Light-induced changes in the fluorescence yield of chlorophyll a in Anacystis nidulans II. The fast changes and the effect of photosynthetic inhibitors on both the fast and slow fluorescence induction

1. The intensity dependence and spectral variations during thefast transient of chlorophyll a (Chl a) fluorescence have beenanalyzed in the blue-green alga Anacystis nidulans. (Unlikethe case of eukaryotic unicellular green or red algae, the fastfluorescence induction characteristics of the prokaryotic blue-greenalgae had not been documented before.)
2. Dark adapted cellsof Anacystis exhibit two types of fluctuationsin the fluorescenceyield when excited with bright orange light(absorbed mainlyin phycocyanin). The first kinetic patterncalled the fast (sec)fluorescence transient exhibits a characteristicoriginal levelO, intermediary hump I, a pronounced dip D, peakP and a transitorysmall decline to a quasi steady state S.After attaining S,fluorescence yield slowly rises to a maximumlevel M. From M,the decline in fluorescence yield to a terminalT level is extremelyslow as shown earlier by Papageorgiou andGovindjee (8). Ascompared with green and red algae, blue-greenalgae seem tohave a small PS decline and a very characteristicslow SM rise,with a M level much higher than the peak P.
3. A prolonged darkadaptation and relatively high intensity ofexciting illuminationare required to evoke DPS type yield fluctuationsin Anacystis.At low to moderate intensities of exciting light,the time forthe development of P depends on light doses, butfor M, thisremains constant at these intensities.
4. Fluorescence emissionwas heterogeneous during the inductionperiod in Anacystis;the P and the M levels were relativelyenriched in short-wavelengthsystem II Chi a emission as comparedto D and S levels.
5. Thefast DPS transient was found to be affected by electrontransportcofactor (methyl viologen), and inhibitors (e.g.,DCMU, NH₂OH)in a manner suggesting that these changes are mostlyrelatedto the oxido-reduction level of intermediates betweenthe twophotosystems. On the other hand, the slow SM changesin fluorescenceyield, as reported earlier (5, 15), paralleloxygen evolution.These changes were found to be resistant toa variety of electrontransport inhibitors (O-phenanthroline,HOQNO, salicylaldoxime,DCMU, NH₂OH and Antimycin a). It issuggested that, in Anacystis,even in the presence of so-calledinhibitors of cyclic electronflow, a "high energy state" isstill produced.
6. Measurementsof Chlorophyll a fluorescence and delayed lightemission inthe presence of both DCMU and NH₂OH indicate thatthe slow SMchanges are not due to the recovery of the reactioncenter IIin darkness preceeding illumination.
7. Our results, thus, suggestthat in Anacystis a net electrontransport supported oxidation-reductionstate of the quencherQ regulates only partially the developmentof the DPS transient,but the development of the slow fluorescenceyield changes seemsnot to be regulated by these reactions.It appears, from datapresented elsewhere, that the slow risein the yield resultsdue to a structural modification of thethylakoid membrane.

¹We are grateful to the National Science Foundation for financialsupport. (Received November 21, 1972; )     

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; )     

Organic Acid Metabolism of Sedum praealtum

1. The organic acids present are citric, isocitric, and l-malic,with a small residue of unidentified acids.
2. The diurnal variationin acidity is due chiefly to changes,in malic acid, with aparallel fluctuation shown by citric acid.Under these conditionsisocitric acid shows little change.
3. The importance of carbondioxide during acidification is confirmed,and it is shown thatat room temperatures or higher the CO₂produced in respirationis sufficient to produce maximum acidification.At lower temperaturesthe supply of CO₂ limits acid production.
4. In the absence ofoxygen no acidification occurs, but even smallquantities (approx.1 per cent.) are sufficient to cause someacid production.
5. Completebalance-sheets are presented for acids, carbohydrates,CO₂ andoxygen for leaves maintained in the dark at high andlow temperatures.As acids are produced there is a correspondingloss of carbohydrate(chiefly starch). A scheme of reactionsis suggested to explainthe experimental results.

