Plant Physiol. (1987) 85, 72-770032-0889/87/85/0072/06/$0 1.00/0

Carbonic Anhydrase and the Uptake of Inorganic Carbon bySynechococcus sp. (UTEX 2380)1

Received for publication March 5, 1987 and in revised form May 28, 1987

CHINGKUANG Tu, HART SPILLER, GEORGE C. WYNNS, AND DAVID N. SILVERMAN*Departments ofPharmacology and Biochemistry, University ofFlorida, Gainesville, Florida 32610

ABSTRACT

We report the changes in the concentrations and "I0 contents ofextracellular CO2 and HCO3- in suspensions of Synechococcus sp.(UTEX 2380) using membrane inlet mass spectrometry. This marinecyanobacterium is known to have an active uptake mechanism for inor-ganic carbon. Measuring iS0 exchange between CO2 and water, we havefound the intracellular carbonic anhydrase activity to be equivalent to 20times the uncatalyzed CO2 hydration rate in different samples of cellsthat were grown on bubbled air (low-CO2 conditions). This activity wasonly weakly inhibited by ethoxzolamide with an Iso near 7 to 10 micro-molar in lysed cell suspensions. We have shown that even with COrstarved cells there is considerable generation of CO2 from intracellularstores, a factor that can cause errors in measurement of net CO2 uptakeunless accounted for. It was demonstrated that use of `C-labeled inor-ganic carbon outside the cell can correct for such errors in mass spectro-metric measurement. Oxygen-18 depletion experiments show that in thelight, CO2 readily passes across the cell membrane to the sites ofintracellular carbonic anhydrase. Although HC03- was readily taken upby the cells, these experiments shown that there is no significant effluxof HCO3- from Synechococcus.

We report the changes in the concentrations and '80 contentsofextracellular CO2 and HCO3 in suspensions ofSynechococcussp. (UTEX 2380). Our goals are to extend our work on Chlorella(18) using membrane-inlet mass spectrometry to cells with activeuptake mechanisms for inorganic carbon and to apply some newtechniques to this problem. This work complements that ofBadger et al. (2) who have reported 180 depletion from CO2 insuspensions of Synechococcus using membrane-inlet mass spec-trometry. We extend that work by measuring also '80 depletionfrom extracellular HC03- and 180 depletion from '3C-containinginorganic carbon to show the potential errors that can arise fromneglect ofCO2 generated from intracellular stores. We also report'80 depletion in the presence of iodoacetamide, an inhibitor ofCO2 fixation.The exchange of 180 between CO2 and water is caused by the

hydration-dehydration cycle, and was first described by Millsand Urey (10):C'O080O + H2160 = HC'80'80'60 + H+ C180'60 + H2180The H2'80 produced is so greatly diluted by H2160 that the backreaction is negligible and the 180 depletion from inorganic carbonis considered irreversible. Since carbonic anhydrase catalyzes theabove reaction, it also catalyzes the depletion of '80 from CO2

'Supported by a grant from the National Science Foundation (PCM-8318753).

(13). In suspensions of cells that contain carbonic anhydrase,depletion of 180 from species of extracellular inorganic carbonreflects the rate of access of the inorganic carbon across the cellmembrane to the sites of carbonic anhydrase and the activity ofthis enzyme in the cells. This method was first used quantitativelyby Gerster (4), and subsequently several studies have used themethod on red cells and algae (2, 14, 18).

It is known that cyanobacteria similar to Synechococcus havean active uptake mechanism for inorganic carbon (7, 9) whichfunctions in the transport of either CO2 or HCO3 (1), althoughat the pH of seawater, about 8.2, the uptake of HCO3 predom-inates. The mechanism of this active transport is uncertain, butit is suggested to function like an electrogenic pump (8). Lowlevels of carbonic anhydrase have been detected in certain ofthese cyanobacteria (2, 6, 17) although the function of thisenzyme in the accumulation of inorganic carbon has not beendetermined. In this work, we have found carbonic anhydraseactivity inhibitable by ethoxzolamide in lysed preparations ofSynechococcus. Ethoxzolamide is a very potent inhibitor ofmanyisozymes of plant and animal carbonic anhydrase but in this caseis weak with an Iso of 7 to 10 uM. Our results using 13C labelingshow considerable generation of CO2 from intracellular sourceseven in Synechococcus cells which had been starved for inorganiccarbon. This is a factor that must be considered in experimentsthat measure changes in external inorganic carbon. Our oxygen-18 exchange experiments show that in the light CO2 readilypasses across the cell membrane to the sites of intracellularcarbonic anhydrase. We also show that although HCO3 is readilytaken up in the light, there is no measurable efflux of HCO3from Synechococcus.

