Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochromeb-559 were studied for membrane fragments of the blue-greenalga Anabaena variabilis. Cytochrome photooxidation was observableat room temperature when the membrane fragments had been preincubatedat room temperature in the dark. A CCCP addition (10^–4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction.The former depended on actinic illumination of short wavelength.The latter showed a dependency on longer wavelength light. Theformer was assumed to be induced by the action of photosystemII and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation,and was enhanced by added DPIP or Ferro. This was interpretedas photosystem II action inducing oxidation as well as reduction;reduction being inactivated during dark incubation or beingsuppressed by added redox reagents which compete for electronacceptance from photosystem II so that oxidation was apparentlyenhanced. The oxidationreduction reactions of this cytochromewith low redox potential were assumed to be almost identicalwith those of the high redox potential form, at least in themembrane fragments of Anabaena variabilis. (Received June 8, 1975; )     

The light-induced oxidation-reduction reaction of cytochrome b-559 in membrane fragments of the blue-green alga Anabaena variabilis

Light-induced oxidation-reduction reactions of cytochrome b-559were investigated with membrane fragments of Anabaena variabilisand supplementarily with Plectonema boryanum. The oxidation-reduction reactions of cytochrome b-559 observedwith membrane fragments were similar in their kinetics to thoseof the cytochrome in aged chloroplasts. The reactions were annihilatedby the addition of Ferro, indicating that the cytochrome ofhigh redox potential (E'_o=+373 mV, pH 6.5) was photoreducedand oxidized. Titration with reducing agents indicated that cytochrome b-559is contained in Anabaena membrane fragments in an amount 1.5times as much as the content of P700 on a molar basis; the contentof the species of high redox potential was estimated to be around70%. Kinetic treatment of the photoreduction indicated that the cytochromewas reduced at some site of the electron transport system betweenthe two photosystems. The photo-oxidation depended on the actionof either photosystem II or I even in the presence of DCMU,indicating that the photooxidation was induced by both photosystems.The oxidation by photosystem I action was inhibited by HgCl₂-treatment,indicating that this reaction is mediated by plastocyanin. EDTA (5?10^-3 M) suppressed the cytochrome photoreduction andenhanced the rapid phase of the photooxidation. The latter effectappeared only when an appropriate dark time (3 min) was insertedafter the cytochrome photoreduction. The phenomenon was interpretedas showing that EDTA modifies the reactivity of the electroncarrier which directly donates electrons to cytochrome b-559.The oxidation, and probably also the reduction of cytochromeb-559, was assumed to be regulated by the oxidation-reductionstate of this carrier. (Received April 26, 1974; )     

Cytochrome components of green alga, Bryopsis maxima: Purification and properties of cytochrome f from membrane fragments

From the membrane fragments of the green alga Bryopsis maxima,a cytochrome which resembles cytochrome f of higher plants wassolubilized with methyl ethyl ketone. The cytochrome was partlypurified by ammonium sulfate fractionation, followed by gelfiltration. Its properties were similar to those of the algalcytochrome f reported by Wood (26). The approximate molar ratioof cytochromes f, c-553 and chlorophyll in B. maxima was 1 :1 : 600–700. ¹ In this communication, according to the recommendation byWood (26), cytochrome f is the membrane-bound c-component andcytochrome c-55 the soluble one. In some references cited, thesechloroplast cytochromes are called algal cytochrome f. (Received February 16, 1978; )     

Comparison of main emissions at--196?C from phycobilisomes of two blue-green algae Anabaena cylindrica and Anacystis nidulans

