Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO₂ after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.     

Light-Dependent Oxygen Uptake, Glycolate, and Ammonia Release in l-Methionine Sulfoximine-Treated Chlamydomonas

Glycolate and ammonia excretion plus oxygen exchanges were measured in the light in l-methionine-dl-sulfoximine treated air-grown Chlamydomonas reinhardii. At saturating CO₂ (between 600 and 700 microliters per liter CO₂) neither glycolate nor ammonia were excreted, whereas at the CO₂ compensation concentration (<10 microliters per liter CO₂) treated algae excreted both glycolate and ammonia at rates of 37 and 59 nanomoles per minute per milligram chlorophyll, respectively. From the excretion values we calculate the amount of O₂ consumed through the glycolate pathway. The calculated value was not significantly different from the component of O₂ uptake sensitive to CO₂ obtained from the difference between O₂ uptake of the CO₂ compensation point and at saturating CO₂. This component was about 40% of stationary O₂ uptake measured at the CO₂ compensation point. From these data we conclude that glyoxylate decarboxylation in air-grown Chlamydomonas represents a minor pathway of metabolism even in conditions where amino donors are deficient and that processes other than glycolate pathway are responsible for the O₂ uptake insensitive to CO₂.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

Glycolate excretion by air-grown Euglena graeilis z

In contrast to reported fact, the air-grown cells of Euglena gracilis z were found to excrete glycolate into the surrounding medium, when placed in an atmosphere of 100% O₂ under illumination at 20000 lux at the same rate of the 5% CO₂-grown cells, but with a lag phase of about 30 min. The lag was eliminated by lowering intracellular CO₂ concentration in the air-grown cells.This paper is the eighth in a series on the metabolism of glycolate in Euglena gracilis.     

Photorespiratory rates in wheat and maize as determined by o-labeling

A method was devised to quantify short-term photorespiratory rates in terrestrial plants using ¹⁸O-intermediates of the glycolate pathway, specifically glycolate, glycine, and serine. The pathway intermediates were isolated and analyzed on a GC/MS to determine molecular percent ¹⁸O-enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, derived rate equations, and nonlinear regression techniques. Glycolate synthesis in wheat (Triticum aestivum L.), a C₃ plant, and maize (Zea mays L.), a C₄ plant, was stimulated by high O₂ concentrations and inhibited by high CO₂ concentrations. The synthesis rates were 7.3, 2.1, and 0.7 micromoles per square decimeter per minute under a 21% O₂ and 0.035% CO₂ atmosphere for leaf tissue of wheat, maize seedlings, and 3-month-old maize, respectively. Photorespiratory CO₂ evolution rates were estimated to be 27, 6, and 2%, respectively, of net photosynthesis for the three groups of plants under the above atmosphere. The results from maize tissue support the hypothesis that C₄ plants photorespire, albeit at a reduced rate in comparison to C₃ plants, and that the CO₂/O₂ ratio in the bundle sheath of maize is higher in mature tissue than in seedling tissue. The pool size of the three photorespiratory intermediates remained constant and were unaffected by changes in either CO₂ or O₂ concentrations throughout the 10-minute labeling period. This suggests that photorespiratory metabolism is regulated by other mechanism besides phosphoglycolate synthesis by ribulose-1,5-bisphosphate carboxylase/oxygenase, at least under short-term conditions. Other mechanisms could be alternate modes of synthesis of the intermediates, regulation of some of the enzymes of the photorespiratory pathway, or regulation of carbon flow between organelles involved in photorespiration. The glycolate pool became nearly 100% ¹⁸O-labeled under an atmosphere of 40% O₂. This pool failed to become 100% ¹⁸O-enriched under lower O₂ concentrations.     

Glycolate Excretion and the Oxygen to Carbon Dioxide Net Exchange Ratio during Photosynthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells were grown in high (5% v/v) or low (0.03% v/v) CO₂ concentration in air. O₂ evolution, HCO₃⁻ assimilation, and glycolate excretion were measured in response to O₂ and CO₂ concentration. Both low- and high-CO₂-grown cells excrete glycolate. In low-CO₂-grown cells, however, glycolate excretion is observed only at much lower CO₂ concentrations in the medium, as compared with high-CO₂-adapted cells. It is postulated that the activity of the CO₂-concentrating mechanism in low-CO₂-grown cells is responsible for the different dependence of glycolate excretion on external CO₂ concentration in low- versus high-CO₂-adapted cells.     