EXPERIMENTAL PROCEDURES

Algae. Synechococcus sp. (UTEX 2380), a marine, unicellular,nonnitrogen fixing cyanobacterium, was obtained from the Uni-versity of Texas Culture Collection (Department of Botany,Austin, TX). These cells were grown in ASN-III medium bufferedwith 25 mm Bicine at pH 8.2, a medium made of inorganic saltsas described by Rippka et al. (12). Cultures were grown in 1 Lflasks under constant illumination by a series ofwhite fluorescentlights providing a light flux of 160 liE m 2 s-' at 24°C, and werebubbled either with air (low-CO2 conditions) or with C02-en-riched air (2-3% C02, v/v, high-CO2 conditions) at a flow rateof 100 ml/min. Cells were grown to a density of 10 to 25 ,l perml pcv2 and in most cases the ratio of pcv to Chl (ml/mg) wasclose to unity.To prepare spheroplast suspensions of Synechococcus sp., we

washed cells once in 0.5 M mannitol medium buffered with 15mM Tricine at pH 7.5, containing 1 mM EDTA, and thenconcentrated cells to 250 ,l/ml pcv. This suspension was incu-bated in the presence of lysozyme at 2 mg/ml, and at 30°C for

2Abbreviation: pcv, packed cell volume.

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CARBONIC ANHYDRASE IN SYNECHOCOCCUS SP.

90 min. The mannitol incubation with lysozyme generates fragilespheroplasts that are enzymically active for several days, if kepton ice ( 16). In cases for which lysate was needed, the spheroplastswere passed twice through a French press set at 17,000 psi andimmediately placed back on ice.

'80-Exchange Method. This method is based on the exchangeof 180 between CO2 and H20 caused by the hydration-dehydra-tion cycle which is catalyzed by carbonic anhydrase. Details ofthe method are described by Silverman (15) and the use of themethod in a study of Chlorella vulgaris is presented by Tu et al.(18). The experimental observation is the depletion of 18O fromCO2 and HCO3 .

The rate of 180 depletion was measured by membrane-inletmass spectrometry. The membrane inlet vessel was based on adesign by Hoch and Kok (5). It is a vessel the bottom of whichholds a membrane permeable to C02, 02, N2 and which issupported by a porous, stainless steel disk. Gases passing acrossthe membrane have H20 removed in a dry ice-acetone trap andthen enter a mass spectrometer (Dycor MIOOM interfaced withan IBM XT computer). The time lag between the inlet vesseland the mass spectrometer was less than 2 s.The measured variable was the 180 atom fraction in 13C02:

180 atom fraction in 13C02(13C'80160) + 2(13CI80180)

2('3C02)

(47) + 2(49)2(45 + 47 + 49)

where 45, 47, and 49 are the heights of the corresponding masspeaks. We have also measured the 180 atom fraction in H 33COf:

0 atom fraction in H3C03-

(H'3C160160180) + 2(H'3CI60180180) + 3(H'3C180'80'80)3(H'3CO3O

This was determined by taking a sample from the solution,acidifying it rapidly, and measuring the 180 atom fraction in theresulting '3C02. Knowing the pH of the solution and the 180

atom fraction in 13C02 at the time the sample was taken fromthe membrane inlet vessel, we were able to determine the 180content of H'3C03-, as described by Mills and Urey (10). Usingsuspensions of Synechococcus sp., we measured these '80 atomfractions in the solution surrounding the cells. With the appro-priate calibrations made from solutions of known CO2 and 02

content, we were also able to measure the concentration of CO2and 02 outside the cells.The uptake of bicarbonate at pH 8.2 was monitored using

H'4C03- (total bicarbonate at 0.35 mM) with cell material de-pleted ofinternal pools ofinorganic carbon by illumination priorto the start of the experiment. '4C was added at 50 MCi/mmol at0°C. Aliquots taken at timed intervals were passed immediatelythrough a 0.2 ,m filter in 2 s, and the radioactivity determinedin the filtrate by liquid scintillation counting, using the appro-priate controls.

RESULTSWe have observed the uptake of CO2 into Synechococcus sp.,

(UTEX 2380) in the absence and presence of light using cellsthat had been illuminated prior to experiments until all net 02

evolution had ceased (Fig. 1). It is known that this cyanobacter-ium takes up both CO2 and HCO3- in the light (1), althoughFigure 1 shows only the change in extracellular CO2 and 02. Wediscuss the change in extracellular HCO3 in connection withFigures 2 and 5. Figure 1 also shows the evolution of 02 whichproceeded without a plateau when cells were illuminated. In the

0.

