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.     

Construction of a carbon-conserving pathway for glycolate production by synergetic utilization of acetate and glucose in Escherichia coli

Glycolate is a bulk chemical which has been widely used in textile, food processing, and pharmaceutical industries. Glycolate can be produced from sugars by microbial fermentation. However, when using glucose as the sole carbon source, the theoretical maximum carbon molar yield of glycolate is 0.67 mol/mol due to the loss of carbon as CO₂. In this study, a synergetic system for simultaneous utilization of acetate and glucose was designed to increase the carbon yield. The main function of glucose is to provide NADPH while acetate to provide the main carbon backbone for glycolate production. Theoretically, 1 glucose and 5 acetate can produce 6 glycolate, and the carbon molar yield can be increased to 0.75 mol/mol. The whole synthetic pathway was divided into two modules, one for converting acetate to glycolate and another to utilize glucose to provide NADPH. After engineering module I through activation of acs, gltA, aceA and ycdW, glycolate titer increased from 0.07 to 2.16 g/L while glycolate yields increased from 0.04 to 0.35 mol/mol-acetate and from 0.03 to 1.04 mol/mol-glucose. Module II was then engineered to increase NADPH supply. Through deletion of pfkA, pfkB, ptsI and sthA genes as well as upregulating zwf, pgl and tktA, glycolate titer increased from 2.16 to 4.86 g/L while glycolate yields increased from 0.35 to 0.82 mol/mol-acetate and from 1.04 to 6.03 mol/mol-glucose. The activities of AceA and YcdW were further increased to pull the carbon flux to glycolate, which increased glycolate yield from 0.82 to 0.92 mol/mol-acetate. Fed-batch fermentation of the final strain NZ-Gly303 produced 73.3 g/L glycolate with a productivity of 1.04 g/(L·h). The acetate to glycolate yield was 0.85 mol/mol (1.08 g/g), while glucose to glycolate yield was 6.1 mol/mol (2.58 g/g). The total carbon molar yield was 0.60 mol/mol, which reached 80% of the theoretical value.     

Fixation of O(2) during Photorespiration: Kinetic and Steady-State Studies of the Photorespiratory Carbon Oxidation Cycle with Intact Leaves and Isolated Chloroplasts of C(3) Plants

Mass spectrometric techniques were used to trace the incorporation of [¹⁸O]oxygen into metabolites of the photorespiratory pathway. Glycolate, glycine, and serine extracted from leaves of the C₃ plants, Spinacia oleracea L., Atriplex hastata, and Helianthus annuus which had been exposed to [¹⁸O]oxygen at the CO₂ compensation point were heavily labeled with ¹⁸O. In each case one, and only one of the carboxyl oxygens was labeled. The abundance of ¹⁸O in this oxygen of glycolate reached 50 to 70% of that of the oxygen provided after only 5 to 10 seconds exposure to [¹⁸O]oxygen. Glycine and serine attained the same final enrichment after 40 and 180 seconds, respectively. This confirms that glycine and serine are synthesized from glycolate.

The labeling of photorespiratory intermediates in intact leaves reached a mean of 59% of that of the oxygen provided in the feedings. This indicates that at least 59% of the glycolate photorespired is synthesized with the fixation of molecular oxygen. This estimate is certainly conservative owing to the dilution of labeled oxygen at the site of glycolate synthesis by photosynthetic oxygen. We examined the yield of ¹⁸O in glycolate synthesized in vitro by isolated intact spinach chloroplasts in a system which permitted direct sampling of the isotopic composition of the oxygen at the site of synthesis. The isotopic enrichment of glycolate from such experiments was 90 to 95% of that of the oxygen present during the incubation.

The carboxyl oxygens of 3-phosphoglycerate also became labeled with ¹⁸O in 20- and 40-minute feedings with [¹⁸O]oxygen to intact leaves at the CO₂ compensation point. Control experiments indicated that this label was probably due to direct synthesis of 3-phosphoglycerate from glycolate during photorespiration. The mean enrichment of 3-phosphoglycerate was 14 ± 4% of that of glycine or serine, its precursors of the photorespiratory pathway, in 10 separate feeding experiments. It is argued that this constant dilution of label indicates a constant stoichiometric balance between photorespiratory and photosynthetic sources of 3-phosphoglycerate at the CO₂ compensation point.

Oxygen uptake sufficient to account for about half of the rate of ¹⁸O fixation into glycine in the intact leaves was observed with intact spinach chloroplasts. Oxygen uptake and production by intact leaves at the CO₂ compensation point indicate about 1.9 oxygen exchanged per glycolate photorespired. The fixation of molecular oxygen into glycolate plus the peroxisomal oxidation of glycolate to glyoxylate and the mitochondrial conversion of glycine to serine can account for up to 1.75 oxygen taken up per glycolate.

These studies provide new evidence which supports the current formulation of the pathway of photorespiration and its relation to photosynthetic metabolism. The experiments described also suggest new approaches using stable isotope techniques to study the rate of photorespiration and the balance between photorespiration and photosynthesis in vivo.

