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; )     

Photosynthetic and light-enhanced dark fixation of 14CO2 from the ambient atmosphere and 14C-bicarbonate infiltrated through vascular bundles in maize leaves

1. The capacity of light-enhanced dark fixation of ¹⁴CO₂ from theambient atmosphere decayed following time-course characteristicsof a first-order reaction (half-life, 1–2 min). The levelof phosphoenolpyruvate in maize leaves under CO₂-free air didnot decrease in the dark subsequent to preillumination. Theseresults indicate that phosphoenolpyruvate carboxylase is activatedin light and quickly inactivated in the following darkness.
2. Removal of oxygen from the atmosphere did not exert any effecton the products of light-enhanced dark fixation of ¹⁴CO₂ providedfrom the atmosphere, the major labeled compounds being malateand aspartate. This confirms that the transfer of carboxyl carbonof C₄-acids to form 3-phosphoglycerate is light-dependent.
3. WhenNaH¹⁴CO₃ solution was vacuum-infiltrated through vasculartissuesof maize leaves, the main initial photosynthetic ¹⁴CO₂fixationproducts were phosphate esters. This indicates thatby thistechnique, ¹⁴CO₂ could be directly provided to the bundlesheathcells, and was fixed via the reductive pentose phosphatecycle.On the other hand, the main initial ¹⁴CO₂-fixation productswere malate and aspartate even when ¹⁴CO₂ was provided throughvascular tissues in the dark immediately following preillumination.The possible regulatory mechanisms underlying the above findingsare discussed.

¹ This work was reported at the 4th International Congress onPhotosynthesis, Reading, September 1977. Request for reprintsshould be addressed to S. Miyachi, Institute of Applied Microbiology,University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ² Present address: Okinawa Branch of Tropical Agriculture ResearchCenter, Ishigaki-shi, Okinawa 907, Japan. (Received October 28, 1977; )     

Photosynthesis in Cell Suspension Culture of a C4 Plant, Gisekia pharnaceoides L.

Photomixotrophic cell suspension culture was established fromthe leaf derived callus cells of Gisekia pharnaceoides L., aC₄ dicotyledonous weed. The late log phase cells possessed shade-typecharacters such as low chlorophyll a/b ratio, less pronouncedO₂ evolution and CO₂ fixation, saturation of photosyntheticCO₂ fixation at low intensity. The chloroplasts from these cellscontained granal stacking with high degree of a very few granawhich are characterized by their wide and high degree of stackings. The predominant labelling of 3-phosphoglyceric acid and sugarphosphates (40% of the total ¹⁴C incorporated) during 5 s exposureto ¹⁴CO₂ in light and subsequent decrease in percentage of ¹⁴Cin these compounds with increase in exposure time indicatedthe operation of the C₃ pathway in these cells. The simultaneoussynthesis of malate (23% of the total ¹⁴C incorporated) is relatedto the much pronounced glycolytic and tricarboxylic acid cycleactivities in these cells. The initial proliferation of callimainly from the zones of vascular supplies in the leaf, highstarch content of the cells, presence of large starch grainsin all the chloroplasts, activities of Calvin cycle enzymes,heavy labelling of C₃ type intermediates and less labellingof aspartate as early photosynthates and rapid accumulationof radioactivity into starch during ¹⁴CO₂ assimilation indicatedthat most of the cells in photomixotrophic culture were derivedfrom bundle sheath cells or the leaf cells of Gisekia changetheir function under culture conditions. ¹Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received January 29, 1982; Accepted June 4, 1983)     