2

.09

MINUTES

FIG. 1. Changes in extracellular concentrations of total CO2 ('2CO2and 13CC2) (-), '2CO2 (- -), and 13C02 (- ----) (O2... is righthand coordinate) in a suspension of Synechococcus sp. (UTEX 2380)measured using membrane inlet mass spectrometry. Cells were grownwith bubbled air (low CO2 conditions) and prior to the experiment wereilluminated (about 12 min) until 02 evolution ceased. Cells were thensuspended (packed cell volume 1% of total volume) in a solution in themembrane inlet vessel; the solution was a sea water medium containing25 mm barbital and 4 mm phosphate buffer at pH 7.4. Measurementswere made at 25'C. Arrows indicate the times of turning on and off redlight of intensity 400 ME m-2 s-'.

Synechococcus sp. (UTEX 2380)

2 Add cells 400fi1EEm2s-1 PH 7.9

81Add cells 0oi\7.~.WFWOZ20 ~~f- Ecm.2 0

0 1 o

0 5 0 15 20MINUTES

FIG. 2. (Top) Changes in extracellular concentration of '3C02and total inorganic carbon as measured by 14C (0) and (bottom) in the'8 atom fraction of extracellular 3CO2 (-) and H'3CO3- (0) in asuspension of Synechococcus sp. (UTEX 2380). Cells were grown withbubbled air (low CO2 conditions) and then suspended (packed cellvolume 3.3%) in a solution in the membrane inlet vessel; the solutionwas sea water with minor elements containing also 15 mM Tricine and20 mm phosphate buffers at pH 7.9 (except the experiments measuringtotal inorganic carbon in the top figure which were performed at pH7.5). The total concentration of all species ofCO2 was 2.0 mm with 70%13C content. Measurements were made at 25°C. The arrows indicate theaddition of cells to the inlet vessel and the duration of red light ofintensity 400 ME m2 s-'.absence of light, the cells did not take up CO2 to an extent thatcould be measured by our membrane inlet mass spectrometer.This was true both before and after illumination (Figs. 1 and 2).To determine the rate of uptake of CO2 and to distinguish thisprocess from CO2 generated within the cells, we placed in solu-tion CO2 and HCO3 that were highly enriched in carbon-13 (upto 99% 13C). The natural abundance of 13C iS 1.1% and the cellshad not been exposed to enriched 13C before the experiment.Then we know that the decrease in concentration of 13C02outside the cells is not significantly altered by efflux of 12C02from the intracellular pools. That this procedure is useful isdemonstrated in Figure 1 which shows the increase in 12C02upon illumination. This increase must be due to release of '2CO2from internal pools of organic intermediates in the cells, in alight induced process. This 13C labeling is a necessary procedurewhen kinetic information is to be obtained from measurementsof changes in CO2 concentration.

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Plant Physiol. Vol. 85, 1987

The change in extracellular 3CO2 concentration upon turningon the light was observed to be triphasic (Fig. 1). Upon turningoff the light, there was an increase in extracellular 13C02 concen-

tration to a value less than the initial concentration reflecting netcarbon uptake and fixation.The uptake of '3C02 under slightly different conditions and

without preillumination is shown in Figure 2. In a separateexperiment under similar conditions, we also measured the con-

centration of HC03 outside the cells not by mass spectrometrybut by 14C uptake, the data for which are also shown in Figure2. These data demonstrate the uptake upon illumination of bothC02 and HCO3 . The bottom part of Figure 2 shows the changein the 180 atom fraction of 13CO2 and H'3C03- during theexperiment. Before illumination, there was no appreciable dif-ference between the 180 contents of '3C02 and H'3C03-, as

predicted for these two species in chemical equilibrium (10).Moreover, the rate of depletion of '80 from '3C02 before andafter illumination was (5.7 ± 0.8) x 10-4 s-' which is indistin-guishable from the uncatalyzed 180 exchange rate of 5.6 x 10-4s-' at the pH of these experiments which was 7.9. Upon illumi-nation, there was a lag phase of about 30 s followed by the lossof 180 from 13C02 outside the cells. However, there was no

corresponding decrease in the 180 content of H'3C03-. Uponturning the light off, the 180 content of13C02 returned to a valueclose to that of H'3C03- (Fig. 2). The region in the light from 10to 15 min in Figure 2 in which the 180 content of'3C02 remainedunchanged was coincidental (i.e. caused by a coincidental can-

celing of the depletion of 180 contentof C02 by the cells and thegeneration of labeled C02 from the dehydration of labeled bicar-bonate outside the cells). It was not a general feature of our