Effect of Photon Fluence Rate on Oxygen Evolution and Uptake by Chlamydomonas reinhardtii Suspensions Grown in Ambient and CO(2)-Enriched Air

A closed system consisting of an assimilation chamber furnished with a membrane inlet from the liquid phase connected to a mass spectrometer was used to measure O₂ evolution and uptake by Chlamydomonas reinhardtii cells grown in ambient (0.034% CO₂) or CO₂-enriched (5% CO₂) air. At pH = 6.9, 28°C and concentrations of dissolved inorganic carbon (DIC) saturating for photosynthesis, O₂ uptake in the light (U_o) equaled O₂ production (E_o) at the light compensation point (15 micromoles photons per square meter per second). E_o and U_o increased with increasing photon fluence rate (PFR) but were not rate saturated at 600 micromoles photons per square meter per second, while net O₂ exchange reached a saturation level near 500 micromoles photons per square meter per second which was nearly the same for both, CO₂-grown and air-grown cells. Comparison of the U_o/E_o ratios between air-grown and CO₂-grown C. reinhardtii showed higher values for air-grown cells at light intensities higher than light compensation. For both, air-grown and CO₂-grown algae the rates of mitochondrial O₂ uptake in the dark measured immediately before and 5 minutes after illumination were much lower than U_o at PFR saturating for net photosynthesis. We conclude that noncyclic electron flow from water to NADP⁺ and pseudocyclic electron flow via photosystem I to O₂ both significantly contribute to O₂ exchange in the light. In contrast, mitochondrial respiration and photosynthetic carbon oxidation cycle are regarded as minor O₂ consuming reactions in the light in both, air-grown and CO₂-grown cells. It is suggested that the “extra” O₂ uptake by air-grown algae provides ATP required for the energy dependent CO₂/HCO₃⁻ concentrating mechanism known to be present in these cells.     

Glycolate Metabolism and Excretion by Chlamydomonas reinhardtii

The flux of glycolate through the C₂ pathway in Chlamydomonas reinhardtii was estimated after inhibition of the pathway with aminooxyacetate (AOA) or aminoacetonitrile (AAN) by measurement of the accumulation of glycolate and glycine. Cells grown photoautotrophically in air excreted little glycolate except in the presence of 2 mm AOA when they excreted 5 micromoles glycolate per hour per milligram clorophyll. Cells grown on high CO₂ (1-5%) when transferred to air produced three times as much glycolate, with half of the glycolate metabolized and half excreted. The lower amount of glycolate produced by the air-grown cells reflects the presence of a CO₂ concentrating mechanism which raises the internal CO₂ level and decreases the ribulose-1,5-bisP oxygenase reaction for glycolate production. Despite the presence of the CO₂ concentrating mechanism, there was still a significant amount of glycolate produced and metabolized by air-grown Chlamydomonas. The capacity of these cells to metabolize between 5 and 10 micromoles of glycolate per hour per milligram chlorophyll was confirmed by measuring the biphasic uptake of added labeled glycolate. The initial rapid (<10 seconds) phase represented uptake of glycolate; the slow phase represented the metabolism of glycolate. The rates of glycolate metabolism were in agreement with those determined using the C₂-cycle inhibitors during CO₂ fixation.     

An investigation of glycolate excretion in two species of blue-green algae

Summary The amount of ¹⁴C-glycolate excreted by Oscillatoria sp. and Anabaena flos-aquae is less than 1% of the ¹⁴C fixed by the algae during photosynthesis. Transfer of cells grown on 5% CO₂ in air to a medium of low bicarbonate concentration or treatment of the cells with isonicotinyl hydrazide (INH) during photosynthesis, caused little increase in glycolate excretion. -Hydroxysulfonates failed to stimulate massive excretion of glycolate. Although these blue-green algae excreted little glycolate, a significant proportion of the photosynthetically fixed carbon was excreted in the form of basic, neutral and acidic compounds, and such excretion was greater in 5% CO₂-grown cells than in air-grown cells.     