     

pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts

Experiments in which [1-¹⁴C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C₃ plants.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).     

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration

When the CO₂ concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO₂ in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO₂) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO₂, providing further evidence that the cycle is well regulated.     

Evidence of Reentrance of Glycolate Carbon into the Photosynthetic Carbon Reduction Cycle in Photosynthesizing Euglena gracilis Z

Aminooxyacetate induced excretion of glycolate from air-grown cells of Euglena gracilis in both air and 1% CO₂ atmospheres. The rate of the excretion reached 70% of the photosynthetic rate in the air on a carbon basis, and was 10% in 1% CO₂. The compulsory loss of photosynthetically fixed carbon as glycolate at the high rate in air in the presence of aminooxyacetate caused a decrease of the rate of synthesis of paramylon, the reserve polysaccharide. Analyses of the steady levels of photosynthetic intermediates showed that a decrease of the 3-phosphoglycerate level was the cause of the slow rate of paramylon synthesis under these conditions.     

Rates of synthesis and source of glycolate in intact chloroplasts

Summary Intact chloroplasts capable of high rates of CO₂ assimilation completely oxidized 3-phosphoglycerate and dihydroxyacetone phosphate to glycolate when CO₂ concentrations were low. Bicarbonate was converted first into products of the Calvin cycle and then into glycolate. Under high oxygen and at high pH values CO₂ fixation and glycolate formation ceased before bicarbonate was exhausted. This is interpreted as the consequence of a depletion of ribulose diphosphate (RuDP) at the oxygen compensation point, where oxygen consumption by glycolate formation and oxygen evolution by phosphoglycerate reduction balance each other. Depletion of RuDP by glycolate formation is proposed to play a role in the Warburg effect. The maximum rate of glycolate synthesis observed with dihydroxyacetone phosphate as substrate was 35 mol mg^-1 chlorophyll h^-1 at 20°C. This may not reflect the maximum capacity of chloroplasts for glycolate synthesis. Dithiothreitol and catalase, which prevent accumulation of oxygen radicals or H₂O₂ during carbon assimilation, increased glycolate formation. H₂O₂ was inhibitory. Other inhibitors of glycolate formation were glyceraldehyde and carbonylcyanide p-trifluoro-methoxphenylhydrazone. From the sensitivity of glycolate synthesis to uncoupling and the ATP requirement of RuDP formation it is concluded that glycolate originated from RuDP. Different induction periods of carbon fixation and glycolyte formation suggested that glycolate synthesis is not only regulated by the ratio of oxygen to CO₂ but also by another factor.     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Involvement of glycolate metabolism in acclimation of Chlorella vulgaris cultures to low phosphate supply

Chlorella vulgaris (Beijer.) was grown for 8 d under air in cultures with complete (Control) or with phosphorus-deficient (–P) medium limiting culture growth. The cells assimilated only 5–17 % of orthophosphate supplied from the complete medium, whereas from medium of –P cultures, orthophosphate was almost totally exhausted. Despite limited phosphorus availability, cells in the oldest –P cultures contained the same amount of inorganic orthophosphate as the control cells and only slightly less organic phosphates. The –P cells showed normal chlorophyll concentration and increased V_max and 1/K_0.5 dissolved inorganic carbon (DIC) of photosynthetic O₂ evolution. Phosphorus deficiency enhanced production, excretion and metabolism of glycolate during the whole investigated period. In the initial phase of –P culture growth, medium acidification and low DIC concentration were conducive to glycolate production. With subsequent medium alkalization, DIC content and cell carbonic anhydrase activity increased the photosynthetic O₂ evolution of –P cells two-fold. At that period, the elevated intrachloroplast O₂ concentration might be the main reason of enhancement of glycolate metabolism. The results support the suggestion that involvement of glycolate metabolism in acclimation to low phosphorus supply improves regeneration of inorganic orthophosphate and protects chloroplasts against photoinhibitory damage by consumption of excess of absorbed light energy.     

Rate of Glycolate Formation During Photosynthesis at High pH

The products of C¹⁴O₂ fixation by Chlamydomonas and Chlorella were studied under conditions most favorable for glycolate synthesis. The highest percentage of the C¹⁴ was incorporated into glycolate in the pH range of 8 to 9. After 1 to 2 minutes as much as 40% of the C¹⁴ was found in glycolate products and only a trace of C¹⁴ was present as phosphoglycerate. Below pH 8 the rate of photosynthesis was much faster, but only a small percent of the C¹⁴ was incorporated into glycolate in 1 or 2 minutes, while a high percent of the C¹⁴ accumulated in phosphoglycerate. C¹⁴ labeling of glycolate even at pH 8 or above did not occur at times shorter than 10 seconds. During the first seconds of photosynthesis, nearly all of the C¹⁴ was found in phosphoglycerate and sugar phosphates. Thus glycolate appears to be formed after the phosphate esters of the photosynthetic carbon cycle.