results; that is, a variety of different conditions produces a netloss in the 180 content in this region. In an experiment similarto that of Figure 2 but using cells that had been preilluminatedto deplete pools of inorganic carbon, this region showed net lossof 180 from13C02 with a rate about three times that of theuncatalyzed 180 exchange.To demonstrate further the chemical equilibrium between C02

and HC03 outside the cells and the 180 disequilibrium in thesetwo species, we include the results in Figure 3. This experimentis similar to that of Figure 2 except that bovine red cell carbonicanhydrase (1.4,ug/ml) was added to the suspension at the timeindicated by the arrow. The presence of this enzyme in theextracellular solution causes the rapid attainment of isotopicequilibrium between C02 and'80-labeled HC03 outside thecell by catalyzing the conversion of HCOO180- into CO'80. Theincrease in180 contentof C02 upon adding this enzyme isanother manifestation of the higher 180 content of HC03 rela-tive to CO2 at the time of addition of enzyme. The 180 contentof C02 outside the cell is low because C02 has rapid access tointracellular carbonic anhydrase.

In a tightly coupled light-dependent system, intracellularHC03- would be converted to C02 and get fixed directly intoorganic carbon before C02 can pass out of the cell. If this occurs,the mass spectral data on'80 contentof C02 outside the cellwould not be able to detect the activity of carbonic anhydrase.To overcome this handicap, we repeated experiments usingsolutions containing 4 mm iodoacetamide. This alkylating agentis known to inhibit C02 fixation (11) and probably affects otherprocesses in the cell as well. However, we know from our

experiments (data not shown) that 4 mm iodoacetamide does notinhibit carbonic anhydrase activity in suspensions of lysed Sy-nechococcus. In a parallel experiment, we determined that 4 mmiodoacetamide did not affectlinear electron transport to methylviologen. Furthermore, photosynthetic 02 evolution, completelyblocked by 4 mM iodoacetamide, was restored to 80% of itsmaximum value when ferricyanide was added to the assay. Insolutions containing 4 mmv iodoacetamide, the fixation Of 13C02

o-

-

(' 0.5o 1X~10~~~0

0

O 0.01 BA)

12

-.5

0.05

U.

0SCA

o 0.01 (1.4 gIAomI)0.0

5 10 15 20

MINUTES

FIG. 3. (Top) Changes in extracellular concentrationof 3C02 and'2C02 and (bottom) in the'80 atom fraction in extracellular3C02 in asuspension of Synechococcus sp. Cells were grown with bubbled air (low-CO2 conditions) and just prior to the experiment they were illuminateduntil 02 evolution ceased (about 12 min), and then placed in themembrane inlet vessel for mass spectral measurements. The packed cellvolume was 15,ul per ml of medium containing 10 mm of Tricine bufferat pH 8.0; the initial concentration of all speciesof'3CO2 was 1.0 mm(99%'3C-containing inorganic carbon was used). Arrows indicate thetimes of turning on and off redlight of intensity 400 JEm-2s-'. In thislighting period, a small volume of solution containing bovine red cellcarbonic anhydrase was added (at the time indicated by the arrow)resulting in 1.4 ggm/ml of this enzyme.

was blocked and after about a minute of illumination '3C02generated from intracellular bicarbonate passed out of the cellinto the medium as evidenced by the appearance of13C02 inconcentrations above the equilibrium value (Fig. 4). This isevident upon adding carbonic anhydrase to the external fluidwhich causes a sudden decrease in extracellular ['3CO2]. Thechange in180 contentof C02 is qualitatively similar to that ofFigure 3, showing that the C02 passing out of the cell has an 180content below that of extracellular HCO3.

Evidence from previous work suggests a large pool of inorganiccarbon to be present in Synechococcus and other cyanobacteria.Carbon-l 3 added externally might be exchanged in the dark anddiluted with unlabeled carbon. To check this possibility, wefollowed the uptake and efflux kinetics of low concentrations ofH'4C03-(0.35 mM) using cells with a depleted inorganic carbonpool, suspended in medium of pH 8.2 where 99% of the carbonis present as bicarbonate (Fig. 5). Initially the sample was alsokept on ice in the dark, conditions in which cell membranes arein a deenergized state and incapable of energy-dependent ionuptake. Initially H'4C03- was added and the first sample taken.Upon warming to room temperature a small, but repeatableamount of bicarbonate was taken up, corresponding to a darkpool increase to 2 mm inorganic carbon, and 7 mm in the lightunder our conditions. During the following 8 min in the dark,there was no measurable uptake of bicarbonate from the me-dium. Upon switching the light on, the uptakeof '4C' wasobserved. After the light was switched off, an initial small increase

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CARBONIC ANHYDRASE IN SYNECHOCOCCUS SP.