Glycolate biosynthesis by Scenedesmus and Chlorella in the presence or absence of NaHCO3

Summary Both Scenedesmus and Chlorella excreted comparable quantities of glycolate. Glycolate formation was dependent upon light and oxygen, but occured in the absence of added CO₂ or NaHCO₃ for net photosynthesis. In an environment of 3000 ft. c. light and an atmosphere of oxygen, about 35 g glycolate were excreted per hour per milliliter 1% (v/v) algae without NaHCO₃ or CO₂. Upon addition of NaHCO₃ the rate increased to about 55 g. Glycolate formation in the light in the absence of CO₂ may result from photometabolism of algal polysaccharides.Glycolate excretion by Scenedesmus occurred at all pH values between 6.5 and 9.5 and was not related to utilization of bicarbonate. Scenedesmus obliquus excreted glycolate when existing in plates of four or eight cells, but not when present as small individual cells.At pH 9 ¹⁴C fixation by Scenedesmus was faster than fixation by Chlorella. There was no significant difference in products of ¹⁴C fixation formed by Scenedesmus at pH values between 6.5 and 9.5.For unknown reasons -hydroxy-2-pyridinemethanesulfonate stimulated CO₂ fixation by Scenedesmus by at least 100%. This sulfonate had no effect on glycolate excretion nor upon the distribution of ¹⁴C among the products of ¹⁴CO₂ fixation by Scenedesmus.Supported in part by NSF Grant GB-4154 and published with the approval of the Director of the Michigan Agricultural Experiment Station as journal article No. 3946. The research was initiated during the period when N. E. Tolbert was supported in part by a National Institutes of Health Senior Fellowship at the Biochemisches Institut, Universität, Freiburg/Br., Germany.     

Incorporation of Oxygen into Glycolate, Glycine, and Serine during Photorespiration in Maize Leaves

Glycolate, glycine, and serine extracted from excised Zea mays L. leaves which had been allowed to photosynthesize in the presence of ¹⁸O₂ were analyzed by gas chromatography-mass spectrometry. In each case, only one of the oxygen atoms of the carboxyl group had become labeled. The maximum enrichment observed in glycine and serine was attained after 5 minutes and 15 minutes of exposure to ¹⁸O₂ at the CO₂ compensation point; the labeling was very high, reaching 70 to 73% of that in the applied O₂. Thus, it appears that all or nearly all of the glycine and serine are synthesized in maize leaves via fixation of O₂. In the presence of CO₂ (380 or 800 microliters per liter), ¹⁸O-labeling was markedly slower.

Glycolate enrichment was variable and much lower than that in glycine and serine. It is possible that there are additional pathways of glycolate synthesis which do not result in the incorporation of ¹⁸O from molecular oxygen. An estimation of the metabolic flow of O₂ through the photorespiratory cycle was made. It appeared that less than 75% of the O₂ taken up by maize leaves is involved in this pathway. Therefore, other processes of O₂ metabolism must occur in the light.

     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

Inhibition of photosynthesis by intracellular carbonic anhydrase in microalgae under excess concentrations of CO2

