Photoinhibition of 5-aminolevulinic acid formation under GO2-free condition

5-Aminolevulinate accumulation in the presence of levulinatewas followed in greening Chlorella protothecoides cells. Underthe CO₂-free condition, ALA formation was severely inhibitedby 20 W/m² white light. The inhibition was removed by CMU. Combinedaddition of CMU with N, N'-tetramethyl phenylenediamine plusascorbate again caused photoinhibition of ALA formation, whilethe addition of CMU with dithiothreitol caused severe inhibitionof ALA formation in both light and darkness. Exogenous glucose enhanced ALA formation in darkened algal celb,but not in photo- and DTT-inhibited cells. In either case, glucoseseemed to be metabolized mainly by the algal cells through theglycolysis-citric acid system. It was inferred that ALA formationwas suppressed at the site of, or related to, an enzyme reactionforming ALA. (Received June 27, 1979; )     

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.     

Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added {delta}-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation

Light-induced formation of chlorophyll in "etiolated" cellsof Chlorella protothecoides was studied under various experimentalconditions, (i) Two different types of enhancing effect of lightwere demonstrated: a "long-term" effect lasting for many hoursafter a relatively short illumination of etiolated cells anda "short-term" effect disappearing in a few hours after illumination,(ii) Addition of ALA caused enhancement of chlorophyll synthesisin etiolated cells in darkness as well as in light; the ALA-enhancedrate of dark chlorophyll synthesis, however, was much lowerthan the rate in light without added ALA. ALA was replaceablewith succinic acid plus glycine in light, but not in the dark,for enhancement of chlorophyll formation, (iii) Adding glucose,fructose, galactose, glycerol or acetate—at concentrationsmuch lower than those previously shown to induce "bleaching"of green algal cells-caused a more or less marked suppressionof light-induced greening in etiolated cells, (iv) Added glucosealmost instantaneously and completely stopped chlorophyll synthesisin light as well as in darkness either with or without addedALA. On the basis of these and other results, a tentative schemeis presented for the enhancing effects of light and the suppressiveeffects of glucose on chlorophyll formation in algal cells. (Received April 1, 1970; )     

Role of Light in 5-Aminolevulinic Acid Formation in Wild Strain and Mutant C-2A' Cells of Scenedesmus obliquus

In dark-grown wild strain cells of Scenedesmus obliquus, 5-aminolevulinicacid (ALA) formation was induced by irradiation with a weakblue light, as in its mutant C-2A' cells. The induction wasinhibited by distamycin A, 6-methylpurine, cycloheximide andchloramphenicol. After the light induction, the ALA formationcould proceed in the dark as well as in the light, in such heterotrophicallygrown wild type cells, but not in the greening mutant C-2A'cells. In the latter, ALA formation was dependent on red light,as well as on blue light, in the presence of CMU. The amountsof protochlorophyll in the mutant cells increased upon cessationof illumination and decreased with subsequent irradiation withblue and red light. The possible role of protochlorophyll asa photoreceptor in regulation of ALA formation in the mutantcells is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otuka, Hachioji, Tokyo 192-03, Japan. (Received January 17, 1981; Accepted April 30, 1981)     

Inhibitory Effect of Gabaculine on the Biosynthesis of 5-Aminolevulinate in the Tigrina d12 Mutant of Barley

Six-day-old, dark-grown, seedlings of barley homozygous forthe recessive mutation tigrina d¹² accumulated 5-aminolevulinicacid (ALA) and protochlorophyll (ide) in amounts exceeding thewild type level. Transferring the etiolated mutant to lightresulted in the destruction of pigments and the deteriorationof the ALA forming system. Such deleterious effects did notoccurusing light-grown mutant or etiolated and greened wildtype seedlings. Gabaculine (GAB) at 50 µM inhibited ALAsynthesis by about 85% when etiolated wild type seedlings wereexposed to light. In light-grown leaves of either wild typeor mutant strain, ALA production was also sharply (ca. 75%)inhibited by GAB. During dark incubation, however, the inhibitionof ALA accumulation did not exceed 50% in all types of tissues.The results give further evidence for the operation of the C₅pathway in such seedlings since GAB decreased the biosynthesisof ALA to the same extent in both tigrina d¹² mutant and wildtype of barley. (Received July 2, 1990; Accepted May 7, 1991)     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells II. Blue Light Effect on 5-Aminolevulinic Acid Formation

