Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Modulated Degradation of Ribulose Bisphosphate Carboxylase in Leaves on Top-Pruned Shoots of the Mulberry Tree (Morus alba L.)

Yamashita, T. 1987. Modulated degradation of ribulose ftisphosphatecarboxylase in leaves on top-pruned shoots of the mulberry tree(Morus alba L.).—J. exp. Bot. 38: 1957–1964. The effects of pruning shoot tops on the synthesis and degradationof ribulose 1,5–Wsphosphate carboxylase (RuBPCase) inleaves on remaining shoots were investigated in mulberry trees.Leucine labelled with ¹⁴C was fed to leaf discs from field-grownmulberry trees and ¹⁴C incorporation into RuBPCase was examined.Proportion of ¹⁴C in RuBPCase to leucine–¹⁴C absorbedby leaf discs was remarkably lowered by top-pruning, thoughoccasionally a slight increase was observed soon after pruning.Yet RuBPCase content in leaves on top-pruned shoots became progressivelyhigher than that in leaves on intact shoots. Changes in ¹⁴Cin Ru1BPCase in leaves of mulberry saplings previously fed ¹⁴CO₂were followed. Following ¹⁴CO₂ feeding, the attainment of themaximal level of ₁₄C in RuBPCase was retarded by top-pruning.The highest level of ₁₄C in RuBPCase was maintained in leaveson top-pruned shoots but decreased in leaves on intact shoots.Specific radioactivity in RuBPCase continued to increase inleaves on top-pruned shoots even after attaining a maximum levelin the control leaves. These facts suggest that the increasein RuBPCase by top-pruning results from a cessation of its degradationfor the remobilization of nitrogen for newly developing leaveson shoot tops. Key words: RuBP carboxylase, shoot pruning, mulberry (Morus alba)     

Transport and Fixation of Inorganic Carbon during Photosynthesis in Cells of Anabaena Grown under Ordinary Air: III. Some Characteristics of the HCO3--Transport System in Cells Grown under Ordinary Air

In cells of cyanobacterium Anabaena variabilis grown under ordinaryair (low-CO₂ cells), the transport of both CO₂ and HCO₃^–was significantly enhanced by Na⁺. This effect was pronouncedas the external pH increased. When low-CO₂ cells were treatedwith an inhibitor of carbonic anhydrase (CA), only CO₂ transportbut not HCO₃^– transport, was inhibited. The initial rateof photosynthetic carbon fixation as a function of the concentrationof internal inorganic carbon (IC) was practically the same irrespectiveof whether CO₂ or HCO₃^– was externally supplied. Theseresults suggest that IC is actively transported through theplasma membrane in a form of HCO₃^– probably by some transporterand that the transmembrane Na⁺ gradient is involved in thisIC transport system. Free CO₂ may be hydrated by CA to HCO₃^–and then transported to the cells by this transporter. On the other hand, CO₂ is actively taken up by cells grown withair containing 5% CO₂ (high-CO₂ cells) though the enhancingeffect of Na⁺ was much smaller in high- CO₂ cells than in low-CO₂cells. The initial rate of fixation as a function of internal IC concentrationindicated that the rate of the carboxylation reaction of accumulatedIC is higher in I0W-CO₂ cells than in high-CO₂ cells. The studieswith ethoxyzolamide indicated that even in low-CO₂ cells, CAdoes not function inside Anabaena cells. These results suggestthat inside the low-CO₂ cells of Anabaena, some mediator(s)facilitates the transport of IC to RuBPCase. (Received January 23, 1987; Accepted April 24, 1987)     

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase(RuBPCase) was estimated after rapidly extracting it from intactwheat leaves pretreated under different light and CO₂ levels.No HCO₃^– was added to the extraction buffer since it isshown to inhibit RuBPCase. The activity increased as light intensityor CO₂ concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased.Light activation did not affect the affinity of RuBPCase forCO₂. A K_m of 30 µM CO₂ under air level O₂ was determined.CO₂, light and temperature are three main limiting factors ofphotosynthesis. It seems that the activity of RuBPCase is regulatedby these factors according to the requirements for CO₂ fixation.     