ENZYMIC FORMATION OF GLUTAMINE IN RICE-PLANT SEEDLINGS

1. Purified preparation from rice-plant seedling catalyses a stoichiometricreaction between ATP, glutamate, and NH₂OH in the presence ofMg⁺⁺ to form glutamyl hydroxamate, ADP and inorganic phosphate.
2. The method of purification and some of the properties of theenzyme are described. Co⁺⁺ can be substituted for Mg⁺⁺. Mn⁺⁺,NaF, and PCMB inhibit the enzyme strongly.
3. Inorganic orthophosphateis liberated from ATP by the additionof or cysteine in the presence of glutamate, Mg⁺⁺ andthis preparation. Glutamine was detectedin the reaction productsby paperchromatography.
4. The same preparation catalyses a reactionbetween gluta mineand NH₂OH in the presence of Mg⁺⁺ or Mn⁺⁺,ADP and inorganicphosphate, to form glutamyl hydroxamate.

¹ Present address: The Department of Chemistry, Faculty of Science,Kanazawa University, Kanazawa. (Received October 31, 1960; )     

Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L

The pattern for primary products of CO₂-fixation and the chloroplaststructure of Amaranthus retrqflexus L., a species which incorporatescarbon dioxide into C₄ dicarboxylic acids as the primary productof photosynthesis, were compared in various chlorophyll containingtissues,i.e., foliage leaves, stems, cotyledons and pale-greencallus induced from stem pith. Despite some morphological differencesin these assimilatory tissues, malate and aspartate were identifiedas the major compounds labelled during a 10 sec fixation of¹⁴CO₂ in all tissues. Whereas, aspartate was the major componentin C₄-dicarboxylic acids formed in foliage leaves, malate predominatedas the primary product in stems, cotyledons and the pale-greencallus. The percentage of ¹⁴C-radioactivity incorporated intoPGA and sugar-P esters increased and ¹⁴C-sucrose was detectedin the prolonged fixation of ¹⁴CO₂ in the light, not only infoliage leaves, but also in stems and cotyledons. ¹ This work was supported by a Grant for Scientific ResearchNo. 58813, from the Ministry of Education, Japan. ² Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. ³ Present address: Department of Biochemistry, University ofGeorgia, Athens 30601. Georgia, U. S. A. (Received July 10, 1971; )     

Use of inhibitors to distinguish between C4 acid decarboxylation mechanisms in bundle sheath cells of C4 plants

Both malate and aspartate were decarboxylated at the 4-carbonposition by isolated bundle sheath strands of C₄ plants butto different extents depending upon the species. In Digitariasanguinalis, an NADP-malic enzyme (NADP-ME) species, 100 µMoxalic acid blocked malate decarboxylation through NADP-ME withoutaffecting aspartate decarboxylation which apparently occursthrough NAD-ME. In several phosphoenolpyruvate carboxykinase(PEP-CK) type C₄ species, 200 µM 3-mercaptopicolinic acid(3-MPA), an inhibitor of PEP-CK, specifically inhibited themalate decarboxylation and partially inhibited aspartate decarboxylation.The aspartate decarboxylation insensitive to 3-MPA may occurthrough NAD-ME. Neither inhibitor prevented C₄ acid decarboxylationin bundle sheath cells of NAD-ME species. The inhibitors thusserved to differentiate between the decarboxylation of C₄ acidsin PEP-CK and NADP-ME type C₄ species through their major decarboxylasefrom that of their less active decarboxylation through NAD-ME. ¹ Present address: Department of Biochemistry and Microbiology,Rutgers University, New Brunswick, NJ 08903, U. S. A. (Received January 28, 1977; )     

ORGANIC ACID SYNTHESIS IN RESPONSE TO EXCESS CATION ABSORPTION IN VACUOLATE AND NON-VACUOLATE SECTIONS OF CORN AND BARLEY ROOTS

1. ¹⁴CO₂ fixation into organic acids in tips and proximal sectionsof both corn and barley roots was studied as a function of thenature and concentration of the salt in the external solution.
2. In comparison with the level of ¹⁴CO₂ fixation by vacuolateproximal sections in KCI, incorporation was markedly enhancedin K₂SO₄ and diminished in CaCk. By contrast, non-vacuolateroot tips were indifferent to the type of external salt withrespect to UCO₂ incorporation into organic acids.
3. The effectof salt type on organic acid formation from ¹⁴CO₂was most pronouncedat relatively high concentrations.
4. The conclusion was reachedthat organic acid synthesis in responsetoexcess cation uptakeis the result of cation movement into thevacuole, and thattransport into the vacuole is mediated bythe low-affinity componentof the dual mechanisms involved inion absorption.