     

Transport pathways in canine lingual epithelium involved in sweet taste

The responses of canine lingual epithelium to D-glucose weremeasured in an Ussing chamber to determine the possible contributionof the osmotic changes of taste cells to the response of saccharides.With the mucosal solution containing 50 mM NaCl, 2 mM HEPES,pH 7.4 (solution A) and the serosal solution containing Krebs—Henseleit(KH) buffer the addition of up to 0.5 M D-glucose in the mucosalsolution increased the short circuit current (I_sc) in a sigmoidalmanner. The D-glucose-stimulated I_sc was inhibited by 0.1 mMamiloride or 1 mM ouabain added to either the mucosal or theserosal solution, and partially inhibited by 5 mM BaCl₂ addedto the serosal solution. The inhibition by these three compoundswas also observed in the presence of 0.5 M NaCl. Ouabain alsoinhibited transport when added to solution A. These experimentssuggest that in canine lingual epithelium the paracellular pathwaypermits molecules as large as ouabain (mol. wt 586) to diffusefrom the mucosal to the serosal solution and vice versa underall osmotic conditions. These results may explain the phenomenonof intravascular taste. Such is not the case in rat tongue whereouabain only inhibited transport when added to the serosal solution.Increasing the osmolality of the serosal KH buffer by additionof relatively membrane-impermeable saccharides such as sucroseor L-glucose did not significantly alter the I_sc, whereas makingthe serosal KH solution hypo-osmotic resulted in a transientdecrease in I_sc. These data suggest that the increase in I_scinduced by saccharides, such as D-glucose, is not simply anosmotic response of the epithelium but more likely the consequenceof saccharides binding weakly to receptors. That the responseto both salts by themselves and in the presence of saccharidesexhibits the same cation selectivity, and that both are inhibitedby amiloride, ouabain, BaCl₂ and LaCl₃ suggest that in caninelingual epithelia, in contrast to rat epithelium, the responsesto hyperosmotic concentrations of salts and saccharides mightoccur via the same transcellular pathways.     

INFLUENCE OF INORGANIC NITROGENOUS COMPOUNDS ON TOBACCO SEED GERMINATION INDUCED BY GIBBERELLIN A3, KINETIN AND AMMONIUM SALTS OF ORGANIC ACIDS

1. Investigations were made on the influence of inorganic nitrogenouscompounds upon the the germination of tobacco seeds (Nicotianatabacum L. var. virginica (AGDH.) COM. "Bright Yellow") inducedby GA₃, kinetin and ammonium salts of organic acids. Potassiumnitrate, ammonium chloride, ammonium sulfate and ammonium nitrategreatly increase the germination of the seeds induced by theabove reagents, while these inorganic salts, given alone, arealmost ineffective in causing germination.
2. Kinetin was shownto induce germination of tobacco seeds inthe dark. The discrepancywith the results of previous investigationsin this respectwas discussed.
3. It was inferred that nitrogenous metabolismis involved in theprocess of dark-germination of tobacco seedsas induced by theabove-stated stimulating factors and promotedby inorganic nitrogenoussubstances.

(Received July 17, 1961; )     

PARTIAL PHOTO-REPRESSION OF THE GLYOXYLATE BY-PASS IN EUGLENA

1. Addition of exogenous acetate or ethanol to autotrophic culturesof Euglena gracilis strain Z induces formation of the glyoxylateby-pass.
2. Visible light decreases the activity of malate synthasein greenEuglena by about 50%. No such effect was found in apermanentlybleached mutant.
3. Aconitase activity parallelsthat of malate synthase, but isocitricdehydrogenase activityis constant under all conditions examined.
4. Oxygen consumptionis proportional to the activities of malatesynthase and aconitase,but not to that of isocitric dehydrogenase.
5. The results ofsimilar studies with other growth substrates(pyruvate, malate,succinate) suggest that some of the oxygenconsumed by C₂-grownEuglena may not be associated with energyproduction.

(Received March 25, 1966; )     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

INTERACTION OF GIBBERELLIN A3, AND INORGANIC PHOSPHATE IN TOBACCO SEED GERMINATION

1. Investigation was made on the influence of inorganic phosphateupon the germination of positively photoblastic tobacco seed(Nicotiana tabacum L. var. uirginica (AGDH.) COM. "Bright Yellow")induced by GA₃, GA₃M, kinetin, red light, and ammonium saltsof various organic acids.
2. Inorganic phosphate increases theGAs-induced germination, andinhibits the germination causedby ammonium citrate, while itdoes not influence the germinationbrought about by GA₃M, kinetin,and red light.
3. The optimumpH for the GA₃-induced germination lies in the acidicpH range,indicating that the undissociated form of GA₃ is operative.The stimulatory effect of phosphate is, however, not ascribedmerely to the pH control in the mediurr. Phosphate exerts somespecific influence for which the presence of the free carboxylgroup of GAs is required.
4. The observed contrasting effectsof phosphate on the GA₃-inducedgermination (i.e., acceleration),on the one hand, and on theammonium citrate-induced germination(i.e., inhibition), onthe other, were explained by assumingthat the phosphate effectsultimately consist in acceleratingthe uptake of the carboxylicacid into the seeds.
5. GA₃M alsohas an activity of inducing the germination of tobaccoseedwithout light.