When cells of Chlorococcum littorale that had been grown in air (air-grown cells) were transferred to extremely high CO₂ concentrations (>20%), active photosynthesis resumed after a lag period which lasted for 1–4 days. In contrast, C. littorale cells which had been grown in 5% CO₂ (5% CO₂-grown cells) could grow in 40% CO₂ without any lag period. When air-grown cells were transferred to 40% CO₂, the quantum efficiency of PS II (Φ_II) decreased greatly, while no decrease in Φ_II was apparent when the 5% CO₂-grown cells were transferred to 40% CO₂. In contrast to air-grown cells, 5% CO₂-grown cells showed neither extracellular nor intracellular carbonic anhydrase (CA) activity. Upon the acclimation of 5% CO₂-grown cells to air, photosynthetic susceptibility to 40% CO₂ was induced. This change was associated with the induction of CA. In addition, neither suppression of photosynthesis nor arrest of growth was apparent when ethoxyzolamide (EZA), a membrane-permeable inhibitor of CA, had been added before transferring air-grown cells of C. littorale to 40% CO₂. The intracellular pH value (pH_i) decreased from 7.0 to 6.4 when air-grown C. littorale cells were exposed to 40% CO₂ for 1–2 h, but no such decrease in pH_i was apparent in the presence of EZA. Both air- and 5% CO₂-grown cells of Chlorella sp. UK001, which was also resistant to extremely high CO₂ concentrations, grew in 40% CO₂ without any lag period. The activity of CA was much lower in air-grown cells of this alga than those in air-grown C. littorale cells. These results prompt us to conclude that intracellular CA caused intracellular acidification and hence inhibition of photosynthetic carbon fixation when air-grown C. littorale cells were exposed to excess concentrations of CO₂. No such harmful effect of intracellular CA was observed in Chlorella sp. UK001 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glycolate Formation and Excretion by Chlorella pyrenoidosa and Netrium digitus

Conditions are described whereby suspensions of Chlorella pyrenoidosa and Netrium digitus photosynthetically biosynthesize and excrete glycolate continuously in high yields. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate-linked enzymes, increased the excretion of glycolate approximately 4-fold in 1 hour (8 millimolar) and 20-fold in 4 hours (40 millimolar) in the presence of 0.2% CO₂ in air. The amount of glycolate excreted in the presence of aminooxyacetate and an atmosphere of 0.2% CO₂ in air equaled or exceeded the amount excreted in 0.2% CO₂ in O₂ minus aminooxyacetate. CO₂ and light were required for glycolate excretion. Aminooxyacetate also stimulated photosynthetic glycolate excretion in an atmosphere of 0.2% CO₂ in nitrogen or helium, although the stimulation was not as great as when air or O₂ was present.

The excreted glycolate was converted to H₂ and CO₂ by the combined action of glycolic oxidase and the formic hydrogenlyase complex found in Escherichia coli in total conversion yields of 80%.

     

Photoexcretion and Fate of Glycolate in a Hot Spring Cyanobacterial Mat

Photosynthesis by Synechococcus lividus, the sole oxygenic phototroph inhabiting the surface of the 55°C cyanobacterial mat in Mushroom Spring, Yellowstone National Park, causes superoxic and alkaline conditions which promote glycolate photoexcretion. At O₂ concentrations characteristic of the top 2 mm of mat during the day, up to 11.8% of NaH¹⁴CO₃ fixed in the light was excreted, and glycolate accounted for up to 58% of the excreted photosynthate. Glycolate was neither incorporated nor metabolized by S. lividus, but it was incorporated by filamentous microorganisms in the mat. Incubation of mat samples with NaH¹⁴CO₃ resulted in labeling of both S. lividus and filaments, but the addition of nonradioactive glycolate increased the level of ¹⁴C in the aqueous phase and decreased the extent of labeling of filaments. This suggests that cross-feeding of glycolate from S. lividus to filamentous heterotrophs occurs and that underestimation of the extent of photoexcretion is probable.     

Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa

Oxygen inhibition of photosynthesis and CO₂ evolution during photorespiration were compared in high CO₂-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO₂ cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O₂, which appeared to be competitive and similar in magnitude to that in higher C₃ plants. With increasing time after transfer to air, the photosynthetic rate in high CO₂ cells increased while the O₂ effect declined. Photorespiration, measured as the difference between ¹⁴CO₂ and ¹²CO₂ uptake, was much greater and sensitive to O₂ in high CO₂ cells. Some CO₂ evolution was also present in air-grown algae; however, it did not appear to be sensitive to O₂. True photosynthesis was not affected by O₂ in either case. The data indicate that the difference between high CO₂ and air-grown algae could be attributed to the magnitude of CO₂ evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae.     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)     

