Effects of chloride ion on HILL reactions in Euglena chloroplasts

Effects of Cl^– and other anions on the rate of HILL reactionin Euglena chloroplasts were investigated. Cl^– acceleratedthe reaction rate with ferricyanide as HILL oxidant; Br^–,F^– and I^– were also effective; NO₃^–, PO₄^2–and SO₄^2– were less effective. Divalent cations, Ca²⁺and Mg²⁺, were also highly effective. The promoting effectsof these ions were highly dependent on pH and the nature andconcentration of the HILL oxidant used. Accelerating effectsof the ion increased with decreasing concentrations of ferricyanide.Generally, the stimulating effect of Cl^– was much moremarked at pH 7–7.5, with little effect at pH 5. Thus,the pH-activity relationship in the HILL reaction is more orless markedly modified by addition of ions. Cl^–, and other anions, accelerated the reaction by affectingonly the dark rate-limiting portion of the HILL reaction; thelight reaction constant remained uninfluenced. We inferred thatsome reaction step, at which ferricyanide receives electronfrom photosystem 2, is accelerated by Cl^– and other ions.Cl^– effects were rather small, or undetectable, with DPIPor p-benzoquinone as oxidants. (Received January 8, 1970; )     

SYNCHRONIZATION OF BUDDING CYCLE IN YEAST CELLS, AND EFFECT OF CARBON MONOXIDE AND NITROGEN-DEFICIENCY ON THE SYNCHRONY

1. A procedure was devised to synchronize the budding cycleof baker's yeast. The method consists of preliminary selectionof the cells by differential centrifugation, combined with treatmentat 40° for 1 hr. On further incubation of the pre-treatedcells in the fresh medium at 27°, a synchronized buddingof the whole population occurs. followed by equally well synchronizedmaturation and separation of the buds formed. 2. The effect of CO on the budding cycle was investigated byintroducing the gas at various stages of the cycle. The mostmarked effect was observed when it was introduced in the inductionperiod of the cycle, resulting in a prolongation of the inductionperiod. Anaerobiosis was found to cause inhibition similar tothat caused by CO described above. 3. 2, 4-Dinitrophenol, when added at the beginning of the inductionperiod, at a low concentration (1.8x 10^–4M), was foundnot to abolish the first budding, but to prolong bud-maturationmarkedly. At higher concentrations of the poison, the firstbudding was markedly retarded, followed by the suppression ofbud-maturation. The budding was completely suppressed at l.8x10^–3M of the poison. 4. The absence of N-source in the incubation medium did notsignificantly affect the first budding, but completely abolishedthe capacity of the cells (both mothers and daughters) for thesecond budding. The lost capacity could be recovered by supplementingthe deficient medium with an N-source (urea), but when the timingof the supply was delayed, the recovery became less and lesssatisfactory. The presence of CO in the gas atmosphere was foundto enhance the deleterious effect of N-deficiency. (Received September 2, 1961; )     

Ferricyanide-induced absorbance change of carotenoid in chromatophores of Rhodopseudomonas spheroides correlated to the oxidation of bacteriochlorophyll 885

Addition of high concentrations (e.g., 1–100 mM) of ferricyanideto a chromatophorc suspension of Rhodopseudomonas spheroidescaused a change in the absorption spectrum of carotenoid (spheroidene),which was completely reversed by adding reducing reagents suchas ferrocyanide and ascorbate. The spectral change is representedby a shift in the absorption spectrum of carotenoid by 2 to2.5 nm towards the longer wavelength side. The presence of piericidinA, o-phenanthroline or Cl-CCP in the reaction mixture did notaffect the ferricyanide-induced absorbance change. Triton X-100markedly suppressed the magnitude of the change. The additionof ferricyanide also caused simultaneous absorbance changeswith maxima at 590 and 885 nm. These are ascribed to oxidationof the (bulk) bacteriochlorophyll, BChl 885. There was no absorptionchange at other peaks of bacteriochlorophyll in the infraredregion (i.e., 800 and 855 nm). Therefore, the ferricyanide-inducedabsorbance change of carotenoid did not represent an oxidation-reductionreaction of carotenoid but was intimately correlated with oxidationof BChl 885 in the chromatophores, as judged from similaritiesobserved with respect to the time course patterns, midpointpotential (545–555 mv) in the ferriferrocyanide reactionsystem, as well as behavior towards various reagents and inhibitorsadded. A similar change of carotenoid (i.e., 2–2.5 nmshift of absorption spectrum) was caused by addition of MgCl₂to the chromatophores, but this did not induce any change inthe absorption spectrum of bacteriochlorophyll. The nature ofthe spectral change of carotenoid in chromatophores is discussed. (Received April 16, 1970; )     