2.0

60r

0r .5

40

20

01a' 0.650

z

z 0.1

I-o 0.01

IL

0-

0.0101- 0.01

5

105

C.)a)(I)

on0xCbUZ

1.0

1

0

n n . .

5 10 15 20MINUTES

FIG. 4. Identical to Figure 3 with the exception that 4.0 mM iodoa-cetamide was present in the medium.

400 FE m-28-1

3000-0

txI~ ~ ~ ~

0 10 20 30

MINUTE8

FIG. 5. Uptake and retention of '4C-bicarbonate in whole cells ofSynechococcus depleted of inorganic carbon. Algae were suspended inASN III medium, buffered with Tricine 10 mm, phosphate 5 mM at pH8.2 at a cell density of 18 sl/ml pcv. '4C-Labeled bicarbonate was addedat 0.35 mm after preillumination of algae kept in ice in the dark. Aliquotswere withdrawn and immediately filtered to monitor the disappearanceof labeled carbon in the filtrate. The first sample (t = 0) was withdrawnin the cold, all others were taken at room temperature.

of 14C label was observed in the external solution, possibly thediffusion of CO2 back into the medium. This is clarified by thedata in Figure 1 which show the increase in external CO2 afterthe light is turned off. The new level of 14C is the result ofHC03-uptake as well as CO2 fixation in the light period. These kineticssuggest that bicarbonate is usually not taken up in the dark; itsretention in internal pools is the subject of further study in thislaboratory.The 180 exchange experiments showed clear evidence of a

small carbonic anhydrase activity in suspensions of lysed, low-CO2 grown Synechococcus, an activity that could be inhibited bythe sulfonamide ethoxzolamide with an 150 in the range 7 to 10,M (Fig. 6). This activity is equivalent, upon multiplying by thedilution factor, to a carbonic anhydrase activity in the cell whichis 20 times the uncatalyzed activity ofthe hydration ofCO2. The

00'1

I 00° 1o 102 103

Ethoxzolamide [gM]FIG. 6. Inhibition by ethoxzolamide of '8 exchange between CO2

and H20 in a suspension of Synechococcus sp. (UTEX 2380) in whichcells were lysed by treatment with lysozyme and using a French press.The amount of lysed cells used was equivalent to a packed cell volumeof 10% had the cells been intact. O. is the first-order rate constant forthe depletion of '80 from CO2 catalyzed by carbonic anhydrase. Thetotal concentration ofall species ofCO2 was 1.0mm at pH 7.4 maintainedby 10 mM barbital and 6 mM phosphate buffers. Temperature was 25C.The Synechococcus sp. cells were grown using bubbled air (low CO2conditions).

carbonic anhydrase activities of air-grown cells were twice aslarge as that for high C02-grown cells, using the '80-exchangemethod. Similar results were obtained using spheroplasts ofSynechococcus in a CO2 hydration assay in which we measuredchanges in pH.

DISCUSSIONWe report here several techniques in measuring and interpret-

ing 180 exchanges in suspensions of Synechococcus sp. (UTEX2380) that allow information to be obtained from experimentsusing a membrane inlet to a mass spectrometer. The membraneinlet measured isotopic content of CO2 outside the cells and theexchanges are between '80-labeled CO2 and water. This work on180 exchange from CO2 in suspensions of Synechococcus sp. ismeant to complement the published work of Badger et al. (2) onthe same topic. The catalyzed mechanism of '80 depletion, inthese cases of Synechococcus with no external carbonic anhy-drase, is that inorganic carbon passes into the cell and is depletedof 180 by catalyzed hydration-dehydration cycles and passes outof the cells as CO2. It is useful to distinguish between thismechanism of isotope dilution and the generation of unlabeledCO2 from the internal pools of the cells, which is natural abun-dance 99% '2C02. One technique to do this is to add '3C enrichedinorganic carbon to the solution outside the cells and measurethe change in the concentration and 180 content of extracellular'3CO2. Examination of Figure 1 shows this effect, demonstratingthe generation of '2CO2 in the light from intracellular sources.The inorganic carbon in the external solution was 85% 13C atthe beginning of the experiment. The three phases in the changein the concentration of CO2 can be attributed to: first, the initialflux of inorganic carbon into the starved cells upon turning onthe light; second, a short period of net CO2 efflux caused by thedehydration in the cells of accumulated HCO3-; and third, adecline in extracellular CO2 concentration resulting from the netfixation of CO2. It is in the third phase that the difference in -rateof decrease of extracellular ([12C02] + [13C02]) and [13C02] ismost apparent. Clearly, the latter, [13C02J, is a more accuratemeasure of one-way uptake by Synechococcus of extracellular