Main emissions at—196?C from phycobilisomes of two blue-greenalgae Anabaena cylindrica and Anacystis nidulans were studiedwith special reference to allophycocyanin (APC) B content. Supplementaryexperiments were done with Anabaena variabilis (M-2 and M-3).The main emission from phycobilisome of Anacystis nidulans richin APC B was located at 681 nm. The location was identical tothat of the main emission from APC B but at a shorter wavelengththan that of in vivo emission (685 nm). Results indicate thatAPC B acts as the energy output of phycobilisomes, but thatthe in vivo 685 nm emission is not attributed to APC B. The main emission of the phycobilisome of Anabaena cylindricawas always located at 685 nm irrespective of the preparationmethod; 0.75 M phosphate buffer [Plant Physiol., 63: 615–620(1979)] or 30% polyethylene glycol [Special Issue of Plant &Cell Physiol., No. 3, p. 23–31 (1977)]. This alga alsocontained a special form of APC, but its content was very low.The location of its emission band (681 nm) was identical tothat of APC B, but shorter than that of the main band of phycobilisomes(685 nm). The 685 nm emitter in phycobilisomes showed a charactersimilar to chlorophyll a but not phycobiliproteins in treatmentsfor aqueous extraction or methanol extraction. Results indicatethat the pigment is probably chlorophyll a as we assumed previously.The 685 nm emission from phycobilisomes of Anabaena variabilis(M-2 and M-3) showed the same character. Results were interpreted as indicating that (i) the contentof the special form of APC varies with the species or strainof blue-green algae and (ii) the energy at the phycobilin levelis transferred directly from APC to pigment system II chlorophylla when the amount of the special form of APC is low. (Received October 24, 1979; )     

Pyrone derivatives: effective inhibitors of photosynthetic electron flow system

The inhibitory effects of the pyrone derivatives, 6-()-decenyl-2,3-dimethyl--pyrone(DDP) and 6-farnesyl-2,3-dimethyI--pyrone (FDP), on the photosyntheticelectron flow system was investigated using the blue-green algaAnabaena variabilis and the green alga Chlorella pyrenoidosa. Both reagents inhibited photosynthesis in intact cells; 50%inhibition occurred at 2.7 x 10^–5 M with DDP and at 4.3 lO^–6 M with FDP in Anabaena photosynthesis. The reagentssuppressed the photosystem II reaction [water to 2,6-dichlorophenolindophenol (DCIP)] of Anabaena membrane fragments, but werefar less inhibitory on the photo-system I reaction (DCIPH₂ tomethyl viologen). The kinetics of the fluorescence inductionindicated that the reagents do not block Q-reduction, but dosuppress the oxidation of reduced Q indirectly. Oxygen evolutionunder repetitive flashes at a low repetition rate (5 Hz) wasinsensitive to the reagents even at concentrations which inducedmore than 50% inhibition. These results are evidence that DDPand FDP inhibit the plastoquinone reaction by slowing down itsturnover rate. The advantages of pyrone derivatives are that they are inactivein the oxidation-reduction reaction and do not quench the fluorescenceof chlorophyll in vivo. (Received April 14, 1980; )     

Oxidation Rate of Monomeric Radish Cytochrome f with Plastocyanin

The reaction rate of reduced monomeric cytochrome f with oxidizedplastocyanin, both purified from Japanese radish, was determinedby a stopped-flow method. The oxidation rate constant was 6.0x 10⁷ M¹sec¹ at pH 7.0 and 25°C, which is slightly higherthan the value reported by Wood [(1974) Biochim. Biophys. Acta357 : 370] for oligomeric parsley cytochrome f Thermodynamicparameters also were determined to be 56 KJ M^–1 for activationenthalpy and 90 J M^–1 K^–1 for activation entropy.Neither a pH from 6 to 9 nor the addition of NaCl, polylysine,histone or polyaspartate affected the rate constant. ¹Present address: The National Institute for Environmental Studies,Yatabe, Ibaraki 305, Japan. (Received October 9, 1980; Accepted November 17, 1980)     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

COMPONENTS OF THE ELECTRON-TRANSFERRING SYSTEM IN ACETOBACTER SUBOXYDANS AND RECONSTRUCTION OF THE LACTATE OXIDATION SYSTEM

1. Cytochromes a₁⁵⁹⁰, b⁵⁶⁰, c₁⁵⁵⁴ and c₁⁵⁵² were isolated andpurifiedfrom a strain of Acetobacter suboxydans. The proceduresusedwere described in detail.
2. The main cytochrome band at550-560 mµ in intact cellssplitted at liquid air temperatureinto two bands, 551 mµ(strong) and 559 mµ (weak).
3. Optical and physiological properties of the four cytochromeswere investigated. Lactic dehydrogenase activity was found tobe associated with cytochrome c₁⁵⁵⁴. The two c₁-type cytochromes,especially cytochrome c₁⁵⁵⁴, persisted in their reduced formafter the purification through many steps.
4. By some combinationsof isolated components reconstruction ofthe oxygen uptake systemcould be realized.
5. The oxygen-consuming activity of purifiedoxidase preparationswas accelerated by a-tocopherol but notby Emasoll 4130 andTween 80.
6. Some discussions were made onthe nature of terminal oxidase,the role of cytochrome c₁⁵⁵²in the electron-transport system,and persistence of reducedstate of c₁-type cytochromes.
7. A possible scheme of the electron-transferringsystem of Acetobactersuboxydans was presented.