¹Present address: Government Forst Experiment Station, Meguro,Tokyo.     

Transient Changes in the Energy State of Adenylates and the Redox States of Pyridine Nucleotides in Chlorella Cells Induced by Environmental Changes

The unicellular green algae Chlorella ellipsoidea was used tostudy transient changes in the energy state of adenylates andthe redox states of pyridine nucleotides induced by environmentalchanges. The transition from anaerobic to aerobic conditionsin the dark induced a sharp rise in the ATP ratio [ATP/(ATP+ADP+AMP)],a sudden decrease in the NADH ratio [NADH/(NAD⁺+NADPH)] anda transient drop in the NADPH ratio [NADPH/(NADP⁺+NADPH)]. Illuminationafter a dark period under anaerobic, CO₂-free conditions inducedsharp increases in the ATP and NADPH ratios and a slower decreasein the NADH ratio. Illumination under aerobic conditions, ineither the presence or absence of CO₂, caused a sharp increasein the NADPH ratio, a small increase in the ATP ratio and aslower increase in the NADH ratio. In the presence of CO₂, asubsequent large drop in the NADPH ratio occurred. Darkeningunder anaerobic, CO₂-free conditions induced a sudden decreasein the ATP ratio, a temporary fall in the NADPH ratio and aslow increase in the NADH ratio. Darkening under aerobic conditionsinduced transient drops in the ATP and NADPH ratios and a suddendrop in the NADH ratio. The addition of CO₂ to the atmospherewith illumination produced a decrease in all three parameters. These results are discussed in relation to current theoriesof the interaction between photosynthesis and respiration. Ourobservations indicate that the energy and reducing potentialsgenerated by photochemical processes are used for and controlother processes besides CO₂ fixation in photosynthetic cells. (Received December 3, 1981; Accepted May 4, 1982)     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Long chain fatty acid synthesis in developing castor bean seeds I. The operation of the path from acetate to long chain fatty acids in a subcellular particulate system

The path of LFA synthesis from acetate in developing castorbean seeds was associated with subcellular 10,000 g particles.Further fractionation of these particles by a stepwise densitygradient method showed the high possibility that the site ofLFA synthesis is the proplastid. A study on cofactor requirementswhen [1-¹⁴C]acetate predominantly incorporated into LFAs indicatedthat synthesis would be achieved by acetyl-CoA carboxylation,malonyl-ACP condensation. ATP, CoA, HCO₃^– and Mg⁺⁺ orMn⁺⁺ were essential for synthesis from acetate by the 10,000gparticulate system. Results of inhibhitor experiment suggestedthat the supply of ATP to the LFA synthesizing system is broughtabout by mitochondrial oxidative phosphorylation, when acetateis the sole precursor for LFA synthesis in this system. TheNADPH generating system was contained in the paticles, althoughthe addition of NADP⁺ and G-6-P increased synthesis. NADH markedlystimulated LFA synthesis from acetate. The primary role of NADHseems to be as a direct reductant in both steps involving thereduction and oxidative desaturation of fatty acid chains; particularly,in the former step, although NADH partially contributes to thesupply of ATP as a respiratory substrate. It is unlikely thatNADH works as a hydrogen donor to NADP⁺. LFA synthesis by thecastor bean particulate system was not stimulated by light,thus differing from that by leaf chloroplasts. (Received July 23, 1973; )     