¹Present address: Department of Vegetable Crops, Universityof California, Davis, California, U.S.A. (Received March 12, 1962; )     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

METABOLISM OF GLUCOSE IN THE PROCESS OF "GLUCOSE-BLEACHING" OF CHLORELLA PROTOTHECOIDES

1. As previously demonstrated, normal cells of Chlorella protothecoidesare bleached with degeneration of chloroplasts when they areincubated, under aerobic conditions—either in the lightor in darkness—, in a glucose-containing medium withoutadded nitrogen source ("glucose-bleaching"). It was found inthe present study that under the atmosphere of N₂, neither bleachingnor growth of algal cells occurs in the dark, while in the lighta significant growth of cells takes place with formation ofa certain amount of chlorophyll.
2. Studies on the effects ofvarious inhibitors (ammonium ion,DNP, CMU, -hydroxysulphonates,arsenate, cyanide, azide, andantimycin A) under different conditionsshowed that oxidativephosphorylation is a necessary processfor the occurrence ofthe glucosebleaching as well as the assimilationof glucose(cellular growth). Under light-anaerobic conditionsin the presenceof glucose, assimilation of glucose (cellulargrowth) takesplace being supported by photophosphorylation,but no bleachingoccurs.
3. When the algal cells in the courseof bleaching were transferredto the glucose-free mineral medium,the cell growth ceased immediatelybut the cell bleaching proceededfor several hours before itscessation. The respiratory activity,which was high in the glucose-containingmedium, became loweron transferring the algal cells into theglucose-free medium.The lowered level of respiration was maintained,for more than8 hr after the transfer of cells to the glucose-freemedium.
4. When the cells in the course of bleaching were placed underthe atmosphere of N₂, the cell bleaching ceased almost instantaneously.
5. Based on these observations and other inhibition experiments,it was inferred that a certain intermediate(s) produced by theaerobic respiration of glucose is closely associated with theoccurrence of cell bleaching, and that an O₂-requiring stepmay be involved in the process of chlorophyll degradation.

(Received September 9, 1965; )     

The Effects of Gibberellins on the Growth of Excised Tomato Roots

1. At appropriate concentrations both gibberellic acid (GA) and1-naphthalene-acetic acid (NAA) enhance the main axis growthof excised tomato roots grown in culture media containing sucroseat concentrations below 1 per cent. Lateral root extension growthis enhanced by GA at all sucrose concentrations tested; onlyat the lower sucrose concentrations is this effect observedwith NAA. Both GA and NAA increase the number of emergent lateralroots and this effect is most marked in media of low sucrosecontent. Both GA and NAA at higher concentrations inhibit rootgrowth but NAA exhibits its full range of growth effects overa much narrower concentration range than GA.
2. GA, like NAA,speeds up the loss of meristematic activity whichoccurs whenindividual meristems are repeatedly subculturedin media containing1 per cent, or higher concentrations ofsucrose.
3. The promotionof main axis growth by both GA and NAA involvesenhanced cellelongation and cell division. At a moderatelyinhibitory concentrationGA reduces both cell elongation andcell division; this is notthe case with NAA.
4. Gibberellins A₁, A₂, and A₄ resemble GA(gibberellin A₃) intheir growth effects. Allogibberic acidlike G A promotes lateralroot extension growth but causes markedinhibition of root growthat a much lower concentration thanGA.

     

Further Studies on the Acid Metabolism of Aspergillus niger: THE FORMATION AND UTILIZATION OF OXALIC ACID