(Received May 16, 1960; )     

Lipid Biosynthesis in the Blue-Green Alga (Cyanobacterium), Anabaena variabilis III. UDPglucose:Diacylglycerol Glucosyltransferase Activity in vitro

The synthesis of glyceroglycolipids was studied in membraneand soluble fractions of Anabaena variabilis. The membrane fractionexhibited a high activity of UDPglucose: diacylglycerol glucosyltransferase,but practically no activity of UDPgalactose: diacylglycerolgalactosyltransferase. The glucosyltransferase activity wasmaximal at about pH 7.0 and dependent on Mg²⁺ The Michaelisconstant (K_m) for UDPglucose was 45?10^–6 M. The solublefraction catalyzed the incorporation of galactose from UDP galactoseinto digalactosyl diacylglycerol. These in vitro results werecompatible with the biosynthetic pathway of glyceroglycolipidsin this alga that we previously elucidated on the basis of tracerexperiments in vivo. ¹ Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received June 1, 1982; Accepted July 1, 1982)     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

On the causes of interspecific differences in the growth-irradiance relationship for phytoplankton. II. A general review

The causes of interspecific differences in the µ-l relationshipare examined in the context of a mechanistic model which relatesµ to irradiance in terms of six factors:, k_c photosyntheticquotient (PQ), Chl a:C, respiration and excretion. The effectof cell size on the light saturated growth rate is also considered.It is shown that photosynthetic efficiency and PQ exhibit remarkablylittle interspecific variability, and average 0.024 ±0.005 µg C(µg Chl a)^–1 h^–1 (µEm^–2 s^–1)^–1 and 1.5 ± 0.2 mol 02 molC^–1 (when NO₃^– is the nitrogen source) respectively.Two useful relationships were derived: (i) between growth efficiency,_g and Chl a:C at µ. = 0; (ii) between the compensationintensity, I_c and the Chl a-specific maintenance respirationrate. Both relationships were independent of temperature anddaylength. Species best adapted to growth at low light werefound to exhibit high Chl a:C ratios and low maintenance respirationrates. As a group, diatoms were consistently the best adaptedfor growth at low irradiance. Chiorophytes, haptophytes, chrysophytesand cryptophytes were intermediate in their performance at lowirradiance. Dinoflagellates exhibited extreme diversity, withspecies spanning the spectrum from very good performance atlow irradiance to very poor. A new µ_max-cell carbon relationshipis given based on growth rates normalized to 15°C. Evidenceis presented to show that noise in this relationship can besignificantly reduced by using only carbon-specific growth ratesand using only data for species grown at the same daylength.     

Comparison between Electron Transfers through Plastocyanin in Spinach Chloroplasts and Cytocbrome C2 in Rhodopseudomonas sphaeroides

Photosynthetic electron transfers through the water-solubleperipheral membrane proteins of plastocyanin and cytochromec₂, were studied in spinach chloroplasts and the photosyntheticbacterium Rhodopseudomonas sphaeroides. In spinach chloroplasts,the rate of flash-induced oxidation of cytochrome f was highlydependent on the salt concentration in the suspending medium.The maximum rate with a half time of 200 µs was observedin the presence of 50 mas KCl or 5 mM MgCl₂. The salt effectwas similar to that on the reaction rate between P700 in thylakoidfragments and externally added plastocyanin. On the other hand,in intact cells of R. sphaeroides, in which cytochrome c₂ islocated in the periplasmic space exposed to the outer ionicenvironment, the rate of cytochrome c₁ oxidation via cytochromec₂ was almost independent of salt concentration. This independencewas a contrast to the strong dependence on salt concentrationof reactions between isolated reaction centers and cytochromec₂ These results suggest that plastocyanin reacts collisionallywith the photosystem I reaction center and cytochrome b₆f complexin a manner that is controlled by the surface electrostaticpotential. Cytochrome c₂, on the other hand, reacts with thebacterial reaction center and cytochrome bc₁ complex probablyby forming a complex prior to activation of the reaction center. ¹ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan.     

