Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

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)     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO₂, but its synthesis was induced under low concentrations of CO₂ (air levels of CO₂). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO₂ to growth on air levels of CO₂, (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Mechanism of Inorganic Carbon Uptake in Chlorella saccharophila: The Lack of Involvement of Carbonic Anhydrase

The acid-tolerant green alga Chlorella saccharophila maintainedphotosynthesis and accumulated intracellular pools of inorganiccarbon over a a range of external pH from 4.0 to 7.5. This accumulationwas unaffected by treatment of cells with 10 mol m^–3 acetazolamide(AZA). Cells grown at alkaline pH had extracellular carbonicanhydrase (CA), but CA activity was repressed when cells weregrown at pH 5.0. Acid-grown cells retained a high affinity forCO₂, both at acid and alkaline pH, and the ability to accumulateinorganic carbon. Rates of photosynthesis of acid-grown cellsand alkaline-grown AZA-treated cells at pH 8.0 were 2.5-foldhigher than the rate of CO₂ supply from the uncatalysed dehydrationof , indicating that the cells can take up as a source of substrate for photosynthesis. Isotopic disequilibrium experiments with acid-grown cells maintainingsteady-state photosynthesis at pH 7.5 demonstrate that ¹⁴C from¹⁴CO₂ was taken up more rapidly than from H¹⁴. This uptake takes place against a concentration gradient. Theseresults demonstrate that C. saccharophila cells have activetransport systems for the uptake of both CO₂ and and both operate without the mediation of CA. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, Chlorella saccharophila, inorganic carbon accumulation     

Role of Carbonic Anhydrase and Identification of the Active Species of Inorganic Carbon Utilized for Photosynthesis in Chora corallina

Carbonic anhydrase (CA, EC. 4.2.1.1 [EC] ) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O₂ evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent K_m for external inorganic carbon (C_i) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent K_m(CO₂)was about three times larger at pH 6.0 than at pH 7.6. The K_m(C_i)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of ¹⁴CO₂ at 20 µMand of H¹⁴CO₃^– at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic ¹⁴CO₂-fixation but it stimulated the H¹⁴CO₃^–-fixation.This result indicates that free CO₂ is an active species ofC_i that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO₂ isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO₂ in photosynthesisby facilitating the supply of CO₂ from the plasmalemma to thesite of CO₂-fixation. ³Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989)     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

Correlation between Carbonic Anhydrase Activity and Inorganic Carbon Internal Pool in Strain Synechocystis PCC 6174

Existence of an internal carbonic anhydrase was demonstrated in the cyanobacterium Synechocystis PCC 6714. The enzyme, present at a low specific activity, was inducible by limitation in inorganic carbon and inhibited both in vivo and in vitro by acetazolamide. The internal inorganic carbon pool as determined by mass spectrometry, was similarly modulated by the actual inorganic carbon growth regime; its building up was also sensitive to acetazolamide. A possible role of carbonic anhydrase in inorganic carbon metabolism regulation through the control of the dimension and nature of the inorganic carbon pool is discussed.     

Inorganic Carbon Uptake by Chlamydomonas reinhardtii

The rates of CO₂-dependent O₂ evolution by Chlamydomonas reinhardtii, grown with either air levels of CO₂ or air with 5% CO₂, were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO₂ required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO₂. This is consistent with the hypothesis that these cells take up CO₂ but not HCO₃⁻ from the medium and that their CO₂ requirement for photosynthesis reflects the K_m(CO₂) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO₂ concentration of only 0.4 to 3 micromolar for half-maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO₃⁻ concentration is very low (40 nanomolar). However, at high external pH, where HCO₃⁻ predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO₃ to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO₂ is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO₂. If active HCO₃⁻ accumulation is a step in CO₂ concentration by air-grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma.     