Washing Chlamydomonas with water 2 or 3 times resulted in the loss of most of their free phosphate. When a small aliquot of NaHC¹⁴O₃ was added to washed algae in the absence of this buffering capacity, the pH of the algal medium became 8 or above and much of the fixed C¹⁴ accumulated in glycolate.

     

Different metabolic fate of two carbons of glycolate in its conversion to serine in Euglena gracilis z

In our preceding work (A. Yokota, Y. Nakano, and S. Kitaoka, 1978, Agric. Biol. Chem. 42, 121-129), extensive decarboxylation of glycolate carboxyl carbon during its metabolism in Euglena gracilis suggested occurrence of a metabolic pathway of glycolate different from that of higher C3 plants. In the present report, we establish the Euglena glycolate pathway from characteristics of the decarboxylation of the carboxyl carbon and from the metabolic fate of hydroxymethyl carbon of glycolate. The ratio of the decarboxylation of the carboxyl carbon of glycolate to the total metabolized carbon increased with increasing metabolic rate in an asymptotic fashion. Thus, the ratio was 20% at the metabolic rate of 0.05 nmol of glycolate/10(6) cells/min, but it was over 60% at the rate of more than 0.35 nmol/10(6) cells/min after 2 min of incubation. Metabolic products were also changed depending on the rate of metabolism of glycolate; glycine was the main product at the low rate of glycolate metabolism and the contribution of glycine was reversed by the increased contribution of evolved CO2 at the high rates. At the metabolic rate of 1.5 nmol of glycolate/10(6) cells/min, the rate of the decarboxylation was 1.0 nmol of CO2/10(6) cells/min, which could not be explained by the extremely low activity of glycine synthase in Euglena. Experiments with [2-14C]glycolate showed that exogenously added formate and methionine caused accumulation of radioactive formate. Based on these results, we have proposed that the glycolate metabolism of E. gracilis consists of glycine and formate pathways and that the relative contribution of both pathways to the glycolate metabolism depends on the metabolic rate of glycolate.     

Photorespiration and Glycolate Metabolism: A Re-examination and Correlation of Some Previous Studies

Some previous studies of photorespiration and glycolate oxidation were re-examined and correlated by infra-red CO₂ analysis. Data about rate of photosynthesis and oxygen sensitivity indicated that complete inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1 dimethyl urea (DCMU) allowed dark respiration to continue in the light. Photorespiration was also inhibited. The oxygen sensitivity of glycolate-stimulated CO₂ production was found to be compatible with the proposal that glycolate is a substrate of photorespiration. Both `in vivo' and `in vitro' studies of the alga Nitella flexilis have revealed a pathway of glycolate oxidation similar to that of higher plants. DCMU inhibition of photosynthesis by Nitella gave results similar to those for the monocotyledons tested. Under very low light intensity, carbon dioxide compensation in corn was measurable but was not sensitive to high oxygen concentration. It appears that the lack of photorespiration in this plant is not the end result of efficient internal recycling of CO₂ to photosynthesis.     

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO₂-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO₂. Adaptation to air levels of CO₂ by Chlamydomonas involves induction of a CO₂-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO₂-enriched cells. Unlike WT, the high-CO₂-requiring mutant, cia-5, does not respond to limiting CO₂ conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO₂ which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.     

Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis

In plants, glycolate oxidase is involved in the photorespiratory cycle, one of the major fluxes at the global scale. To clarify both the nature of the mechanism and possible differences in glycolate oxidase enzyme chemistry from C₃ and C₄ plant species, we analyzed kinetic parameters of purified recombinant C₃ (Arabidopsis thaliana) and C₄ (Zea mays) plant enzymes and compared isotope effects using natural and deuterated glycolate in either natural or deuterated solvent. The ¹²C/¹³C isotope effect was also investigated for each plant glycolate oxidase protein by measuring the ¹³C natural abundance in glycolate using natural or deuterated glycolate as a substrate. Our results suggest that several elemental steps were associated with an hydrogen/deuterium isotope effect and that glycolate α-deprotonation itself was only partially rate-limiting. Calculations of commitment factors from observed kinetic isotope effect values support a hydride transfer mechanism. No significant differences were seen between C₃ and C₄ enzymes.     

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.     

Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate

The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The K_m values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH₄)₂SO₄ fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O₂ concentration. Glycolate oxidation increased 3.6-fold when the O₂ atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O₂ concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate.     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

Induction of glycolate oxidase by SO2 in Nicotiana tabacum

In contrast to the inhibitory action of sulfite on glycolate oxidase, the specific activity of the enzyme in tobacco leaves exposed to SO₂ for 18 hr increases in proportion to the SO₂ concentration. This increase is strongly reduced by pretreatment with cycloheximide. As a consequence of induced de novo synthesis of glycolate oxidase the glycolate content of the leaves is markedly reduced after 18 hr exposure to SO₂.