LIGHT-INDUCIBLE ABSORBANCE CHANGES AND VANADATE-SENSITIVE ATPase ACTIVITY ASSOCIATED WITH THE PRESUMPTIVE PLASMA MEMBRANE FRACTION FROM CAULIFLOWER INFLORESCENCES

Sucrose density gradient centrifugation of a microsomal membrane fraction of cauliflower inflorescences showed a strong correlation between a blue light mediated cytochrome b reduction (LIAC) and an ion stimulated nitrate-insensitive but a vanadate-sensitive ATPase activity at 38-40% sucrose. LIAC activity and vanadate-sensitive ATPase might be assigned to the same type of membrane different from ER, Golgi, tonoplast and mitochondria. The Mg²⁺-dependent ATP-hydrolytic activity obtained after purification of the microsomal fraction on an aqueous polymer two phase system was partially characterized. Temperature optimum (40°C), pH optimum (pH 7.0), vanadate inhibition (I₅₀ at 20 μ M ), substrate kinetics ( K _m= 1.37 m M Mg.ATP) and inhibitor studies all point to the presence of the frequently described plasma membrane ATPase. Potassium and Na⁺ stimulated the enzyme activity (20-40%). In general our data arc strongly in favour of the hypothesis that LIAC activity is localized on the plant plasma membrane. The cytochrome b involved in the light reaction has a midpoint potential near +150 mV. This cytochrome which has been previously shown in a cauliflower microsomal fraction is a constituent of the plasma membrane.     

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.     

THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

Abstract— In isolated respiratory multienzyme complexes of beef heart mitochondria the b -type cytochromes can be photoreduced in presence of flavin via the superoxide anion. O^-₂ does not reduce cytochrome c ₁. In an anaerobic system, FMNH₂ formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c ₁ The possible implication of O^-₂ in the electron transfer from flavin/flavoprotein to cytochrome b in blue light-controlled biological processes is discussed.     

PHOTOREDUCTION OF CYTOCHROME b551 OF PARTIALLY PURIFIED NEUROSPORA NITRATE REDUCTASE VIA ITS INTERNAL FLAVIN

Abstract— Nitrate reductases (NR) from NR-normal Neurospora crassa mutant albino band and from NR-defective mutantsnit–1 andnit–3 were isolated and partially purified in order to test the photo-reducibility of their cytochrome b ₅₅₇ via the NR-internal FAD. Photoreducibility with blue light of the isolated enzyme was observed as absorbance increase at 423, 524 and553–557 nm. It was independent of NADPH-nitrate reductase activity and could be induced if the dissociable FAD was not lost in the isolation procedure. The photoreduction of cytochrome b ₅₅₇ was readily reversible due the high autoxi-dation rate of this cytochrome. Therefore, anaerobic conditions are required for photoreduction with low light intensities. If aerobic conditions are applied, high intensities become necessary to overcome the simultaneous cytochrome h₅₅₇ oxidation.     

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.     

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

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.     

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).     

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.     

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.     

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.     

PHOTOLYSIS OF POLYRIBOBROMOURIDYLIC ACID

Abstract— Photolysis of polyribobromouridylic acid with 313 nm light at neutral pH caused extensive debromination and a loss of A₂₈₀ (280–nm absorbance) without comparable increase in A₂₆₀. At an exposure of 190μE/cm ² , strand breakage occurred on the average of one break every 170 BrU residues. Little if any pyrimidine hydrate was produced. Exhaustive RNase hydrolysis of photolysed polymer gave a mixture of mononucleotides and oligonucleotides. The mononucleotide fraction was found to be composed of unaltered BrUMP and contained little if any UMP. Irradiation of the polymer at alkaline pH caused little or no debromination or spectral change.     

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (¹O₂) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of ¹O₂ from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in ¹O₂ generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0₂ quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by ¹O₂ which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of ¹O₂ was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of ¹O₂.     

OXIDATION-REDUCTION AND EPR PROPERTIES OF A CYTOCHROME COMPLEX FROM Chromatium vinosum

A cytochrome b₅₆₀-c₅₅₃ containing fraction from the photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to contain equimolar amounts of the two cytochromes and also to contain ubiquinone. The oxidation-reduction midpoints (at pH 7.5) of cytochrome b₅₆₀ and c₅₅₃ in this fraction are −5 and +20 mV, respectively, essentially identical to the values found for the cytochromes in chromatophores. The fully oxidized fraction exhibits EPR features at g = 2.93 and 3.35. The former had previously been identified as originating from ferricytochrome c₅₅₃, while the latter appears to arise from ferricytochrome b_S60.     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

ALKALI-LABILE PHOTOLESIONS MAPPING TO PURINE SITES IN ULTRAVIOLET-IRRADIATED DNA

Gel sequencing experiments with the 5'- and 3'-end-labeled oligonucleotides d(A₃GA₄GA₅GA₆GA₃G) and d(AT) ₁₀ have demonstrated that dimeric adenine photoproducts and thymine-adenine photoadducts constitute alkali-labile lesions in UV-irradiated DNA. On treatment with hot piperidine, DNA strand breakage occurs predominantly at the sites of 5'-adenines in the dimeric photoproducts and of 3'-adenines in the thymine-adenine photoadducts. With 5'-end-labeled oligonucleotides of mixed sequence, major UV-induced loci for alkaline cleavage map to purine bases flanked on their 5'-side by two pyrimidines. This behavior does not arise from enhanced photoreactivity of purines in this sequence context as has been inferred from photofootprinting studies. Instead, as shown by 3'-labeling and selective substitution with 5-methylcytosine, it results from the anomalous electrophoretic mobility of 5'-end-labeled fragments produced by alkaline cleavage of DNA at adjacent pyrimidine (6-4) pyrimidone photoproducts.     

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.     

EVIDENCE AGAINST THE PRODUCTION OF SUPEROXIDE BY PHOTOIRRADIATION OF HEMATOPORPHYRIN DERIVATIVE

Abstract Experiments were performed to ascertain whether superoxide anion (O₂⁻) was produced by the photodynamic activation of hematoporphyrin derivative (HPD). Three different systems were utilized to detect formation of O₂⁻, oxidation of epinephrine to adrenochrome, reduction of cytochrome c and reduction of nitro blue tetrazolium (NBT). The effects on these detectors under identical conditions for HPD + h ν were compared to those obtained with two O₂⁻ generating systems, riboflavin + by and xanthine-xanthine oxidase, and to a singlet oxygen generating system, photoradiation of methylene blue. The results indicated that HPD + hv differed from the two O₂⁻ generating systems in failing to reduce cytochrome c or NET, and that HPD + h ν was similar to the behavior of methylene blue + h ν . In addition, HPD + h ν but not the O₂⁻ generating systems could inhibit mitochondrial cytochrome c oxidase activity. We conclude that the photodynamic activation of HPD does not produce O₂⁻ as a major oxygen radical and that the effects of HPD + h ν on mitochondrial cytochrome c oxidase are not caused by O₂⁻.     

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.