Light-induced absorption spectrum changes of carotenoid in chromatophores of photosynthetic bacterium, Rhodopseudomonas spheroides II. Rapid and slow absorption changes of carotenoid in chromatophores

Time courses of light-induced absorption changes of carotenoid(spheroidene) in chromatophores of the photosynthetic bacterium,Rhodopseudomonas spheroides, consisting of an 8–10 nm-shiftin the carotenoid absorption spectrum towards the longer wavelength,were investigated. The whole time course of absorption changecan be expressed as the sum of two first order reactions; arapid change accomplished in several msec after the onset ofillumination with actinic light (800–900 nm) and a slowchange extending over the whole period (200 msec). The rapidchange required a high light intensity, whereas the slow changewas saturated at relatively low light intensity (ca. 5xl0³ erg/cm²sec). Electron transport inhibitors (HOQNO, piericidin A and o-phenanthroline)and Cl-CCP, at concentrations uncoupling photophosphorylation(10^–M), inhibited slow change but did not affect rapidchange. The rapid change was inhibited by higher concentrationsof Cl-CCP (10–4 M) and by o-phenanthroline in the concomitantpresence of an uncoupling concentration of Cl-CCP. The rapidand slow changes have midpoint potentials of 440 and 420 mv,respectively; as calculated from absorption changes in the presenceof ferri- and ferrocyanide mixtures. Relationships between absorptionspectrum changes and other reactions in the chromatophores wereanalyzed. Slow absorption change is closely related to the highenergy intermediate, or state, of photophosphorylation. Rapidabsorption change, with a midpoint potential of 440 mv, is relatedto the electron flow mediated by P870; although it does notdirectly reflect the state of P870, itself. ¹ This article is the second of a series published under thesame title in this journal, volume 11 (1970) p. 519–530. ² Present address: Department of Biology, Faculty of Science,Toho University, Narashino City, Chiba 275, Japan. (Received October 30, 1970; )     

EFFECT OF PLASTOCYANIN ON PHOTOPHOSPHORYLATION IN ISOLATED CHLOROPLASTS

The effect of addition of plastocyanin on photophosphorylationin isolated chloroplasts was studied in comparison with itseffect on photoreduction. Catalytic amounts of added plastocyaninstimulated the HILL reaction with NADP as oxidant, but the photophosphorylationcoupled to this reaction was not influenced by the additionof plastocyanin. The addition of NH₄+ or the complete phosphorylatingsystem did not affect the plastocyanin-induced increase in rateof the photoreduction. The rate of photoreduction of NADP, FMN,or indigo carmine in the presence of DPIP and ascorbate wasmuch accelerated by added plastocyanin, but these reactionswere not coupled to ATP formation. As reported earlier, isolated chloroplasts can utilize plastocyaninas oxidant in the HILL reaction. This reaction was found tobe accompanied by ATP formation, but the efficiency in thiscase was somewhat lower than in photophosphorylation coupledto the HILL reaction with the usual oxidants. The possible mechanism of these reactions is described brieflyand a scheme for reactions of plastocyanin in the electron transferin chloroplasts is presented. (Received July 20, 1965; )     

CHANGES IN EMISSION SPECTRA OF PHOTOSYNTHETIC PIGMENTS IN VIVO

1. The effects of 3-(4'-chlorophenyl)-1, 1-dimethylurea (CMU)onthe fluorescence of photosynthetic pigments in vivo wereinvestigatedin blue-green, red and brown algae and in isolatedspinach chloroplasts.CMU caused an increase in steady statelevel of fluorescenceof chlorophyll a, but did not influencethe fluorescence ofphycobilins. The spectrum of the fluorescenceincrement hada peak at 685 m/µ and a shoulder at 730–740mµ.These two bands probably arise from chlorophyll a(C_f684) belongingto pigment system II.
2. On excitation of chlorophylla in a red alga, Porphyra yezoensis,a fluorescence band witha peak at 720 mµ was observedbesides a shoulder at 685mµ. The 720 m band is inferredto arise from chlorophylla (probably, C_f-1) in pigment systemI.
3. On addition of CMUto the algal cells, the induction of fluorescencewas modifiedto take a simple time course. The induction wasobserved onlywith respect to the fluorescence of chlorophylla, but not inthe fluorescence of phycobilins. The spectrumof the "transient"fluorescence showed two emission bands ofchlorophyll a at 685mµ and 740 mµ, and was quitesimilar in form tothe spectrum of the CMU-caused increase insteady state fluorescence.
4. These facts were interpreted in terms of the correlation offluorescence of chlorophyll a and the photochemical reactionsof photosynthesis