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Plant Physiol. Vol. 85, 1987

C02, especially initially when the cell has not accumulatedappreciable '3C-containing species. The rates of these twochanges in concentration differ by nearly a factor ofthree, whichapproximates the error in rate of CO2 uptake that would accrueif extracellular [CO2] were measured without accounting for theCO2 generated intracellularly.Another technique that adds to interpretation is to measure

the '80 content of H'3C03 outside the cells. This has theadvantage of showing the difference in 18Q content of extracel-lular CO2 and HCO3 , a difference that arises because ofdifferentrates ofpermeability ofCO2 and HCO3 into and out ofthe cells.Inside the cells, the catalyzed hydration-dehydration cycle occursand 18' loss to H20 is rapid. In Figure 2, the more rapid loss of180 from CO2 than from HCO3 means that the process 'influx-180 depletion-efflux' is faster for CO2 than for HCO3-.Figure 2 shows 180 depletion from extracellular 13C02 and

H'3CO3 in suspensions of Synechococcus sp. We comment firston the region of data taken in the dark. In this region of theexchange, we observed a rate of depletion of '80 from '3C02which was indistinguishable from the uncatalyzed rate eventhrough Synechococcus cells were present in suspension. Onefirm conclusion from this result is that there is no detectable andactive carbonic anhydrase on the outer surface of this Synecho-coccus. This is of further interest because we know from otherexperiments that carbonic anhydrase is present in the cells at ameasurable activity and that CO2 is rapidly accessible to thisintracellular enzyme. Our interpretation is based on the knowl-edge that the Synechococcus cells we used had been grown inlow-CO2 conditions and starved for inorganic carbon. Upon thestart of our experiment, these cells were placed in a suspensionof 2 mm total inorganic carbon. One possible reason that noenhanced 180 depletion is observed in the dark is that CO2 takenup by the cell is converted to HCO3 and retained; perhaps aspart of the internal inorganic carbon pool, and does not effluxfrom the cell. This uptake would have to be slower than theuncatalyzed generation of CO2 from HCO3 outside the cellssince we do not see any change in total CO2 concentration in thedark outside the cells (Figs. 1 and 2).A region of large changes in 180 content and concentration of

CO2 followed the irradiation of the inlet vessel with red light at400 yE m 2 s-' (Figs. 1 and 2). These were also reported byBadger et aL (2) and our results do not differ qualitatively withtheirs. We observed the influx of both CO2 and HCO3 into thecells upon turning on the light, an uptake which was rapid onthe rather slow time scale of these experiments (Fig. 2, top).About 30 s after illumination, there was a rapid depletion of 180from '3C02 outside the cells with a rate constant of 8.4 x 10-3sec-', but the '80 content of H'3C03- outside the cells changedvery slowly (Fig. 2, bottom). We believe, and support with laterexperiments using iodoacetamide (Fig. 4), that this depletion isdue to the efflux from the cells of '80-depleted 13C02. Thequestion then is whether this depletion of '8 is due to the pHin the cells or to the carbonic anhydrase. In a solution containingjust CO2 and HCO3 with no enzyme, the rate of uncatalyzed180 depletion from CO2 increases as the pH is lowered from pH7.9. To be consistent with the suggestion that 180 depletion inthis region is not caused by carbonic anhydrase but only by lowpH in the cells would require an intracellular pH less than 6 inSynechococcus; this is contrary to other work on cyanobacteriawhich under conditions similar to ours found values of cyto-plasmic pH to be near 7.5 to 7.8 (3, 7, 9). Moreover, ourobservations are not consistent with the suggestion that all '8depletion is due to pH as low as 5 in the intrathylakoid space,which accounts for about 7% ofthe cellular volume. We concludethat our observations require that carbonic anhydrase be activein the cells. This is supported by our measurement of carbonicanhydrase activity, inhibitable by ethoxzolamide, in suspensions

of lysed Synechococcus (Fig. 6). The rate constant we observedfor 180 depletion from 13C02 in suspensions of whole cells uponturning on the light is consistent with our results using lysed cellswhich indicated a catalyzed CO2 hydration activity in the cellsof 20 times the uncatalyzed hydration rate. This is substantiallylower than the 290- and 350-fold intracellular increase in CO2hydration rates for low-CO2 grown cells reported by Badger etal. (2) and is possibly due to differences in the strains of Syne-chococcus used or in growth conditions.Badger et al. (2) were not able to observe inhibition of photo-