Isolation of Native Pyrenoid Core Matrix Having Ribulose 1,5-bisphosphate Caxrboxylase Activity from the Green Alga Bryopsis maxima

The native pyrenoid core matrix of the green alga Bryopsis maximawas isolated by diethyl ether treatment and sucrose densitygradient centrifugation using 1.8 M phosphate buffer. The purityof the pyrenoids was examined by microscopy, polyacrylamidegel electrophoresis and marker materials. The purified pyrenoidscontained the large subunit and the small subunit of ribulose1,5-bisphosphate carboxylase (RuBPCase) and more than 10 minorpolypeptides. They also showed RuBPCase activity when solubilizedon being transferred to a low-concentration buffer. The specificactivity was 0.62 µmol CO₂ fixed (mg protein)^–1min^–1. This isolation method is suitable for obtainingintact pyrenoids not covered by starch sheaths or membraneswithout the need for chloroplast fixation. (Received July 27, 1987; Accepted October 20, 1987)     

Activities of Carboxylation Enzymes in Freshwater Macrophytes

Fifteen species of freshwater macrophytes, mainly from cool,temperate waters, were assayed for ribulose bisphosphate carboxylase-oxygenase(RuBPCase) and phosphoenolpyruvate carboxylase (PEPCase) activities.In extracts from all the species RuBPCase was the most activecarboxylation enzyme, and the RuBPCase/PEPCase ratio was atleast 2·0, even for the submersed species Isoetes lacustrisL. and Littorella unifiora (L.) Aschers. which have been reportedto show Crassulacean Acid Metabolism (CAM) activity. The PEPCaseactivity in I.lacustris was lower than that found in some non-CAM-likespecies. In this respect, I.lacustris and L unifiora differfrom most terrestrial CAM plants. However, these two species,along with Potamogeton praelongus Wulf. and Juncus bulbosusvar.fluitans L., had the lowest RuBPCASE/PEPCase ratios, lowerthan found in terrestrial C₃ species; suggesting that the potentialfor substantial photosynthetic metabolism of C₄ acids existsin some temperate, submersed plants. In the three amphibiousspecies (Potamogeton polygonifolius Pourr., Mentha aquaticaL., and Hippuris vulgaris L.) examined, the aerial leaves exhibitedhigher RuBPCase activities than the submersed leaves. Key words: Ribulose bisphosphate carboxylase-oxygenase, phosphoenolpruvate carboxylase, freshwater macrophytes     

Photosynthetic carbon metabolism in photoautotrophically and photomixotrophically cultured tobacco cells

Labeling patterns of light and dark ¹⁴CO₂-fixation in photoautotrophicallyand photomixotrophically cultured tobacco cells were determined.During short term ¹⁴CO₂ fixation under light, malate(C₃–C₃carboxylation) was heavily labeled as were phosphoglyceric acidand sugar phosphates(C₁–C₅ carboxylation). Dark fixationcould not account for this high ¹⁴CO₂ incorporation into theC₄ compounds linked to PEPCase. Two carboxylation pathways linkedto the RuBPCase and PEPCase were indicated in ¹⁴CO₂-fixationin light in photoautotrophically and photomixotrophically culturedcells. (Received October 25, 1979; )     

Photoinhibition under Atmospheric O2, the Activation State of RuBP Carboxylase and the Content of Photosynthetic Intermediates in Soybean and Wheat