5-Aminolevulinic acid (ALA) accumulation in dark-grown tobaccocallus cells in the presence of levulinic acid (LA) was followedunder blue or red light or in continuous darkness. Significantformation of ALA continued in the dark. The protochlorophyll-(ide) (Pchl) content of dark-incubated cells remained low becauseof its turnover. We inferred that the feedback inhibition ofALA synthesis by Pchl would not occur in darkincubated calluscells. ALA formation was enhanced by blue light, and this effectreached saturation at an intensity of about 800 mW.m^–2.Neither weak nor strong red light affected ALA formation. Fullenhancement of ALA formation by blue light was attained afterfairly long continuous illumination of the callus cells. Thisblue lightenhanced activity of ALA synthesis declined very slowlyduring the subsequent dark incubation. The blue light enhancement of ALA formation was observed incallus cells supplied with sucrose over a wide range of concentrations.Pchl regeneration in carbon-starved callus cells, supplied withglutamate at various concentrations, was also markedly enhancedby blue light. Respiration of the callus cells was not enhancedby blue light. A possible role of blue light in regulating ALAformation in callus cells is discussed. ¹Dedicated to the late Professor Joji Ashida. (Received September 3, 1982; Accepted April 5, 1983)     

Wavelength effects on photosynthetic carbon metabolism in Chlorella

To study the wavelength-effect on photosynthetic carbon metabolism,¹⁴C-bicarbon-ate was added to Chlorella vulgaris 1 lh suspensionunder monochromatic blue (456 nm) and red (660 nm) light. Thelight intensities were so adjusted that the rates of ¹⁴CO₂ fixationunder blue and red light were practically equal. Analysis of¹⁴C-fixation products revealed that the rates of ¹⁴CO₂ incorporationinto sucrose and starch were greater under red light than underblue light, while blue light specifically enhanced ¹⁴CO₂ incorporationinto alanine, aspartate, glutamate, glutamine, malate, citrate,lipid fraction and alcohol-water insoluble non-carbohydratefraction. Pretreatment of the algal cells in phosphate mediumin the dark, which was essential for the blue light enhancementof PEP carboxylase activity, was not necessary to induce theabove wavelength effects. Superimposition of monochromatic bluelight at low intensity (450 erg.cm^–2.sec^–1) on thered light at saturating intensity caused a significant decreasein the rate of ¹⁴CO₂ incorporation into sucrose and increasein incorporation into alanine, lipid-fraction, aspartate andother related compounds, indicating that the path of carbonin photosynthesis is regulated by short wavelengdi light ofvery low intensity. Possible effects of wavelength regulationof photosynthetic carbon metabolism in algal cells are discussed. ¹ Part of this investigation was reported at the XII InternationalBotanical Congress, Leningrad, 1975 and the Japan-US CooperativeScience Seminar "Biological Solar Energy Conversion", Miami,1976. Requests for reprints should be addressed to S. Miyachi,Radioisotope Centre, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. ⁴ Present address: Department of Chemistry, Faculty of PharmaceuticalSciences, Teikyo Univ., Sagamiko, Kanagawa, Japan. (Received August 6, 1977; )     

Further studies on the metabolism of glucose in the process of "glucose-bleaching" of Chlorella protothecoides

Previous studies have demonstrated that when cells of Chlorellaprotothecoides are incubated in a medium containing glucosebut no nitrogen source, they are profoundly bleached with degenerationof chloroplast structure and photosynthetic activity. When anitrogen source (urea) is added to the glucose medium, bleachingof algal cells is greatly suppressed. In this work the metabolismof glucose in the process of glucose-induced bleaching was studiedusing ¹⁴C-glucose as tracer. Changes in algal cell activityfor ¹⁴CO₂-evolution and ¹⁴C-incorporation into various cellularsubstances from ¹⁴C-glucose were followed. Most conspicuouswere increases in cellular activities for assimilating ¹⁴C-glucoseinto lipids (fatty acids) and glucose polymer. When urea wasadded to the glucose medium, the incorporation of ¹⁴C by algalcells into fatty acids was greatly reduced, while the assimilationof ¹⁴C into glucose polymer was increased. These and previous observations suggest that the formation oflarge amounts of lipids (fatty acids) probably is causally relatedto the induction of algal cell bleaching. (Received March 5, 1969; )     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 1. The Time Course of 14CO2 Production