synthetic 02 evolution by the sulfonamide inhibitor of carbonicanhydrase ethoxzolamide (up to 100 gM) using whole cell sus-pensions. This may be due to slow entry of the inhibitor into thecells or due to the carbonic anhydrase catalysis not being a rate-contributing step in the 180 depletion and hence requiring nearlycomplete inhibition to observe a change in 180 depletion rates.In our lysed cell experiments, we observed a high I5o for inhibitionof Synechococcus carbonic anhydrase by ethoxzolamide (7-10,gM), suggesting that very high concentrations of this inhibitorwould be required for near complete inhibition of carbonicanhydrase inside the cell.The pattern of 180 depletion we have observed upon turning

on the light is quite similar to that for Chlorella vulgaris (18).The 180 content of CO2 falls almost immediately below that ofHCO3-, and the 180 content of HCO3 is altered only slightly(Fig. 2, bottom). This means that the 180 labeled CO2 outsidethe cell is being diluted significantly by unlabeled CO2 passingout of the cell. On the other hand, this is not so for HCO3 . Weknow that HCO3 is taken by up cyanobacteria (1, 7, 9) but ourresults show that there is hardly any efflux of unlabeled HCO3out ofthe cell (none in the dark) to dilute that in the surroundingsolution. Moreover, we have consistently observed that '80 de-pletion of CO2 outside the cells begins about 30 s after the netinflux of CO2 begins (Fig. 2). This must mean that the cells,formerly starved for inorganic carbon, spend this time accumu-lating CO2 and HCO3 before there is considerable efflux ofCO2from the cells. This is demonstrated most clearly in the experi-ments in which we added iodoacetamide (Fig. 4). This alkylatingagent can have many effects on cell function, one prominenteffect being the inhibition of CO2 fixation. We do know that theconcentration of iodoacetamide we used (4 mM) does not inhibitthe carbonic anhydrase in Synechococcus. Moreover, Ogawa etal. ( 11) have shown that iodoacetamide does not block the activeuptake of dissolved inorganic carbon. Figure 4, measured in thepresence ofiodoacetamide, shows the large efflux ofCO2 oflower180 content from the cell as manifested by the large increase inCO2 outside the cells and by the large rate of 'IO depletion ofCO. So efficient is this process of active bicarbonate uptake,carbonic anhydrase catalysis, and CO2 efflux, that the concentra-tion of CO2 outside the cell rose above the equilibrium level inthis well buffered solution (Fig. 4, top). Upon turning out thelight, or adding carbonic anhydrase outside the cells, the CO2concentration returned to its equilibrium level. Comparison ofFigures 3 and 4 shows the influence of CO2 fixation on theseexperiments. In the absence of iodoacetamide, much less CO2effluxes from the cells since CO2 is fixed. This emphasizes a long-postulated role for carbonic anhydrase to provide a rapid con-version of stored HCO3- into CO2. The efflux of 12C02 in theabsence ofiodoacetamide in the light (Fig. 3) indicates the releaseof CO2 by some photorespiratory process that is inhibited byiodoacetamide, as shown in Figure 4.

After turning the light off, we observed a return to equilibriumconditions (Fig. 2); that is, the CO2 concentration outside thecells returned to its equilibrium value and the 180 content ofCO2 returned to a value close to that ofHC03- outside the cells.Two processes are possible to account for this reequilibration:the uncatalyzed hydration-dehydration cycle in the solution out-

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CARBONIC ANHYDRASE IN SYNECHOCOCCUS SP.

side the cells and the efflux of CO2 from the cells. The half-timefor the return of CO2 to its equilibrium concentration outsidethe cell is, in the fastest region, 26 s. The half-time due touncatalyzed processes alone to reach equilibrium CO2 concen-tration is near 25 s at pH 7.9. Since this is close to that observedin Figures 1 and 2, we conclude that the reequilibration uponturning out the lights is due mostly to uncatalyzed processesoutside the cell. Consequently, measurements ofCO2 efflux fromthe cell based on the data after turning off the lights would bedifficult.