1. Some recent works on the formation of oxalic acid by variousfungi are critically considered.
2. The present work deals withthe role of oxalic acid in the metabolismof Aspergillus niger.
3. When glucose solutions were supplied to preformed mats ofthefungus oxalic acid accumulated, attaining an equilibriumlevelwhich was not exceeded despite the presence of a considerableconcentration of glucose.
4. When the glucose supplies were depletedthe oxalic acid concentrationfell steeply to a low level.
5. Theconcentration of oxalic acid was dependent on the glucoseconcentration.In three separate series of experiments it wasshown that theoxalic acid concentration diminished with increasingglucoseconcentration.
6. Similar results were obtained when the cultureswere rearedfrom spores on culture solutions with the normalamounts ofnutrient salts but different glucose concentrations.
7. In all cases the CO₂ output increased with the glucose concentration.
8. When cultures were supplied with glucose+oxalic acid, theconcentrationof the latter fell steeply to the equilibriumlevel attainedon glucose only. In a culture receiving glucose+oxalicacid,with the oxalic acid concentration somewhat below thenormalequilibrium concentration, the formation of oxalic acidfromthe glucose ceased as soon as the equilibrium level hadbeenattained.
9. When 1 per cent. oxalic acid only was suppliedto the fungusthe concentration gradually diminished to a lowlevel. When3 per cent. oxalic acid was supplied the rate ofacid utilizationsoon fell to low value.
10. In several experimentsit was shown that the rate of CO₂ outputwas higher from culturessupplied with glucose+excess oxalicacid than from culturessupplied with glucose only.
11. The rate of oxalic acid carbonloss was always below that ofthe CO₂ carbon output both incultures supplied with oxalicacid only and in cultures receivingglucose+oxalic acid.
12. The cultures were incapable of utilizingneutral sodium oxalateand the presence of this substance hadno effecft on the ofCO₂ output.
13. The results indicate thatthe utilization of oxalic acid isassociated with the liberationof at least an equivalent amountof CO₂.
14. It is suggested thatthe utilization of oxalic acid is promotedby the presence ofglucose, thus accounting for the lower oxalicacid concentrationsand higher rates of CO₂ output of cultureswith higher glucoseconcentrations.

     

STUDIES ON THE PATHWAY OF SULFIDE PRODUCTION IN A COPPER-ADAPTED YEAST

Metabolism of some sulfur-containing substances was studiedin a copper-resistant strain of yeast (R), its parent strain(P) and respiratory-deficient(RD) mutants from them. The resultsobtained are as follows:

1. Using sulfate, sulfite and thiosulfate as sulfur sources, Rproducedmore H₂S than P, and both of these had the activityhigher than their RD mutants. All of them produced a large amountof H₂S from cysteine, but only little from methionine, cysteinesulfinic acid and S-sulfocysteine.
2. From sulfite and thiosulfate,P and R produced more H₂S inaerobicthan in anaerobic condition.With sulfate and cysteine, however,H₂S production did not differunder those conditions.
3. In both P and R, the sulfate-to-sulfiteand sulfite-to-sulfidereactions were remarkably lowered byiron and zinc deficiencies.But the cysteine-to-sulfide reactionwas not affected by themetal-deficiencies.
4. H₂S productionfrom sulfate was remarkably depressed by highconcentrationsof pantothenate.
5. Rates of reaction steps on a plausible pathway from sulfatetosulfide and to organic sulfur compounds areestimated forthe strainsused. R is characterized by its largecapacity ofthe reaction step from sulfate to sulfite, and excessivesulfitethus formed is liberatedas sulfide not by the way ofcysteine.

¹Present address: Research Reactor Institute, Kyoto University,Kumatori-cho, Sennan-gun, Osaka     

CHANGES IN RESTING POTENTIAL AND ION ABSORPTION INDUCED BY LIGHT IN A SINGLE PLANT CELL

1. Effect of light on ion absorption and resting potential of theinternodal cell of Nitella flexilis was investigated under variousconditions.
2. On illumination, the resting potential increasedby about 30mVin 10^–4 M KCl and by about 60 mV in 10^–4M NaClsolution. A similar photoelectric response was also observedin 10^–3 M KCl, 10^–2 M CaCl₂ and 5 x 10^–2 MCaCl₂ solutions, but not at all in 10^–2 M KCl solution.
3. Absorption of ions by the cell took place in parallel withthelight-induced change in resting potential.
4. Red and bluelights were very effective in increasing the restingpotential,while green light was almost ineffective. These differenteffectsof color lights were in good agreement with their effectsinincreasing the osmotic value of the cell.
5. The photoelectricresponse was not affected by phenylurethane,which, on the otherhand, strongly inhibited the light-inducedion absorption.
6. Theuptake of ions by the cell from the external medium intothevacuole is assumed to proceed in two different steps: thefirstis the process involving the ion movements across theoutermostplasmalemma, and the second is that involved in thetransportof ions through the cytoplasmic layer and tonoplast.The formerprocess is considered to be influenced by the increasein restingpotential probably caused by the light absorbed bychlorophyll.The process was, however, suggested to be independentof photosynthesis.On the other hand, the latter process issupposed to be relatedto photosynthesis. A discussion was madealong this line.