Glucose, either uniformly labelled with¹⁴C, or specificallylabelled in the I, 2, or 6 position, was added to C. vulgaris.Radio-active carbon dioxide was produced initially ten timesfaster from glucose-I-¹⁴C than from glucose-6-¹⁴C. This differencewas found with carbohydrate-starved cultures, exponentiallygrowing cultures, and cultures assimilating ammonia or nitraterapidly. A similar difference was also found with C. pyrenoidosaand Ankistrodesmus. 37 per cent. of the ¹⁴C added as glucose-¹-¹⁴Cto exponentially growing cells was recovered as carbon dioxidebut generally the recovery was less than this. Only 5 per cent.of ¹⁴C added as glucose-6-¹⁴C was recovered as carbon dioxide.The specific activity of the carbon dioxide produced was considerablylower than that of the carbon in the added glucose.     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Suppressive effect of light on the formation of DNA and on the increase of deoxythymidine monophosphate kinase activity in Chlorella protothecoides

As previously demonstrated, chlorophyll-less cells of Chlorellaprotothecoides are obtained when the alga is grown in a mediumrich in glucose and poor in a nitrogen source (urea). When thesecells are incubated in a medium enriched with a nitrogen source,there occurs, besides greening of algal cells, an active formationof DNA followed by synchronous cellular division. The DNA formationand cellular division are markedly suppressed by light of acomparatively low intensity. Blue light is most effective andred light least effective in suppression. The effect of light on the level of dTMP kinase activity inthe algal cells was investigated in relation to the photoinhibitionof DNA formation. It was found that light suppresses the increaseof dTMP kinase activity in the algal cells which starts in advanceof active DNA synthesis, and that blue light has a strongersuppressive effect than red light. ¹Present address: Institute of Medical Science, University ofTokyo, Tokyo.     

Spectral effectiveness in chlorophyll and 5-aminolevulinic acid formation during regreening of glucose-bleached cells of Chlorella protothecoides

Regreening of glucose-bleached cells of Chlorella protothecoidesis stimulated by light. Spectral effectiveness in the processshowed maxima around 370, 440 and 480 nm, suggesting a flavoproteinas primary photoreceptor. Action spectra of ALA synthesis provedto be similar to those of chlorophyll formation, indicatingthat light stimulation of greening in this alga is regulatedat the first step of chlorophyll biosynthesis. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 27, 1978; )     

Effects of 5-Aminolevulinic Acid on the Accumulation of Chlorophyll b and Apoproteins of the Light-Harvesting Chlorophyll a/b-Protein Complex of Photosystem II

The effects were examined of 5-aminolevulinic acid (ALA) onthe accumulation of Chl and apoproteins of light-harvestingChl a/b-protein complex of photosystem II (LHCII) in cucumbercotyledons under intermittent light. A supply of ALA preferentiallyincreased the accumulation of Chl a during intermittent illumination.However, when cotyledons were pretreated with a brief exposureto light or benzyladenine (BA), the stimulatory effect of ALAon the increase in the level of Chl b was greater than thatin the level of Chl a, resulting in decreased ratios of Chla/b. Time-course experiments with preilluminated cotyledonsrevealed that LHCII apoproteins accumulated rapidly within thefirst 30 min of intermittent illumination with a decline duringsubsequent incubation in darkness. A supply of ALA did not affectthe accumulation of LHCII apoproteins during the intermittentlight period, but it efficiently inhibited the decline in theirlevels during the subsequent darkness. After exposure to a singlepulse of light of BA-treated cotyledons, the prompt increasein levels of LHCII apoproteins was not accompanied by the formationof Ch b, which began to accumulate later. The pattern of changesin levels of LHCII apoproteins was quite similar to that inlevels of Chl a. These results suggest that LHCII apoproteinsare first stabilized by binding with Chl a and that an increasedsupply of Chl a and the accumulation of LHCII apoproteins areprerequisites for the formation of Chl b. ¹Present address: Department of Chemistry, Faculty of Scienceand Technology, Meijo University, Aichi, 468 Japan.     