It is useful at this point to compare the function of carbonicanhydrase in Chlorella vulgaris and Synechococcus. Chlorellahas no active uptake ofHCO3 but does have moderate intracel-lular amounts of carbonic anhydrase possibly to trap as HC03-the CO2 that passes into the cell; this alga has an intracellularcarbonic anhydrase activity 160 times the uncatalyzed CO2 hy-dration rate (18). On the other hand, Synechococcus has an activebicarbonate uptake and needs a smaller activity of carbonicanhydrase mainly for rapid access to the pool of stored HCO3 .Synechococcus sp. (UTEX 2380) has an intracellular activity ofcarbonic anhydrase equivalent to 20 times the uncatalyzed rateof hydration of C02, under the conditions we report here. Thislow intracellular activity is consistent with a number ofmeasure-ments on cyanobacteria. Low levels of carbonic anhydrase weremeasured in low-CO2 grown Coccochloris peniocystis (6) andcarbonic anhydrase has been purified from Anabaena variabilisalthough not detected in whole cells (17). It is pertinent to pointout that one of the properties of carbonic anhydrase purifiedfrom Anabaena (17) is quite different from that we found inSynechococcus: the I50 for inhibition by ethoxzolamide of car-bonic anhydrase from Anabaena is 3 x 10-' M while that forSynechococcus is greater, 1 x 10-5 M in lysates.

LITERATURE CITED

1. BADGER MR, TJ ANDREWS1982 Photosynthesis and inorganic carbon usageby the marine cyanobacterium Synechococcus sp. Plant Physiol 70: 517-523

2. BADGER MR,M BASSm, HN COMINS 1985 A model for HCO3 accumulationand photosynthesis in the cyanobacterium Synechococcus sp. Plant Physiol

77: 465-4713. FALKNER G, F HORNER, K WERDAN, HW HELDT 1976 pH changes in the

cytoplasm of the blue-green alga Anacystis nidulans caused by light-depend-ent proton flux into the thylakoid space. Plant Physiol 58: 717-718

4. GERSTER R 1971 Essai d'interpretation des cinetiques d'echange isotopiqueentre C'802 et eau d'une feuille: experiences a l'obscurite. Planta 97: 155-172

5. HOCH GB, B KOK 1963 A mass spectrometer inlet system for sampling gasesdissolved in liquid phases. Arch Biochem Biophys 101: 160-170

6. INGLE K, B COLMAN 1976 The relationship between carbonic anhydraseactivity and glycolate excretion in the blue-green alga Coccochloris peniocys-tis. Planta 128: 217-223

7. KAPLAN A, MR BADGER, JA BERRY 1980 Photosynthesis and the intracellular,inorganic carbon pool in the blue-green alga Anabaena variabilis responseto external CO2 concentration. Planta 149: 219-226

8. KAPLAN A, D ZENWIRTH, L REINHOLD, JA BERRY 1982 Involvement of aprimary electrogenic pump in the mechanism for HCO3- uptake by thecyanobacterium Anabaena variabilis. Plant Physiol 69: 978-982

9. MILLER AG, B COLMAN 1980 Active transport and accumulation ofbicarbon-ate by a unicellular cyanobacterium. J Bacteriol 143: 1253-1259

10. MILLS GA, HC UREY 1940 The kinetics of isotopic exchange between carbondioxide, bicarbonate ion, carbonate ion, and water. Am Chem Soc 62: 1019-1026

11. OGAWA T, T OMATA, A MIYANO, Y INOUE 1985 Photosynthetic reactionsinvolved in the C02-concentrating mechanism in the cyanobacterium Ana-cystis nidulans. In WJ Lucas, JA Berry, eds, Inorganic Carbon Uptake byAquatic Photosynthetic Organisms. The American Society of Plant Physiol-ogists, Rockville, MD, pp 287-304

12. RIPPKA R, J DERUELLES, JB WATERBURY, M HERDMAN, RY STANIER 1979Generic assignments, strain histories, and properties of pure cultures ofcyanobacteria. J Gen Microbiol 11 1: 1-61

13. SILVERMAN DN, CK Tu, S LINDSKOG, GC WYNNS 1979 Rate of exchange ofwater from the active site of human carbonic anhydrase. J Am Chem Soc101: 6734-6748

14. SILVERMAN DN, CK Tu, N ROESSLER 1981 Diffusion-limited exchange of 'IObetween CO2 and water in red cell suspensions. Resp Physiol 44: 285-298

15. SILVERMAN DN 1982 Carbonic anhydrase: oxygen-18 exchange catalyzed byan enzyme with rate-contributing proton-transfer steps. Methods Enzymol87: 732-752

16. SPILLER H 1980 Photophosphorylation capacity of stable spheroplast prepara-tions ofAnabaena. Plant Physiol 66: 445-450

17. YAGAWA Y, Y SHIRAIWA, S MIYACHI 1984 Carbonic anhydrase from the blue-green alga (Cyanobacterium) Anabaena variabilis. Plant Cell Physiol 25:775-783

18. Tu CK, M ACEVEDO-DUNCAN, GC WYNNs, DN SILVERMAN 1986 Oxygen-18exchange as a measure of accessibility of CO2 and HCO3- to carbonicanhydrase in Chlorella vulgaris. Plant Physiol 80: 997-1001

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