporation at all stages of S phase. The magnitude of this suppression appeared dose dependent. Maximum suppression was reached at 5-7 h after UVB exposure and directly after UVA exposure, and normal levels were reached 25 h after UVB and 7 h after UVA exposure. The lowered BrdU uptake corresponded with a lengthening of the S phase. No dramatic changes in percentages of cells in G₁, S and G₂/M were seen after the various UV irradiations. Apparently, UVA irradiation, like UVB and UVC irradiation, can temporarily inhibit DNA synthesis, which is indicative of genotoxic damage.     

FLUORESCENCE BEHAVIOUR OF SEQUENTIAL TYROSYL POLYPEPTIDES

Abstract— Fluorescence behaviour of three sequential tyrosyl copolypeptides, (Gly-Tyr)₃₅, (Ala-Tyr)₂₉ and (Val-Tyr)₂₂, was studied in alkaline solutions, in which ionisation of tyrosyl hydroxyl was 80%. Irradiation at 280 nm in all the three systems lead to the formation of a photoproduct which fluoresced intensely at 410 nm. The facility of the photoreaction depended markedly on the nature of the comonomer of tyrosine and the conformation of the polypeptide chain. As a result of photoproduct formation, the polypeptide backbone is hindered in acquiring more ordered structure at a lower degree of tyrosyl ionisation.