The Kinetics of Isocitrate Lyase Formation in Chlorella: Evidence for the Promotion of Enzyme Synthesis by Photophosphorylation

The addition of acetate to aerobic Chlorella pyrenoidosa indarkness was followed by the formations of isocitrate lyaseactiity. After a lag period of 40 minutes the formation proceededat a constant rate. By use of actylamide gel electrophoresisit was shown that the increase in enzyme activity was accompaniedby the formation of a new protein which, after separation byelectrophoresis, contained isocitrate lyase activity. The formationof isocitrate lyase was repressed by glucose; it was repressedby light in the presence of carbon dioxide, but not when DCMUwas added. In light, plus DCMU, isocitrate lyase was formedanaerobically and the capacity for photo-formation of isocitratelyase was saturated at 500 ergs/cm²/sec. In this respect theprocess resembled the photo-conversion of glucose to polysaccharidebut differed from the photo-assimilation of carbon dioxide whichbecame saturated at a heigher light intensity. Monochromaticlight of 706 mµ wavelength supported both isocitrate layseformation and the conversion of glucose to polysaccharide butnot carbon dioxide fixation. It is concluded that ATP generatedby cyclic photophosphorylatin can provide the energy for isocitratelyase synthesis in Chlorella.     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

Studies on the Cell Wall of Chlorella IV. Incorporation of Glucose-Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

Uniformly ¹⁴C-labeIled glucose was fed to synchronously growingChlorella cells in the dark or in light. The rate of ¹⁴C-incorporationinto hemicellulose showed two maxima one in the growth phaseand one in the reproductive phase. Significant ¹⁴Cincorporationinto a "rigid wall" was found only in the reproductive phase. (Received April 14, 1983; Accepted June 15, 1983)     

Photoinhibition of Glucose Uptake in Chlorella

In colorless mutant cells of Chlorella vulgaris (M125), endogenousrespiration in the dark was not affected by 30-min preilluminationwith white light (9,000 mW?m^–2), while exogenous respirationof glucose or fructose was inhibited significantly by the sametreatment in air, but not under N₂. This light effect on exogenousrespiration was accompanied by an inhibition of hexose uptake. When autotrophically grown wild-type cells of Chlorella vulgaris(211-11h) were incubated in glucose medium with DCMU, lightalso greatly inhibited glucose uptake and growth. Blue lightwas very effective, while red light had only a slight effect.This photoinhibitory effect was also observed in algal cellsthat had been grown in a glucose-containing medium in the dark. Using SDS-gel electrophoresis, a new protein peak with a molecularweight of 35–40 kDa was detected in plasma membrane-richcell wall fractions when Chlorella vulgaris (211-11h) cellswere transferred to a glucose-containing medium. This peak disappearedafter the algal cells were returned to the glucose-free medium.These findings suggest that this protein includes the hexose-carrierprotein. Blue light significantly inhibited the formation ofthis protein during incubation in a glucose-containing medium. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)     

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)     

Links between Glucose Uptake and Metabolism in Nitella translucens

The uptake of ¹⁴C-glucose into cells of Nitella translucenshas been investigated under experimental conditions previouslyused in studies of the ionic relations of these cells. Glucoseentry was considerably stimulated by light, and under aerobicconditions the fluxes remained constant for many hours. Theinflux of glucose was inhibited by over 80 per cent at low temperature(4° C) and by over 90 per cent by the uncoupler carbonylcycanide-m-chlorophenylhydrazone. 2-Deoxy-D-glucose was a non-competitiveinhibitor of glucose uptake both in light and darkness. Cyclicphotophosphorylation promoted the influx (with decreasing efficiency)for several hours. It is suggested that an ATP-dependent transportprocess controls glucose entry to the cells, and that passivediffusion is of little significance.     

The Inhibitory Effect of Light on Proton-Coupled Hexose Uptake in Chlorella

The addition of 3-O-methyl-D-glucopyranose to wild-type cellsof Chlorella vulgaris (211-11h) grown in glucose medium in thedark induced a rapid alkalization of the external medium (protonuptake), whereas this pH shift did not occur in autotrophicallygrown cells. Light-irradiation inhibited the sugar-induced protonuptake, making the blue end of the visible spectrum very effectiveand the red end only slightly effective. This spectral dependencecorresponds to that of photoinhibition on hexose uptake in thesealgae. A similar photoinhibitory effect was observed in cellsof a colorless mutant of Chlorella vulgaris (M125). The activity of nitrate-proton symport in Chlorella vulgaris(211-11h) was also enhanced by the addition of glucose. Illuminationhad no inhibitory effect on this increased transport. The effectof light on the hexose uptake system is discussed. ¹ Present address: Laboratory of Chemistry, Faculty of PharmaceuticalSciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan. (Received July 31, 1986; Accepted March 12, 1987)