(Received July 26, 1962; )     

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; )     

CHANGES IN LEVELS OF PYRIDINE NUCLEOTIDES IN CHLORELLA CELLS DURING THE COURSE OF PHOTOSYNTHESIS

1. Using intact cells of Chlorella, the effects of CO₂ on thelevelsof oxidized and reduced forms of DPN and TPN in the lightandin the dark were investigated.
2. It was found that the light-inducedchanges of the DPNH-levelwere not affected by the presenceor absence of CO₂. On theother hand, the light-induced increaseof TPNH was suppressedin the presence of CO₂ and the levelof TPNH which was raisedon illumination in the absence of CO₂was lowered by the provisionof CO₂.
3. On the basis of thesefindings, it was concluded that TPNH,but not DPNH, is participating,in some way, in the mechanismof photosynthesis.
4. Discussionswere made on the difference in the sites of participationofTPNH and of the photogenic reducing agent (R) in the pathofcarbon in photosynthesis.

(Received February 28, 1960; )     

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; )     

Studies on nucleic acids in chloroplasts isolated from Chlorella protothecoides

1. 1. Subcellular fractions of Chlorella protothecoides were separatedby fractional centrifugation of the algal cell homogenate inmixtures of cyclohexane and CCl₄. The base composition, meltingprofiles and IRC-50 column chromatographic patterns of DNA preparationsfrom the chloroplast and non-chloroplast fractions were examined.It was shown that the algal chloroplast contains at least oneDNA species which is different from the nuclear DNA.
2. 2. RNApreparations from the subcellular fractions were subjectedtoMAK column chromatography, sucrose density gradient centrifugationand analysis for base composition. It was demonstrated thatthe chloroplast contains ribosomal RNA and soluble RNA. Twocomponents of the chloroplast ribosomal RNA were found to havethe same patterns as those of the E. coli ribosomal RNA in MAKcolumn chromatography and zone centrifugation. The major componentof the chloroplast ribosomal RNA was distinctly different fromthat of the non-chloroplastic (cytoplasmic) ribosomal RNA inall properties examined.

¹This work was partly reported at the Symposium on Mitochondriaand Chloroplasts as Self-duplicating Units sponsored by theBotanical Society of Japan in August, 1966, and at the Symposiumon Biogenesis of Subcellular Particles, the 7th Internatl. Congressof Biochemistry, Tokyo, 1967.     

STUDIES ON THE METABOLISM OF A SULFUR-OXIDIZING BACTERIUM II. THE SYSTEM OF CO2-FIXATION IN THIOBAC1LLUS THIOOXIDANS

1. In the early stage of CO₂-fixation by Thiobacillus thiooxidans,which was incubated aerobically in the presence of sulfur, mostpart of the fixed carbon was found in the phosphate ester fraction.
2. The fixation was inhibited by NaF, picolinic acid, PCMB, azide,dipyridyl, o-phenanthroline, monoiodoacetic acid, and arsenite,each in the concentration range where the sulfur oxidation wasnot affected strongly.
3. The crude extract of this organismcould fix CO₂ in the presenceof ATP, R-5-P and Mg⁺⁺. Most partof the fixed carbon was foundin PGA.
4. The crude extract showedthe CO₂-fixation coupled with the H₂S-oxidationin the presenceof ADP.
5. An appreciable reduction of PGA could not be detectedin thepresence of reducing systems, involving TPNH and DPNH.

(Received March 6, 1962; )     

Microbiological Synthesis of Fat: THE EFFECTS OF NITROGEN LEVEL ON THE METABOLISM OF PENICILLIUM LILACINUM THOM IN A SUCROSE MEDIUM

1. A study has been made of the relationships between the synthesesof carbohydrate, protein, and fat by Penicillium lilacinum Thomin presence of different amounts of sodium nitrate us a definedsucrose salts medium.
2. Under the defined experimental conditionsincreases in the concentrationof NO^–₂ in the medium werefollowed by increases in therates at which nitrogen and sugarwere taken up by the fungus,in the quantities assimilated,and in total and protein nitrogenin the felt. These conditionsprevailed so long as unassimilatedsugar was available.
3. Mediaof lower NO^–₃ concentration (for example, 0·32or 0·64 per cent. (w/v) NaNO^–₂;) yielded feltsricher in carbohydrate than were those grown in media of higherNO^–₂; content (0·96 or 1·28 per cent. (w/v)NaNO₃ The carbohydrate content of the felts increased graduallyuntil the sugar in the medium was exhausted; carbohydrate contentthen decreased.
4. Media of lower NO^–₃; concentration weremore conduciveto fat synthesis than those of higher NO^–₃;content.