Rates of Photosynthesis in Characean Cells: II. PHOTOSYNTHETIC 14CO2 FIXATION AND 14C-BICARBONATE UPTAKE BY CHARACEAN CELLS

Photosynthetic ¹⁴C fixation by Characean cells in solutionsof high pH containing NaH¹⁴CO₃ gave a measure of the abilityof these cells to take up bicarbonate (H¹⁴CO₃^–). Whereascells of Nitella translucens from plants collected and thenstored in the laboratory absorbed bicarbonate at 1–1.5µµmoles cm^–2 sec^–1, rates of 3–8µµmoles cm^–2 sec^–1 were obtained withN. translucens cells from plants grown in the laboratory. Influxesof 5–6 µµmoles cm^–2 sec^–1 wereobtained with Chara australis, 3–8 µµmolescm^–2 sec^–1 with Nitellopsis obtusa, and 1–5µµmoles cm^–2 sec^–1 with Tolypella intricata.It is considered that these influxes represent the activityof a bicarbonate pump, which may be an electrogenic process. In solutions of lower pH, H¹⁴CO₃^– uptake would be maskedby rapid diffusion of ¹⁴CO₂ into the cells: the four Characeanspecies fixed ¹⁴CO₂ at maximum rates of 30–40 µµmolescm^–2 sec^–1 (at 21° C).     

Photosynthetic Units and Carbon Assimilation in Leaves of Grain Sorghum under Different Light Intensities

The Cyt f and P700 contents in leaves of three Sorghum, varietieswere measured, in relation to their carbon assimilation, underdifferent light intensities during growth. At the maximum irradiationused (1,800 µE m^–2 s^–1) the ratio of P700to Cyt f was close to unity, whereas under low irradiation (450µE m^–2 s^–1) the ratio of P700 to Cyt f rangedfrom two to three. A strikingly positive correlation existedbetween the P700 contents of the leaves and their rates of carbondioxide fixation, dry matter production and Cyt f contents,only when the plants were grown under high light intensities.The P700 content of the leaves in plants grown under low irradiationwas unrelated to the contents of Cyt f. Thus, at a high lightintensity there is a close relationship between the Cyt f andP700 levels, but at low intensities the amounts of electroncarriers and the reaction centre are independent. (Received March 7, 1983; Accepted August 24, 1983)     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

Carotenoid photobleaching induced by the action, of photosynthetic reaction center II: DGMU-sensitivity

Carotenoid photobleaching in photosynthetic membrane fragmentsof the blue-green alga Anabaena variabilis was studied withspecial reference to DCMU-sensitivity. Carotenoid photobleaching supported by CCCP is strongly enhancedby Ferri, and, at the same time, becomes less sensitive to DCMU(cf. 5). The DCMU-insensitive reaction was found to show characteristicsvery similar to those of DCMU-sensitive reaction in (i) thedependence on the excitation of pigment system II chlorophylla, (ii) the stimulation by CCCP and NaNa and the suppressionby antimycin A, and (iii) the partial dependence on molecularoxygen. In our membrane fragments Ferri was found to act asan electron acceptor for the photosystem II reaction bypassingthe DCMU-sensitive site. We concluded that (i) carotenoid photobleachinginsensitive to DCMU is also driven by reaction center II, and(ii) in the presence of Ferri, Ferri accepts electrons ejectedby reaction center II bypassing the DCMU-sensitive site. (Received January 20, 1977; )     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

The productive and optical status of the oligotrophic waters of the Southern Aegean Sea (Cretan Sea), Eastern Mediterranean

Primary production, pigment concentrations and spectral measurementsof downwelling irradiance were made at four stations in fourseasons (spring, summer, autumn, winter) during 1994 in thewaters of the South Aegean Sea (Cretan Sea), Eastern Mediterranean.Rates of production were determined using in Situ incubationtechniques and included measurements at the surface microlayer.Depth-integrated values averaged over season were 5.66 mg Cm^–2 h^–1 for primary production and the correspondingchlorophyll (Ch1) a and phaeophytin (Phaeo) a values had meansof 4.87 and 1.21 mg m^–3 respectively. The assimilationratio remained very low (mean over season: 1.19 mg C mg^–2Chl a h^–1 as did the Phaeo a/Chl a ratio (mean over season:0.24). The annual production for the area was estimated to yield24.79 g C m^–2 year^–1. Primary production and Chla estimates showed statistically significant seasonal, spatialand depth variations. The spectral values of the attenuationcoefficient Kd (