EFFECT OF {alpha}-HYDROXYSULFONATES ON PHOTOCHEMICAL REACTIONS OF SPINACH CHLOROPLASTS AND PARTICIPATION OF GLYOXYLATE IN PHOTOPHOSPHORYLATION

1. The effect of -hydroxy sulfonates and sulfite, inhibitors ofglycolate oxidase, on the photochemical reactions of spinachchloroplasts was studied. The photo reduction of ferricyanideand NADP was not affected by the poisons, whereas the photophosphorylationand ¹⁴CO₂ fixation were inhibited.
2. Glyoxylate was photoreducedby the chloroplasts in the presenceof PPNR and glyoxylate reductase,and this reduction was acceleratedby the addition of NADP.ATP formation accompanied with thereduction of glyoxylate bythe illuminated chloroplasts wasobserved. It is supposed thatglyoxylate oxidizes the photoreducedNADPH₂ or PPNR and thusthe photophosphorylation is stimulated.

¹A part of this paper was presented at the annual meeting ofAgricultural Society of Japan, in August, 1964. ²Present address: Radiation Center of Osaka Prefecture, Sakai,Osaka.     

Regulation of Steady-State Photosynthesis in Isolated Intact Chloroplasts under Constant Light: Responses of Carbon Fluxes, Metabolite Pools and Enzyme-Activation States to Changes of Electron Pressure

The reactions of isolated intact spinach chloroplasts at saturatinglight and CO₂ to changes in steady-state electron flow werefollowed at the various stages of photosynthesis. Alterationsin the rate of electron flow were induced by the addition ofoxaloacetate (OAA), nitrite or methyl viologen (MV). Two typesof effect can be distinguished: (1) When a small fraction ofthe electrons produced are accepted by OAA or nitrite (up to20% of the electrons produced in the light), the activationstate of the NADP⁺-dependent malate dehydrogenase (NADP-MDH)was strongly decreased, whereas qP and the rate of O₂-productionwere increased. qN, the stromal metabolite pools and the [¹⁴C]-CO₂-fixationrate were only marginally influenced. (2) Higher amounts ofnitrite or MV decreased O₂ production and strongly inhibited[¹⁴C]CO₂ fixation. This treatment further increased the ATP/ADPratio, but had little effect on the NADPH + H⁺/NADP⁺ ratio.The stromal concentrations of 3PGA, DHAP and FBP, and the ratesof 3PGA and DHAP export were drastically changed. In particular,the DHAP/3PGA ratio increased, and the rate of 3PGA export wasdecreased by minor changes in the rate of electron flow. Additionof high amounts of nitrite or MV, but not of OAA decreased theactivation states of NADP-MDH and fructose 1,6-bisphosphatase(FBPase), while the activation states of NADP⁺-dependent glyceraldehyde3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK)remained unchanged under all conditions. (Received February 10, 1997; Accepted September 2, 1997)     

The Organic Acid Metabolism of Bramley's Seedling Apple Peel1

1. The effect of various Krebs cycle acids on the respirationofdisks of apple peel at various stages of maturity was measuredin a Warburg respirometer.
2. Peel tissue from apples at thepre-climacteric and early post-climactericstages apparentlycontain sufficient of the Krebs cycle acidsused, with the exceptionof succinate, to maintain oxidativeprocesses at a maximum.
3. The addition of malate causes a large increase in the CO₂-outputof peel from post-climacteric and senescent fruit but not frompre-climacteric fruit, and a close correlation exists betweenthe climacteric and this decarboxylation of malate. The decarboxylationof malate does not affect the rate of O₂-uptake of peel tissue.The possible part played by the decarboxylation of malate inthe increased CO₂-output at the climacteric is discussed.
4. Addedpyruvate is decarboxylated by the tissue at all stagesof storagelife.
5. The decarboxylation of added malate is an aerobic fermentation,resulting in the quantitative production of acetaldehyde. Althoughthe presence of oxygen is necessary, the rate of O₂-uptake isnot affected by the reaction. Pyruvate decar boxylation doesnot require the presence of oxygen.
6. The O₂-uptake of peelfrom senescent apples can be stimulatedby addition of malate,succinate, and a-ketoglutarate. No evidencewas obtained, however,of oxidation of fumarate, citrate, orpyruvate. The additionof malate to senescent tissue restoresthe lower endogenousrate of O₂-uptake to that of early postclimacteric tissue.
7. Succinate and fumarate are toxic to peel tissue at concentrationabove 0.02M.