Ward, D. A. and Drake, B. G. 1987. Photoinhibition under atmosphericO₂, the activation state of RuBP carboxylase and the contentof photosynthetic intermediates in soybean and wheat.—J.exp. Bot. 38: 1937–1948. Associations between photosynthesis, the activation state ofRuBP carboxylase and the contents of photosynthetic intermediateswere compared in soybean and wheat leaves before and after exposureto photoinhibitory treatments in the presence of atmosphericO₂. Exposing attached leaves to a supra-saturating irradiance(3 800 µmol quanta m^{– 2} s^–1) for 2 h in CO_2-freeair decreased carboxylation efficiency and the light-saturatedphotosynthetic rate in air by approximately 50%. Exposure tothe photoinhibitory treatment for periods in excess of 2 h didnot cause a further decrease of photosynthesis in soybean. Althoughphotosynthesis was reduced, the initial and total (fully-activated)activities of ribulose 1,5-bisphosphate carboxylase (RuBPCase)in leaf extracts were unaltered in each species by the photoinhibitorytreatment. This was true for leaves sampled under both air andat a rate-limiting intercellular CO₂ partial pressure (C_i) of75 µPa Pa^–1. The contents of ribulose l,5-bisphosphate(RuBP) and 3-phosphoglyceric acid (3-PGA) were reduced by thephotoinhibitory treatment in soybean leaves sampled in air andat a rate-limiting C_i, although the RuBP/3-PGA ratio was unaffected.The relative reduction of RuBP content in soybean leaves atrate-limiting C₁ was similar to the corresponding reductionof carboxylation efficiency. For wheat,the relative reductionof RuBP content at rate-limiting C_i (–19%) caused by thephotoinhibitory treatment was considerably less than the correspondingdecrease of carboxylation efficiency (–49%).The RuBP/3-PGAratio of wheat was also increased significantly by the photoinhibitorytreatment The significance of these observations to the regulationof CO₂-limited photosynthesis in leaves experiencing photoinhibitionunder atmospheric oxygen is discussed. Consideration is alsogiven to the previous contention that contemporary measurementsof initial activity in crude extracts may provide a spuriousindication of the amount of the enzyme-CO₂-Mg_{2 +} form of RuBPcarboxylase present in the leaf. Key words: Carboxylation efficiency, RuBP carboxylase, photoinhibition, RuBP, 3-PGA     

Changes in the Rate of Photosynthesis during Grain Filling and the Enzymatic Activities Associated with the Photosynthetic Carbon Metabolism in Rice Panicles

Photosynthesis is known to occur in rice panicles, but littlehas been reported about the photosynthetic or biochemical characteristicsof such panicles. The estimated gross amount of photo-syntheticallyassimilated CO₂ in a panicle is 30% of that in a flag leaf.This result and the good light-intercepting characteristicsof the panicle in the canopy suggest that photosynthesis inthe panicle may contribute significantly to grain filling. Therice panicle is composed of spikelets and of rachis-branchesincluding rachis which have estimated gross rates of photosynthesisduring the 30-day period after anthesis of 130 to 180 and 50to 100 µmol CO₂.(mg Chl)^–1.h^–1, respectively.The corresponding rate for the flag leaf is 180 to 230 µmolCO₂.(mg Chl)^–.h^–. On the basis of Chl, spikeletshave a high photosynthetic capability which is similar to thatof the flag leaf. The activities of ribulose-l,5-bisphosphate carboxylase (RuBPCase),phosphoenolpyruvate carboxylase (PEPCase), and pyruvate.P_i dikinase(PPDK) in spikelets were 129, 220, and 87 µmol.(mg Chl)^–.h^–,respectively. The activities of PEPCase and PPDK in spikeletswere considerably higher than those in the flag leaf or rachis-branches.Oxygen-insensitive photosynthesis was found only in spikelets.The K_m of NaHCO₃ for photosynthesis by slices of spikelets inan aqueous solution (0.6 mM) was considerably lower than thatfor slices of flag leaf (4.2 mM). All these results indicatethat spikelets have different photosynthetic characteristicsfrom those of the flag leaf and rachis-branches. The possibilityof C₃–C₄ intermediate photosynthesis or C₄-like photosynthesisin spikelets is discussed. ⁴Present address: Department of Biochemistry, Faculty of Science,Saitama University, Urawa, 338 Japan (Received February 14, 1990; Accepted June 12, 1990)     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

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; )     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Variation of Phosphoenolpyruvate Carboxylase Activity in Dunaliella Associated with Changes in Atmospheric CO2 Concentration

In Dunaliella tertiolecta, D. bioculata and D. viridis the activitiesof phosphoenolpyruvate carboxylase and carbonic anhydrase werehigher in the cells grown in ordinary air (low-CO₂ cells) thanin those grown in air enriched with 1–5% CO₂ (high-CO₂cells), whereas in Porphyridium cruentum R-1 there was no differencein phosphoenolpyruvate carboxylase activity between these twotypes of cells. Apparent K_m(NaHCO₃) values for photosynthesisin low-CO₂ cells of all species tested were smaller than thosein high-CO₂ cells. Most of the ¹⁴C was incorporated into 3-phosphoglycerate,sugar mono- and di-phosphates during the initial periods ofphotosynthetic NaH¹⁴CO₃ indicating that both types of cellsin D. tertiolecta are C₃ plants. (Received May 27, 1985; Accepted June 25, 1985)     