The Role of Carbonic Anhydrase 9 in Regulating Extracellular and Intracellular pH in Three-dimensional Tumor Cell Growths

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of ∼300 μm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH_i) and pH in the restricted extracellular spaces (pH_e). With no CA9 expression, spheroids developed very low pH_i (∼6.3) and reduced pH_e (∼6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH_i = ∼6.6), whereas extracellular acidity was enhanced (pH_e = ∼6.6), so that radial pH_i gradients were smaller and radial pH_e gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH_e indicates the enzyme is facilitating CO₂ excretion from cells (by converting vented CO₂ to extracellular H⁺), rather than facilitating membrane H⁺ transport (such as H⁺ associated with metabolically generated lactic acid). This latter process requires titration of exported H⁺ ions with extracellular HCO₃⁻, which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH_e will depend on the cellular CO₂/lactic acid emission ratio (set by local oxygenation and membrane HCO₃⁻ uptake). Our results suggest that CO₂-producing tumors may express CA9 to facilitate CO₂ excretion, thus raising pH_i and reducing pH_e, which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.The carbonic anhydrases (CAs)³ are a family of enzymes that reversibly catalyze CO₂ hydration to H⁺ and HCO₃⁻ (1, 2). Recent studies have identified several CA isoforms, such as CA4, CA9, CA12, and CA14, with extracellular-facing catalytic sites (2). Many cells express extracellular CA (CA_e) isoforms, but their physiological role remains unclear. In particular, the strong link between cancer and CA9 expression (1–5) has provoked great interest in the role of CA_e in tumor biology.Based on their topology, CA_e isoforms are likely to regulate the concentration of extracellular H⁺, CO₂, and HCO₃⁻. Cell metabolism drives transmembrane fluxes of H⁺ ions, CO₂ and HCO₃⁻, and can provide substrate for the CA_e-assisted reaction. For example, CO₂ is released from aerobically respiring cells. By consuming or producing H⁺ ions, the CA_e-catalyzed reaction will affect extracellular pH (pH_e). Many membrane proteins are modulated by pH_e (6–8). Some of these are acid/base transporters that regulate intracellular pH (pH_i) (9). Such modulation allows pH_e to cross-talk with pH_i (10, 11), thus helping to shape the plethora of effects that pH_i has on cellular physiology (3, 9, 12, 13). Extracellular pH can also affect tissue structure through the release or modulation of proteolytic enzymes that act on the extracellular matrix (14, 15). In addition, the pH_e-pH_i difference is important in determining the distribution of membrane-permeant weak acids/bases, which include many drugs used clinically (e.g. doxorubicin).A complete understanding of pH regulation at tissue level requires characterization of events occurring within cells, at their surface membrane, and in the surrounding extracellular space. To date, many pH studies have treated the extracellular space as an infinite, well-stirred, and equilibrated compartment of constant pH. This condition is compatible with experimentally superfused, isolated cells, but it may not apply to all cells in situ. Blood plasma is a major component of extracellular fluid. In health, plasma pH is regulated to ∼7.4 by the lungs and kidneys, acting in concert to remove excess acid/base that has been added to blood from dietary or cellular sources. Tissue fluid occupies the gap between plasma and cells (with the exception of blood-borne cells). Under conditions of ideal diffusive coupling between cells and capillaries, pH_e in tissue fluid would be held close to plasma pH. However, pH_e close to the cell surface can diverge from 7.4, particularly when the cell-capillary distance is increased (e.g. as a result of poor blood perfusion), when the excreted acid/base load is elevated, or when the local buffering capacity is compromised.Regulation of pH_e is particularly important in tumors because these are characterized by a high metabolic rate (16, 17) and abnormal blood perfusion (18, 19). Studies have shown that tumors develop low pH_e (∼6.9) in response to the mismatch between metabolic demand and the capacity to remove metabolic waste products (14, 18, 20). Tumors can survive in considerably more acidic interstitium than their non-neoplastic counterparts, partly because of their ability to maintain a favorably alkaline pH_i for growth and development (21). It has been argued that tumors can survive selectively by maintaining a level of pH_e that is lethal to normal cells but not sufficiently acidic to kill the tumor itself (2, 14, 22).A major fraction of cell-derived acid is excreted in the form of CO₂, generated directly from the Krebs cycle or from titration of intracellular H⁺ with HCO₃⁻. To maintain a steep outward gradient for CO₂ excretion, extracellular CO₂ must not accumulate. This can be achieved by venting CO₂ to the nearest capillary or by reacting CO₂ locally to produce H⁺ and HCO₃⁻. The balance between these two fluxes is set by the diffusion distance and CO₂ hydration kinetics, respectively. Diffusion is anecdotally considered to be fast. However, over long distances, CO₂ diffusion may be slower than its local reactive flux. Assuming a CO₂ diffusion coefficient, D_CO2, of 2500 μm²/s and a spontaneous CO₂ hydration rate, k_f, of 0.14 s⁻¹ (23), local CO₂ consumption by reaction will be faster than CO₂ diffusion over distances >190 μm (√(2 × D_CO2/k_f)). The reactive flux can be augmented enzymatically by CA_e, to increase further the importance of reactive versus diffusive consumption of CO₂. If, for instance, hydration is catalyzed 10-fold, reactive CO₂ removal would exceed diffusive CO₂ removal over distances of >60 μm.The remainder of transmembrane acid efflux takes the form of lactic acid, generated from anaerobic respiration or aerobic glycolysis (Warburg effect) (16). Lactic acid efflux can be accelerated if its extracellular concentration is kept low by diffusive dissipation or by CA_e-catalyzed extracellular titration of H⁺ with HCO₃⁻. It is important to note that CA_e-catalyzed hydration of extracellular CO₂ will reduce pH_e, whereas titration of extracellular lactic acid by HCO₃⁻ (to form CO₂, a weaker acid) will raise pH_e. Therefore, the capacity of CA_e to regulate pH_e will depend on the chemistry of the excreted acid. In most healthy tissues at rest, the majority of cellular acid is emitted as CO₂. Recent work on tumors also suggests a dominance of CO₂ over lactic acid (22, 24).The role for CA_e in facilitating CO₂ removal has been demonstrated for CA4 in skeletal muscle (25) and proposed for CA9 in tumors (2, 26). Furthermore, CA9 expression is strongly up-regulated in hypoxia (5), providing a mechanism by which CA9 levels are linked to diffusion distance. A consequence of facilitated CO₂ removal is the attainment of a more uniformly alkaline pH_i across the tissue. We demonstrated this recently in three-dimensional in vitro tissue models imaged for pH_i (23). One prediction from that study is that CA9, although reducing pH_i nonuniformity, will give rise to local extracellular acidity, particularly at the core of multicellular growths.If pH_e is indeed acidified by CA9, the enzyme expression may be doubly beneficial for CO₂-excreting tumors: it will help to attain (i) a favorable alkaline pH_i for growth and (ii) an acidic pH_e to facilitate invasiveness. Clinically, CA9 may serve as a target for drugs. In the present work, we image pH_e using a novel, membrane-impermeant fluorescent pH dye in multicellular spheroid growths (∼35,000 cells) derived from the colon carcinoma cell line HCT116. We demonstrate a key role for CA9 in regulating both pH_i and pH_e. Furthermore, we show that, even in the hypoxic core of spheroids, the principal substrate for CA9 is cell-excreted CO₂ and that the precise effect of CA9 on pH_e depends on the relative efflux from cells of lactic acid versus CO₂.     