     

Short-Term Fixation of 14Carbon by the Submerged Aquatic Angiosperm Potamogeton pectinatus

In ¹⁴C fixation experiments, 3-phosphoglyceric acid was thefirst product of carbon assimilation in the light in Potamogetonpectinatus. The pattern of early ¹⁴C-labelled compounds wasthe same over a range of pH values of the medium from 3.5 to8.1. Rates of ¹⁴C incorporation declined with increasing pHof the medium indicating that free CO₂ is the major exogenoussource of carbon for photosynthesis in Potamogeton pectinatus.     

INHIBITION OF THE PHOTOSYNTHETIC CARBON DIOXIDE FIXATION OF GREEN PLANTS BY {alpha}-HYDROXYSULFONATES, AND ITS EFFECTS ON THE ASSIMILATION PRODUCTS

1. Several kinds of a-hydroxysulfonates, the bisulfite additioncompounds of aldehydes and ketones, were found to inhibit thephotosynthetic carbon dioxide fixation of the barley and wheatseedlings, tobacco leaf and Chlorella cells. Bisulfite additioncompounds of glyoxal, glyoxylate and benzaldehyde were moreeffective in this respect than those of formaldehyde and acetaldehyde.
2. The presence of -hydroxysulfonate causes an increase in ratiosof :¹⁴CO₂ incorporated in glycolate and alanine, and a decreasein incorporation in serine, malate, isocitrate and citrate.It was inferred that these changes are caused by the blockingof the formation of glyoxylate through inhibition of glycolicacid oxidase by the poison.
3. A reaction scheme was proposedto account for the above-statedresults, and the bearing ofthese findings on the possible roleof glycolic acid oxidasein the photosynthetic carbon dioxidefixation and in the formationof amino and organic acids wasdiscussed.

(Received December 8, 1961; )     

Oxygen enhancement of photosynthesis in Anacystis nidulans cells1

Oxygen enhanced photosynthetic ¹⁴CO₂ fixation in Anacystis nidulanscells. Results obtained under different conditions revealedthe following properties of the oxygen enhancement:

1. The enhancement was most significant at ca. 10% O₂. Furtherincrease in oxygen concentration decreased the enhancing effect.The rate under 100% O₂ was equivalent to or a little higherthan that under N₂ gas.
2. b) With the increase in CO₂ concentration,the magnitude ofthe enhancing effect decreased. No oxygen enhancementwas observedwhen the CO₂ concentration. was raised to 9,000ppm.
3. c) The enhancement was observed only at high light intensities.No enhancement was observed when the rate of photosynthesiswas limited by light intensity.
4. Ribulose 1,5-diphosphate (RuDP)carboxylase activity was demonstratedin the extract obtainedfrom A. nidulans cells. We also foundthat the RuDP carboxylaseactivity in this extract was competitivelyinhibited by oxygen.
5. Based on the above-mentioned results, the possible mechanismunderlying the observed enhancing effect of oxygen was discussed.