Changes in Photosynthetic Characteristics Induced by Transferring Air-Grown Cells of Chlorococcum littorale to High-CO2 Conditions

When air-grown cells of Chlorococcum littorale was enrichedwith CO₂, growth was enhanced after a lag period of one to twodays at 20% CO₂, and 3 to 6 days at 40% CO₂. Changes in therate of photosynthesis measured as oxygen evolution and CO₂fixation, were similar to those observed for growth. Duringthe initial inhibition of photosynthesis in 40% CO₂, the activityof PSII was suppressed. In contrast, PSI activity was greatlyenhanced. Air-grown cells of C. littorale possessed comparatively highcarbonic anhydrase (CA) activity which was localized insidethe cells and on the cell surface. Under high CO₂ concentrationsextracellular CA activity was greatly suppressed and intracellularactivity almost completely abolished. Phosphoenol pyruvate carboxylaseactivity was also suppressed in high CO₂-grown cells. Ribulose-l,5-bisphosphatecarboxylase activity was higher in high-CO₂ grown cells thanin air-grown cells. The above results indicated that the lagphase induced by 40% CO₂ was due to suppression of PSII activity. ¹Part of this work was reported in the International PhotosynthesisCongress, Nagoya, 1992.     

Kinetic Studies on the Active Species of Inorganic Carbon Absorbed by the Cells of Dunaliella tertiolecta

Cells of Dunaliella tertiolecta which had been grown in ordinaryair (low-CO₂ cells) had high carbonic anhydrase (CA) activityon the cell surface and mainly utilized HCO₃^– for photosynthesis.When CA activity on the cell surface was inhibited by Diamoxor subtilisin, the cells utilized CO₂. When bovine CA was added,the subtilisin-treated low-CO₂ cells utilized mainly HCO₃^–.When grown in air containing 2% CO₂, the cells had low CA activityon the cell surface, and preferred CO₂ to HCO₃^–. Kineticanalysis of these results indicated that low-CO₂ cells of D.tertiolecta absorb CO₂ which was converted from HCO₃^–via the CA located on the cell surface. (Received June 29, 1985; Accepted October 9, 1985)     

Role of Carbonic Anhydrase in Photosynthesis of Blue-Green Alga (Cyanobacterium) Anabaena variabilis ATCC 29413

The affinity for NaHCO₃ (CO₂) in photosynthesis of Anabaenavariabilis ATCC 29413 was much higher in the cells grown underordinary air (low-CO₂ cells) than in those grown in air enrichedwith 2–4% CO₂ (high-CO₂ cells) (pH 8.0, 25?C). Ethoxyzolamide(50 µM) increased the K_m(NaHCO₃ in low-CO₂ cells aboutnine times (from 14.3 to 125), while the maximum rate of photosynthesisdecreased about 20%. When high-CO₂ cells were transferred tolow-CO₂ conditions, carbonic anhydrase (CA) activity increased,while K_m(NaHCO₃) in photosynthesis decreased from 140 to 30µM within about 5 h. The addition of CA to the suspensionof both high- and low-CO₂ cells enhanced the rates of photosyntheticO₂ evolution under CO₂-limiting conditions. The rate of ¹⁴CO₂fixation was much faster than that of H¹⁴CO₃^– fixation.The former reaction was greatly suppressed, while the latterwas enhanced by the addition of CA. These results indicate thatthe active species of inorganic carbon utilized for photosynthesiswas free CO₂ irrespective of the CO₂ concentration given duringgrowth. It is suggested that CA plays an active role in increasingthe affinity for CO₂ in photosynthesis of low-CO₂ cells of thisblue-green alga. (Received January 24, 1984; Accepted October 22, 1984)     

Photosynthetic Nitrogen Metabolism in High and Low CO2-adapted Scenedesmus: I. INORGANIC CARBON-DEPENDENT O2 EVOLUTION, NITRATE UTILIZATION AND NITROGEN RECYCLING