Release of Carbonic Anhydrase from the Cell Surface of Chlamydomonas reinhardtii by Trypsin

Treatment with trypsin of Chlamydomonas reinhardtii cells grownin ordinary air (low-CO₂ cells) caused almost complete releaseof carbonic anhydrase (CA) into the suspending medium, but didnot affect the shape and kinesis of the cells. These resultsindicate that most of the CA exists on the cell surface of low-CO₂cells. The released CA has the same molecular weight, specificactivity and susceptibility to various CA inhibitors as thatpurified from non-treated low-CO₂ cells. (Received August 24, 1985; Accepted November 20, 1985)     

Spectral Dependence of Photoregulation of Inorganic Nitrogen Metabolism in Chlamydomonas reinhardii

The utilization of NO₃⁻ by green algae growing photoautotrophically under air, which are growth conditions close to their more habitual situations in nature, is associated with the excretion of NO₂⁻ and NH₄⁺ to the culture medium. The entire process is promoted by blue light and depends on photosynthetically active radiation for the required reducing equivalents. The stimulation of NO₃⁻ utilization and of its associated NO₂⁻ and NH₄⁺ excretions saturated at very low quantum fluxes of blue light (15 microequivalents per square meter per second) in Chlamydomonas reinhardii cells sparged with CO₂-free air and irradiated with 50 microequivalents per square meter per second background red light. The wavelength dependence data of this stimulation correlated closely with the in situ photoactivation of nitrate reductase and also with the light induced increase in its biosynthesis and/or assembly.

These results indicate that the photoregulation of inorganic N metabolism in C. reinhardii is mainly due to the blue light modulation of nitrate reductase. Although flavins are the most suitable candidates to act as physiological photoreceptors, the wavelength dependence data only show a major peak in the blue region between 400 and 500 nanometers.