A study of the control of glycolate excretion in chlorella

Chlorella pyrenoidosa cells grown on 5% CO(2) excreted glycolate when incubated in light with 10 mm bicarbonate, but excreted no glycolate under the same conditions when they were maintained on air for 7 hours prior to the assay. Incubation of 5% CO(2)-grown and air-grown cells with 10 mm isonicotinyl hydrazide or 10 mm alpha-hydroxypyridinemethane sulfonate during the assay stimulated the excretion of glycolate by CO(2)-grown cells severalfold that of air-grown cells.Adaptation of CO(2)-grown Chlorella to growth on air did not affect the levels of glycolate dehydrogenase in the cells and did not affect the rate of dark oxidation and metabolism of exogeneous (14)C-glycolate by the cells. These results indicate that the lack of glycolate excretion by air-grown or air-adapted cells of Chlorella cannot be explained by a concomitant change in the level of glycolate dehydrogenase.     

Evidence That an Internal Carbonic Anhydrase Is Present in 5% CO(2)-Grown and Air-Grown Chlamydomonas

Inorganic carbon (C_i) uptake was measured in wild-type cells of Chlamydomonas reinhardtii, and in cia-3, a mutant strain of C. reinhardtii that cannot grow with air levels of CO₂. Both air-grown cells, that have a CO₂ concentrating system, and 5% CO₂-grown cells that do not have this system, were used. When the external pH was 5.1 or 7.3, air-grown, wild-type cells accumulated inorganic carbon (C_i) and this accumulation was enhanced when the permeant carbonic anhydrase inhibitor, ethoxyzolamide, was added. When the external pH was 5.1, 5% CO₂-grown cells also accumulated some C_i, although not as much as air-grown cells and this accumulation was stimulated by the addition of ethoxyzolamide. At the same time, ethoxyzolamide inhibited CO₂ fixation by high CO₂-grown, wild-type cells at both pH 5.1 and 7.3. These observations imply that 5% CO₂-grown, wild-type cells, have a physiologically important internal carbonic anhydrase, although the major carbonic anhydrase located in the periplasmic space is only present in air-grown cells. Inorganic carbon uptake by cia-3 cells supported this conclusion. This mutant strain, which is thought to lack an internal carbonic anhydrase, was unaffected by ethoxyzolamide at pH 5.1. Other physiological characteristics of cia-3 resemble those of wild-type cells that have been treated with ethoxyzolamide. It is concluded that an internal carbonic anhydrase is under different regulatory control than the periplasmic carbonic anhydrase.     

The relationship between carbonic anhydrase activity and glycolate excretion in the blue-green alga Coccochloris peniocystis

Summary The rate of glycolate excretion by Coccochloris peniocystis Kütz. cells incubated under conditions of low bicarbonate concentration and high light intensity was linear for only the initial 15 min of incubation and no additional glycolate accumulated in the medium after 20 min. Excretion was maximal in cells grown on 5% CO₂ in air when transferred to an incubation medium containing no added bicarbonate. The inhibitor INH (isonicotinyl hydrazide) had no measurable effect on the amount of glycolate released whereas HPMS (-hydroxy-2-pyridyl methanesulfonate) stimulated excretion 3-fold. Cells transferred to air from growth on 5% CO₂ in air increased in carbonic anhydrase activity, while a decrease occurred in the cells' ability to excrete glycolate. Cells grown on air and switched to 5% CO₂ in air showed an increase in their ability to excrete glycolate with a concomitant decrease in carbonic anhydrase activity. Diamox, a specific inhibitor of carbonic anhydrase, was found to stimulate excretion with both airgrown and 5% CO₂-grown cells which had been off 5% CO₂ for approximately 30 min. The rate of carbon fixation by 5% CO₂-grown cells put on air was found to rise over a 110 min period, corresponding to both the induction period of carbonic anhydrase and the period of decline in the ability of the cells to excrete glycolic acid. These results suggest that the absence of carbonic anhydrase in 5% CO₂-grown cells causes a stimulation of glycolate excretion when these cells are transferred to a low bicarbonate medium, because of an increased rate of glycolate formation due to the oxidation of ribulose diphosphate by molecular oxygen at low internal CO₂ concentrations.Abbreviations INH isonicotinyl bydrazide - HPMS -hydroxy-2-pyridyl methanesulfonate