Larsson, M., Larsson, C.-M. and Guerrero, M. G. 1985. Photosyntheticnitrogen metabolism in high and low CO₂-adapted Scenedesmus.I. Inorganic carbon-dependent O2 evolution, nitrate utilizationand nitrogen recycling.—J. exp Bot. 36: 1373–1386 Scenedesmus obtusiusculus Chod. was grown on an inorganic mediumflushed with either air or air supplemented with 3% CO₂. Inair-grown cells, O₂ evolution dependent on low, but not high,HCO^–₃ concentrations was strongly inhibited by the carbonicanhydrase inhibitor acetazolamide. Cells grown with 3% CO² exhibitedlow rates of O2 evolution at low external inorganic C; however,after 30 min in air O2 evolution rates at low inorganic C approachedthose of air-grown cells. These results are compatible withthe view that Scenedesmus develops a ‘CO² concentratingmechanism’ in air, with carbonic anhydrase as an importantconstituent When 3% CO²-grown cells were subjected to air-level of CO²,just a transient decline in NO^–₃ utilization was observed,but in the presence of acetazolamide the rate of the processdecreased drastically in response to the decrease in the CO²level. In CO²-free air NO₃^– was taken up at high ratesbut in a deregulated manner, leading to release of NH₄⁺. A portionof the NO₃^– taken up in the absence of CO² was apparentlyassimilated Cellular nitrate reductase (NR) activity initially decreasedbut subsequently recovered after a transition from 3% CO² toair. In the presence of acetazolamide, a persistent decreasein NR activity was observed. Cellular glutamine synthetase (GS)activity increased after transition from 3% CO² to air, theactivity increase being unaffected by acetazolamide. NH₄⁺ releaseto the medium in the presence of L-methionine-D, L-sulphoximine(MSO) transiently increased in 3% CO²-grown cells in responseto a transfer to air. MSO-induced NH₄⁺ release was in fact higherin air-grown cells than in 3% CO²-grown cells. Glycollate wasinitially released after transition from 3% CO² to air, butthere was no difference in glycollate release between MSO-treatedand untreated cells. In air-adapted Scenedesmus, N recyclingseems to be of minor importance in comparison to primary N assimilation Key words: CO²-fixation, N recycling, nitrate uptake, Scenedesmus     

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)     

Purification and Characterization of Phosphoenolpyruvate Carboxylase of Photomixotrophically Cultured Green Tobacco Cells

Phosphoenolpyruvate (PEP) carboxylase (PEPCase, EC 4.1.1.31 [EC] )was purified to apparent electrophoretic homogeneity from photomixotrophicallycultured tobacco cells by ammonium sulfate fractionation, DEAE-Sephacel-,hydroxylapatite-, Phenyl-Sepharose CL-4B-, and Sepharose CL-6B-chromatography,and fast protein liquid chromatography on Mono Q. The purifiedenzyme had a specific activity of 32 units per mg protein, andits purity was determined by denaturing polyacrylamide gel electrophoresis.The native enzyme, with a molecular weight of about 440,000,was a tetramer of four identical subunits and showed maximumactivity at pH 8.5–9.0. Non-denaturing isoelectric focusingshowed a single band at pl 5.4. Substrate-saturation kineticsof the purified enzyme for PEP, bicarbonate, and Mg^2$ were typicalMichaelis-Menten type, with K_m-values of 60, 200, and 80µM,respectively. Most effectors which are known to influence theactivity of C₄- or bacterial PEPCase had only small effectson the activity of the purified enzyme at optimum pH, whilesome inhibitory effects by organic acids (malate, citrate andoxaloacetate) and.an activating effect by glucose-6-phosphatewere observed at a suboptimal pH of 7.5. (Received September 30, 1987; Accepted December 14, 1987)     

Carbonic Anhydrase in Chlamydomonas reinhardtii II. Requirements for Carbonic Anhydrase Induction

Carbonic anhydrase (CA) activity in wild type cells of Chlamydomonasreinhardtii was low when cells were cultured under 2% CO₃ inthe light. When the gas phase was changed to air, CA activityincresaed as much as 20 fold over the next 24 hours. In contrast,CA activity did not change markedly in cells of the mutantspet 20-8 (PS II-negative), lip 10-2 (photophosphorylation-negative),and F60 (phosphoribulokinase-negative), when they were subjectedto the same induction regimen. DCMU (10^–5 M) and cydoheximide(3 µg/ml) severely inhibited the induction in wild typecells. No induction occured when CO₂ concentration was loweredin darkness. ³Present adress: Photoconversion Research Branch, Solar EnergyResearch Institute, Golden, Colorado 80401, USA. (Received June 7, 1982; Accepted December 25, 1982)