(Received July 20, 1967; )     

Light-induced reactions of ubiquinone in photosynthetic bacterium, Chromatium D. II. Effects of inhibitors and other experimental conditions

Light-induced reactions of ubiquinone in starved cells of ChromatiumD, were investigated under various experimental conditions. The "low amplitude" photoreduction observed on illuminationwithout addition of substrate under aerobic conditions and the"light-off spikes" observed under anaerobic conditions wereshown to be related to the cyclic flow of electrons in the photosyntheticsystem. Both of these were inhibited by o-phenanthroline, HOQNOand piericidin A. The "high amplitude" photoreduction of ubiquinoneobserved in the presence of thiosulfate under aerobic conditionswas inhibited by PCMB, PMA and KCN. The "high amplitude" photooxidationof ubiquinone in the presence of malate under anaerobic conditionswas inhibited by HOQNO, piericidin A, rotenone and PMA. In the presence of KCN and succinate, similar "high amplitude"photooxidation was obtained even under aerobic conditions. Underaerobic conditions, the addition of malate did not affect the"high amplitude" photoreduction due to thiosulfate. On the contrary,under anaerobic conditions, the "high amplitude" photooxidationwith malate was almost completely abolished by the additionof thiosulfate. This apparent counteraction of the oxidationand reduction of ubiquinone was inferred to represent an optimumstate of its efficient turn-over in the noncyclic electron transportchain of photosynthesis inChromatium. ¹This article is the second of a series of studies previouslyreported under the same title in Vol. 8 of this journal (Reference14). (Received August 23, 1968; )     

Enzyme Electrophoretic Evidence for Diploidy in Japanese Woodwardia japonica (L.f.) Sm. (Blechnaceae, Pteridophyte) with 2n= 68

Abstract Enzyme-electrophoretic variation was examined in sporophytes of Woodwardia japonica (2n= 68) and segregation was examined in gametophytes. Despite the high chromosome number, W. japonica displayed disomic segregation of isoenzyme patterns. This and karyotype analyses indicate that W. japonica is a diploid derived from a base number of either x= 17 or x= 34.     

Light-induced absorbance change of carotenoid in chromatophores of photosynthetic bacterium, Rhodopseudomonas spheroides

The nature of the light-induced absorbance change of carotenoid,spheroidene, was investigated with the chromatophores of Rhodopseudomonasspheroides. The experimental results indicate that the changedoes not represent an oxidation-reduction reaction of the carotenoid,but is caused by a change in the state of the chromatophoresclosely related to the high energy state of the photophosphorylation.Since the change almost vanishes at liquid nitrogen temperature,it probably does not represent a primary photochemical reactionin the chromatophores. The values of the quantum yield for thechange of carotenoid were above unity ; 2.5 on an avera (Received November 20, 1969; )     

Two modes of inhibition by 2-heptyl-4-hydroxyquinoline- N-oxide of electron transport associated with photosystem 2 in spinach chloroplasts

2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) exerted two modesof inhibition on the Hill reaction in spinach chloroplasts.The first mode of inhibition by HOQNO, which developed rapidlyand reversibly on addition of the inhibitor to chloroplasts,was observed in the Hill reaction with various electron acceptorsand in photoreduction of methyl viologen with ascorbate as electrondonor, but was not observed in methyl viologen photoreductionsupported by reduced DPIP. Fifty percent inhibition of the Hillreaction was obtained with HOQNO at a low concentration of theorder of 10^–6 M. Besides the fast-developing inhibition, HOQNO caused a progressivedecrease in the Hill activity during the reaction when ferrricyanidewas used as electron acceptor. Similar time-dependent inactivationoccurred when chloroplasts were incubated with HOQNO and ferricyanidein the dark. Chloroplasts, thus inactivated, showed loweredactivity for ascorbate-supported methyl viologen photoreductionbut had unchanged activity for photoreduction with reduced DPIPas electron donor. Progress of the time-dependent inhibitionwas accelerated by increasing the concentration of HOQNO orferricyanide and was suppressed by addition of ferrocyanide.On changing the ratio of ferri- to ferrocyanide in the reactionmedium, fifty percent protection of the Hill activity was obtainedwith a ferri- and ferrocyanide mixture giving the redoxpotentialof 560 mv. A simple kinetic model for the second time-dependentinhibition by HOQNO is presented. ¹ Present address: Department of Botany, Faculty of Science,Toho University, Narashino 275, Chiba, Japan. (Received January 19, 1971; )