(Received May 10, 1976; )     

Glycine-serine transformation in the photosynthetic bacterium Chromatium vinosum

The metabolic transformation of glycine into serine in the photosyntheticbacterium Chromatium vinosum was accompanied by the evolutionof CO₂ due to decarboxylation of glycine. Isonicotinylhydrazideinhibited both ¹⁴CO₂ evolution and the formation of ¹⁴C-serinefrom ¹⁴C-glycine. The results indicate that a glycine-serinetransformation reaction takes place which is analogous to thatoccurring in green leaf tissues. Glycine may be metabolisedthrough serine by this reaction. The light stimulation of ¹⁴CO₂evolution and ¹⁴C-serine formation from 14C-glycine by the Chromatiumcells are judged to be results of the light-induced enhancementof ¹⁴C-glycine uptake by the bacterial cells. ¹This is paper 53 in the series "Structure and Function of ChloroplastProteins" and paper 7 of the series "Biosynthetic Mechanismof Glycolate in Chromatium". Paper 6 of the latter series isRef. 3 by Asami and Akazawa (1978). ²This study was aided by research grants from the Ministry ofEducation, Science and Culture of Japan and the Nissan ScienceFoundation (Tokyo). ³Postdoctoral Fellow (1980) of the Japan Society for the Promotionof Science. (Received May 20, 1980; )     

Rates of Photosynthesis in Characean Cells: I. PHOTOSYNTHETIC 14CO2 FIXATION BY NITELLA TRANSLUCENS

A study has been made of photosynthetic ¹⁴CO₂ fixation by isolated‘mature’ internodes of Nitella translucens. Experimentalconditions were similar to those used in studies of the ionicrelations of these cells. Maximum rates of photosynthesis were33–40µµmoles CO₂, fixed per cm² of surfacearea per second (equivalent to 12–15 /xmoles fixed permg chlorophyll per hour). ^l4CO₂ fixation was inhibited to thedark level by 3(3,4,dichlorophenyl)-1, 1-dimethylurea (at 0-6µM or 10µM) and by the uncoupler carbonyl cyanide-m-chlorophenylhydrazone(SµM). The presence of imidazole or ammonium sulphate(both of which uncouple ATP production in vitro) did not resultin an inhibition of 14CO2 fixation. These results are discussedin relation to published work on solute uptake by Nitella translucens.During photosynthesis there was rapid movement of ¹⁴C-labelledorganic compounds out of the chloroplasts. ¹⁴C-labelled sucrose,ammo-acids, and sugar phosphates were found in samples of vacuolarsap.     

Role of carbonic anhydrase in photosynthesis in Chlorella derived from kinetic analysis of 14CO2 fixation1

Time courses of photosynthetic ¹⁴CO₂ fixation and its simulationare presented for Chlorella cells grown under low CO₂ concentration(low-CO₂ cells) and subsequently exposed to 0.2 mM NaH¹⁴CO₃or 130 ppm ¹⁴CO₂ in the presence or absence of carbonic anhydrase(CA) in the suspending medium. It was shown that Chlorella cells utilized only free CO₂ whenNaHCO₃ was given in the presence or absence of CA, or when CO₂was bubbled in the absence of CA. However, the present simulationindicated that both CO₃ and HCO₃^– were utilized when CO₂was given in the presence of CA. Based on these results, weconcluded that 1) Chlorella cells absorb only free CO₂ and 2)this gas is provided to algal cells in two ways, i.e., by directand indirect CO₂ supply. Usually, the dissolved CO₂ is directlyutilized by the algal cells (direct supply of CO₂). However,when the concentration of dissolved CO₂ is extremely low andwhen there is CA, CO₂ reconverted from HCO₃^– is also utilizedby Chlorella cells (indirect supply of CO₂). The utilizationof HCO₃^– indicated by the above simulation was explainedby the indirect supply of CO₂. We further assumed that the indirectsupply of CO₂ to ribulose 1,5-bisphosphate carboxylase occursmainly in the chloroplasts of low-CO₂ cells containing highCA. Thus, under low CO₂ concentrations, low-CO₂ cells can carryout more efficient CO₂ fixation than high-CO₂ cells, resultingin the lower apparent Km(CO₂). ³Department of Biology, Faculty of Science, Niigata University,Niigata, Japan. (Received April 2, 1980; )     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )