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Ls mflilmlljrl legal 6W3» LIBRARY Michigan State ”Niver‘ity .J This is to certify that the dissertation entitled THE TRANSPORT AND METABOLISM OF GLYCOLIC ACID BY CHLAMYDOMONAS REINHARDTII presented by Barbara Jean Wilson has been accepted towards fulfillment of the requirements for Ph D degreein Biochemistry mtg-7M Major professor Date 771% 343/ [787 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 bV1ESI_J RETURNING MATERIALS: Place in book drop to LlBRARlES remove this checkout from Ala-(3:23;. your record. FINES will be charged if book is returned after the date stamped below. NOV192006 '11 02 06 1APR 232007 l 9423’” THE TRANSPORT AND METABOLISM OF GLYCOLIC ACID BY CHLAMYDOMONAS REINHARDTII by Barbara Jean Wilson A DISSERTATION submitted to Michigan State University in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1987 ABSTRACT THE TRANSPORT AND METABOLISM OF GLYCOLIC ACID BY CLHAMYDOMONAS REINHARDTII by Barbara Jean Wilson In order to understand the excretion of glycolate from Chlamydomonas reinhardtii, the conditions affecting glycolate synthesis and metabolism were investigated. Although glycolate is synthesized only in the light, the metabolism occurs in the light and dark with greater metabolism in the light due to refixation of photorespiratory 002. The amount of internal glycolate will affect the metabolism of externally added glycolate. When glycolate synthesis exceeds the metabolic capacity, glycolate is excreted from the cell. Treatment of leaves from higher plants with aminooxyacetate leads to severe inhibition of CO2 fixation ”:0'” M ,x'" "- "-- --n._ o 1‘ I"- .-_. a—n—an-r gun-'1’" Since CO fixation by isolated chloroplasts was increased by 2 incubation with aminooxyacetate, the inhibition is not due to an effect on reactions within the chloroplasts. Therefore, accumulation of some inhibitory metabolite may be occurring in higher plants and excretion of glycolate by "‘~ algae may be a protective mechanism. The transport of glycolate into the cells occurs very rapidly. Equilibrium is achieved at 4°C within the time cells are pelleted by the silicone oil centrifugation technique through a layer of [14C]glycolate. Glycolate uptake does not show the same time, temperature and pH dependencies as diffusion of benzoate. Uptake can be inhibited by treatment of cells with N—ethylmaleimide and stimulated in the presence of valinomycin/KCl. Acetate and lactate are taken up as quickly as glycolate. The hypothesis was made that glycolate is transported by a protein carrier that transports monocarboxylic acids. The equilibrium concentration of glycolate is dependent on the cell density, implying that there may be a large number of transporter sites and that uptake is limited by substrate availability. A search for a mutant in this transporter was made using survival on fluoroacetate as a screen. All 48 mutants isolated were deficient in acetate metabolism and were unable to utilize acetate heterotrophically. One mutant isolated had reduced acetate and glycolate uptake and is a possible transport mutant. Wild-type Chlamydomonas reinhardtii strain 137 cells do have isocitrate lyase and malate synthase activity when grown photoautotrophically and the activities are increased in the presence of acetate. The mutants were divided into 6 classes based on the levels of isocitrate lyase and malate synthase when grown phototrophically in the presence of acetate. m5g—M—n—.—_ .. - ._ dedicated to Mom and the girls ii ACKNOWLEDGMENTS I would like to acknowledge the financial support provided by Dr. N. E. Tolbert. l Thank you to my committee members: Dr. Shelagh Ferguson—Miller, Dr. Paul Kindel, Dr. Estelle McCroarty and ‘ Dr. Barbara Sears. I would like to express my appreciation to Mike Mulligan who introduced me to photosynthesis by way of the studies on isolated chloroplasts presented in Chapter 3. Special thanks and appreciation go to Cathy Chia and Erin Bell who shared the frustrations and provided much encouragement. A special debt of gratitude goes to the Roberts who instilled determination. The choir at ULC made the music to soothe the savage beast. Many people too numerous to name individually, but expecially Bill, Craig, Dave and Gladys, shared thee day to day adventures. Good luck to you. TABLE OF CONTENTS LIST OF TABLES Vi LIST OF FIGURES Vii LIST OF ABBREVIATIONS ix CHAPTER 1. INTRODUCTION 1 CHAPTER 2. THE METABOLISM AND EXCRETION OF GLYCOLATE FROM CHLAMYDOMONAS REINHARDTII AS RELATED TO GLYCOLATE SYNTHESIS Introduction 6 Methods and Materials 10 Results Glycolate Synthesis 11 Glycolate Metabolism 17 Discussion Glycolate Synthesis 25 Glycolate Metabolism 26 CHAPTER 3. THE EFFECT OF AMINOOXYACETATE ON CO FIXATION AND GLYCOLATE METABOLISM BY ISOLATED LEAVES AND ISOLATED CHLOROPLASTS AS COMPARED TO CHLAMYDOMONAS REINHAREDTII Introduction 32 Methods and Materials 33 Results Effects of Aminooxyacetate on Photosynthesis by Whole Leaves 36 Effects of Aminooxyacetate on Photosynthesis in Isolated Chloroplasts 45 Effects of Aminooxyacetate on GO Fixation by Algae 58 2 iv Discussion Effects of Aminooxyacetate on Photosynthesis by Whole Leaves Effects of Aminooxyacetate on Photosynthesis in Isolated Chloroplasts Effects of Aminooxyacetate on 002 Fixation by Algae CHAPTER 4. THE MECHANISM OF TRANSPORT OF GLYCOLIC ACID BY CHLAMYDOMONAS REINHARDTII Introduction Methods and Materials Results Saturation Time Course and Temperature and pH Dependencies Inhibition Stimulation Discussion Saturation Time Course and Temperature and Ph Dependencies Inhibition Stimulation CHAPTER 5. THE SELECTION BY FLUOROACETATE OF MUTANTS OF CHLAMYDOMONAS REINHARDTII DEFICIENT IN ACETATE METABOLISM Introduction Methods and Materials Results Discussion CHAPTER 6. SUMMARY BIBLIOGRAPHY 58 6O 63 64 69 70 75 82 82 91 92 94 96 100 104 107 116 122 124 LIST OF TABLES Table 1. Effect of Atmosphere on Glycolate Excretion Table 2. Excretion of Glycolate by Mutants and Wild Type Table 3. 14C Distribution Among Products of CO2 Fixation by Leaves Table 4. Effect of Aminooxyacetate on Purified Ribulose—P2 Carboxylase/Oxygenase Activity Table 5. Analysis of Mutants Selected on Fluoroacetate vi 13 16 44 57 108 LIST OF FIGURES Figure l. The C2 Cycle in Algae. Figure 2. The Effect of Bicarbonate Concentration on the Excretion of Glycolate. Figure 3. Saturation of Glycolate Metabolism. Figure 4. Time Course of Glycolate Metabolism in Light and Dark. Figure 5. The Effect of pH on Glycolate Metabolism. Figure 6. Effect of Added Bicarbonate on Glycolate Metabolism by COZ-grown Cells Compared to Air-grown Cells. Figure 7. Percent CO Fixation of Leaves Treated with Aminooxyacetate. Figure 8. Percent CO Fixation of Leaves Treated with Aminooxygcetate at Different Levels of C02. Figure 9. Percent CO Fixation of Control by Leaves Treated with Inhibitors of the C Cycle or Uncouplers of Electron Transport. Figure 10. Chlorophyll Fluorescence Transients of Whole Leaves. Figure 11. Effect of Aminooxyacetate on Ferricyanide/Reduction by Lysed Chloroplasts. Figure 12. Light-dependent pH Change by Lysed Chloroplasts. Figure 13. Time Course of CO Fixation by Isolated Chloroplasts with Aminooxyacetate. Figure 14. Effect of Aminooxyacetate on the Time Course of CO Fixation by Isolated Chloroplasts with Bicgrbonate Concentration. vii 15 19 22 24 30 38 4O 43 47 49 51 54 56 Figure 15. Glycolate Uptake by Chlamydomonas reinhardtii or Erthrocytes. Figure 16. Uptake of Monocarboxylic Acids by Chlamydomonas. Figure 17. Uptake of Glycolate by Different Concentration of Cell Suspensions of Chlamydomonas. Figure 18. Time Course of Glycolate Uptake. Figure 19. Uptake of Glycolate as a Function of External pH. Figure 20. Inhibition of Glycolate Uptake. Figure 21. Stimulation of Uptake by Valinomycin/KCI. Figure 22. Effect of Other Ionophores and Salts on Glycolate Uptake. Figure 23. Dependence of Inhibition or Stimulation of Uptake on Chlorophyll Concentration. Figure 24. Model Proposed for the Stimulation of Glycolate Uptake by High KCl. Figure 25. The Glyoxylate Cycle. Figpse 26. Autoradiograms of Products of CO2 Fixation. Figure 27. Activities of Glyoxylate Cycle Enzymes in Wild Type and Mutants. viii 72 74 77 79 81 84 86 88 90 98 102 111 114 LIST OF ABBREVIATIONS AAN - aminoacetonitrile AOA - aminooxyacetate Bicine — N,N-bis[2—hydroxyethy11glycine CA — carbonic anhydrase Ches - 2[N-cyclohexylamino]ethanesulfonic acid chl — chlorphyll DCMU - 3-(3,4—dichlorophenyl)—1,1—dimethylurea DTT - dithiothreitol EDTA - ethylenediaminetetraacetic acid Epps - N-(2-hydroxyethyl)piperazine-N'-propanesulfonic acid Hepes - N-2-hydroxyethylpiperazine—N'—2—ethanesulfonic acid HPMS - 2-pyridylhydroxymethanesulfonic acid INH — isonicotinic acid hydrazide Mes 2[N—morpholino]ethanesulfonic acid NEM - N-ethylmaleimide PGA - phosphoglyceric acid Rubisco - ribulose-bis-phosphate carboxylase/oxygenase Tes — N-tris(hydroxymethyl)-2—aminoethanesulfonic acid ix CHAPTER 1 INTRODUCTION The C2 oxidative photosynthetic carbon cycle and its physiological significance have been reviewed (1). Glycolate metabolism and the C cycle in unicellular green 2 algae are modified to the extent that the glycolate metabolism is located in the mitochondria rather than the peroxisomes (2). Glycolate metabolism in algae is different from that in higher plants in that the algae have a mitochondrial glycolate dehydrogenase (3). Higher plants have a peroxisomal glycolate oxidase which uses oxygen for the oxidation of glycolate to glyoxylate and forms hydrogen peroxide. Glycolate dehydrogenase does not use oxygen and the electron acceptor for the oxidation step is unknown, except that it is linked to electron transport in the mitochondria. More is known about the synthesis and metabolism of glycolate than about its transport. Phosphoglycolate and phosphoglycerate are the products of the oxygenase activity of Rubisco (4). Phosphoglycolate in the chloroplasts is rapidly hydrolyzed to glycolate by a specific phosphatase. 2 Regulation of the ratio of the carboxylase/oxygenase activities of Rubisco is a broad topic covering characterization of the enzyme and its genetic and chemical regulation and modification. After the discovery of the oxygenase activity of Rubisco (5, 6), the excretion of glycolate by chloroplasts or by algae was not investigated until recently. Algae had commonly been grown in the laboratory with high levels of CC for faster growth and 2 then exposed to air-levels of CO during photosynthetic 2 experimentation. Under these conditions (the change to low atmospheric conditions immediately after growth on high CO glycolate synthesis was increased and consequently 2), the excess glycolate was excreted. Futhermore, biochemical differences between air— and COz-grown algae were found: air—grown cells had the CO -concentrating mechanism with a 2 periplasmic carbonic anhydrase and greater activity of glycolate dehydrogenase. Carbonic anhydrase facilitates the equilibrium of bicarbonate and carbon dioxide. In Chlamydomonas carbon dioxide is the species which diffuses into the cell and is fixed by Rubisco. The expression of carbonic anhydrase activity is repressed in algae grown on high C02. In C4 higher plants the C4 the bundle sheath cells where Rubisco is located. cycle concentrates 002 in Consequently glycolate biosynthesis and photorespiration are reduced. In some unicellular green algae, the ratio of the carboxylase to oxygenase activity of Rubiso is regulated by 3 a C0 - and/or bicarbonate concentrating mechanism, which builds up the inorganic carbon level in cells to about 1 to 3 mM. Both the C cycle and the C0 4 -concentrating mechanism 2 reduce glycolate biosynthesis by increasing the 002 to 02 ratio to favor the carboxylase activity of Rubisco. Research on the excretion of glycolic acid by algae involves three components: synthesis, metabolism and transport of glycolate by the cell. Even though the conditions for the excretion of glycolate were determined (7), the significance was not understood. It has been questioned why an organism would excrete newly fixed carbon that had required energy for its production. The ecological importance of glycolate as a carbon source for other organisms in the medium has been considered (8). However, in recent papers we contend that the algae do not excrete much glycolate in nature because of the C02-concentrating mechanism (9, 10). Paradoxically, even with the C0 ~concentrating 2 mechanism, air-grown cells are more C02 limited than cells grown with 2 to 5% C02. Air-grown cells synthesize and need to metabolize more glycolate. Air consists of 0.04% C02. The C02-concentrating mechanism can concentrate C02 10 fold but 0.4% is still less than 2% which is the lower limit used for high C02—grown cells. By use of inhibitors the total flux of glycolate through the C cycle in air— and C0 —grown 2 2 algae has been determined to be consistent with the amount of glycolate dehydrogenase (10). If the activity of 4 glycolate dehydrogenase is limiting metabolism, the excretion of glycolate may represent nothing more than excess synthesis. There may be no function for glycolate excretion other than maintaining the status quo in the cell. Nevertheless, some regulation of pool size of glycolate must exist to allow loss of expensive carbon atoms by glycolate excretion. Normally other photosynthetic carbon products are not excreted in significant amounts. The mechanism for specific glycolate excretion across the membrane remains unknown. Glycolate will exist predominantly as an anion at cellular pH. It is not known whether diffusion of glycolic acid can provide for the amount of glycolate excreted or whether there is another transport mechanism. Algae had been observed to take up glycolate after a period of excreting glycolate. The term uptake has been used to describe either the actual transport process or the transport and subsequent metabolism of a substrate. This lack of distinction can lead to ambiquity. In determining a mechanism for movement of molecules across the plasma membrane, a distinction must be made between transport and metabolism. The transport process will be obscured if metabolism is very rapid removing substrate from cellular pools as quickly as it is transported. The uptake of substrate is commonly used to measure transport because it is technically easier than efflux, especially if metabolism of preloaded substrate can occur. The transport of substrate has been found to be very rapid in some cases, such as glycolate (11) and glycerate (12). Chapters of this thesis will attempt to address aspects of glycolate excretion. Further introduction to each topic is presented at the beginning of each chhapter. CHAPTER 2 THE METABOLISM AND EXCRETION OF GLYCOLATE FROM CHLAMYDOMONAS REINHARDTII AS RELATED TO GLYCOLATE SYNTHESIS Introduction The purpose of this chapter is to discuss the metabollism and excretion of glycolate as related to glycolate synthesis. The hypothesis is that when glycolate synthesis exceeds glycolate metabolism, glycolate is excreted. Algae possess a concentrating mechanism for inorganic carbon tthat is induced when algae are grown under air levels of C02 and that is repressed when algae are grown under elevated levels of C0 (13). Since the ratio of C0 2 2 to 02 determines the relative activity of the carboxylase to oxygenase of Rubisco, this C02—concentrating mechanism is important in determining the ratio of synthesis of phoshoglycerate to glycolate. The nature of the C02-concentrating mechanism is not completely ellucidated. It is known that periplasmic carbonic anhydrase (CA) is induced during growth under low CO2 levels and that it is an important component of the concentrating mechanism. 6 7 Although this concentrating mechanism increases the internal CO2 concentration, the concentration is not raised enough to prevent glycolate synthesis. Glycolate when added to minimal medium stimulates the growth of Chlamydomonas (14). The metabolism of glycolate in green algae has been reported to be by the glycine/serine pathway similar to that in the C2 cycle of higher plants with some changes as indicated in Figure 1. If the pathway is not saturated with internal glycolate, then external glycolate can be metabolized. The rate of uptake of glycolate has been reported to be saturable in cyanobacteria (15) and green algae (16). It is likely that these data represented the rate of metabolism of glycolate rather than the transport process because of the length of time of incubation with glycolate. Conditons favoring excretion were reported to include high pH, high 02 and low C02 the availability of 002 relative to 02. Glycolate excretion is stimulated by inhibitors of the C (7). These conditions regulate 2 cycle of glycolate metabolism such as aminooxyacetate (AOA) (17, 18). The rate or amount of metabolism and excretion of glycolate will be partly determined by the synthesis of glycolate. Figure 1. The C2 Cycle in Algae. The site of inhibition by aminooxyacetate is shown at the aminotransferases (from Reference (8)). aminotransferose hexose phosphates ‘1 carboxylase COOH RIBULOSE-PZ CO 0 (EHOH 2 Zioxygenose CHZOGD COOH phosphatasej [kmase CH20® COOH P-qucolate CHOH phosphatase CHZOH COOH l CHZOH hydroxypyruvate reductose glycolate dehydrogenase COOH COOH p=o CHO CHZOH ominotronsferose . . . . l l t AOA Inhlbmon g you o e COOH 0HNH , \ COOH l 2 ¢r CHZNHZ CHZOH C02 Figure l. 10 Methods and Materials Algae Chlamydomonas reinhardtii strain 90, was obtained from the UTEX collection. Mutants of Chlamydomonas reinhardtii strain 137 with defects in the inorganic carbon transport (2mg) or carbonic anhydrase (Ea) were obtained from Dr. M. Spalding (U of Iowa). Chlamydomonas reinhardtii strain 137 wild type and other high-CO2 requiring mutants were supplied by Dr. R. Togasaki (U of Indiana). The F-60 strain was a mutation in ribulose-S-P kinase while mutants numbers 7, 112 and 146 were uncharacterized except for their high CO requirement for growth. 2 Growth of algae Chlamydomonas was grown in the minimal medium of Sueoka (19). The F—60 mutant was grown in tris-acetate-phosphate medium (20) with air. Algae were grown at 22°C on an Eberbach shaker under continuous light from fluorescent lamps (100 uE/s/mz) in 1 liter of media in 3—L Fernbach flasks. Cultures were aerated with either air or air supplemented with 3 to 5% CO Cells were harvested 2. by centrifugation at 4°C at 1000g for 5 min. Cells were washed with water and recentrifuged at 4°C at 10,000g for 5 min. Cells were resuspended in designated buffer at about 0.4 mg chl/m1 and stored on ice. Metabolism studies Algae were resuspended in the appropriate buffer and cell concentration. Buffers were to remove C0 and prepared fresh daily and bubbled with N 2 2 02 before being used to resuspend cells. Cell suspensions ll were stirred in vials in a water bath and illuminated from above by a slide projector (700 uE/s/mz). At appropriate times, aliquots were removed and cells separated from the supeernatant by centrifugation. Cells were resuspended according to the purpose of the experiment. Glycolate in the supernatant was determined by the Calkins assay (21). Chlorophyll was determined by the method of Arnon (22). Chromatography Samples were chromatographed on Whatman No. 1 paper in two directions using phenol/water and butanol/propionic acid/water (23). Papers were sprayed with 0.5 M NaHCO3 before they were completely dry to prevent volatilization of glycolic acid. Chromatograms were exposed to X—ray film and compound identification was based upon a standard chromatographic map. Spots were cut out, eluted in water and radioactivity determined by liquid scintillation counting. Materials 14 [ C]glycolate was obtained from ICN and [14C]NaHC0 was obtained from Research Products International, Corp. 3 Results Glycolate synthesis Glycolate synthesis by the oxygenase activity of Rubisco will be sensitive to the composition of the atmosphere since the ratio of C02 to 02 determines how much 12 glycolate is synthesized. During C02 fixation, wild-type 2, 02 or air and the amount of cells were bubbled with N radioactivity in the supernatant was determined (Table 1). The radioactivity in the supernatant has been found to be glycolate (17). With an increase in the amount of oxygen in the atmosphere, the percent radioactivity in the supernatant increased. When wild—type cells were incubated with 10 mM bicarbonate instead of 1 mM, glycolate excretion decreased (Figure 2). In other experiments Moroney, (unpublished) found that about 100 mM bicarbonate is necessary to suppress glycolate excretion to barely detectable levels. The absence of components of the C0 —concentrating 2 mechanism resulted in an increase in glycolate synthesis and excretion by the pmp and EE mutants (Table 2). The data suggest that neither mutant has a complete blockage in the C02—concentrating mechanism because air—grown mutant cells excreted less glycolate than 002—grown mutant cells when tested under atmospheric levels of 00 If blockage of the 2. C02-concentrating mechanism were complete, then there should be no difference between air— and COZ-grown cells. When treated with AOA to block glycolate metabolism, mutants 7 and 112 excreted glycolate at a similar level to wild—type (wt) (Table 2). Mutants 7 and 112 excreted nearly as much radioactivity without AOA as with AOA. MMutant 7 in particular was unusual in that the Calkins determination of ug glycolate in the supernatant was low in proportion to the % radioactivity excreted as compared to wild—type. It is 13 Table 1. Effect of Atmosphere on Glycolate Excretion Wild type cells of 5% (w/v) air—grown Chlamydomonas reinhardtii strain 90 were incubated with and without 2 mM ADA in 3 mM potassium phosphate pH 7. 5 for 60 min with 1 mM C]NaHC03 added every 15 min. Atmosphere Treatment gglic in supernatant N2 control 1.9 N2 AOA 7.5 air control 20.1 air AOA 40.1 02 control 34.9 0 AOA 31.3 14 Figure 2. The Effect of Bicarbonate Concentration on the Excretion of Glycolate. Glycolate excretion from C02-grown Chlamydomonas reinhardtii strain 90 (5% w/v) cells at 1 and 10 mM bicarbonate with and without 2 mM AOA in 25 mM Hepes pH 7.2 was measured. (1 mM Bicarbonate (0»), 1 mM bicarbonate + 2 mM AOA (I ) , 10 mM Bicarbonate (o) and 10 mM bicarbonate + 2 mM AOA (C1)) )Jg Glycolate/ml 15 lmMHCO-+AOA 'l 3 120.0- 80'0'" 10 mM H003_ + AOA 1 mM H003— 40.0% o : 5 . O . 10 mM HCO " 14; O O ///’U ' l l "l O 30 45 60 16 Table 2. Excretion of Glycolate by Mutants and Wild Type Chlamydomonas reinhardtii strain 137 wild type and mutant cell suspensions (5% w/v) in 3 mM potassium phosphate pH 7.5 were incubated with4and without 2 mM AOA for 60 min in the presence of 1 mM [ C]NaHC03 added every 15 min. Radioactivity in the supernatant is expressed as percent of total radioactivity fixed and the ug of glycolate/ml supernatant was determined by the Calkins assay. % radioactivity glycolate cell type in supernatant ug/ml wt 3.3 2.5 wt + AOA 43 8 49.2 pmp 20.3 26.2 pmp + AOA 47.7 87.2 ca 17.4 11.3 ca + AOA 67.3 65.6 wt (TAP) 8.0 wt + AOA (TAP) 29.7 F—60 12.0 F—60 (TAP) 12.8 7 26.3 1.5 7 + AOA 37.0 4.5 112 34.2 2.6 112 + AOA 39.4 15.5 146 5.8 0.4 146 + AOA 16.4 6.4 17 possible that both 7 and 112 may be excreting some other 14C labeled product such as malate rather than glycolate. Malate has been observed as a minor component of products excreted by wild type. Mutant 146 with and without AOA excreted a lower fraction of total radioactivity than did wild—type. Because of the magnitude of the glycolate excretion, it had been suggested that glycolate might be synthesized by a route other than the oxygenase activity of Rubisco. If this were true, mutants in the photosynthetic cycle might still excrete glycolate. The F—6O mutant had only 1% of the CO2 fixation of the wild—type. While 12% of the small amount of radioactivity fixed was found to be in the supernatant, there was no AOA stimulation of excretion (Table 2). The excreted product may be malate from the dark fixation of C0 . Growth on acetate medium did not alter the amount of 2 glycolate excreted by wild—type with or without AOA. Glycolate metabolism Conditions for growth stimulation by glycolate. Glycolate stimulated the growth of Chlamydomonas reinhardtii strain 137 as described by Spencer and Togasaki (14). This stimulation was not observed with strain 90 when grown in the same medium (minimal medium of Sueoka). When agar plates of minimal medium with and without glycolate were used, neither strain showed a growth differential. However with agar plates of tris—phosphate (TMP) medium, glycolate Figure 3. Saturation of Glycolate Metabolism. C02—grown Chlamydomonas reinhardtii strain 137 were resuspended to 50 ug chl/ml in 25 mM Hepes pH 7.5. 1-[14C1glycolate was given at indicated concentrations. At 10 sec, 4, 10, 20, 30 and 45 min samples were removed for determination of radioactivity in the cells and the slopes of the time courses for the different concentrations of external glycolate determined. The radioactivity is expressed in terms of [l4C]glycolate internal concentration even though glycolate had been converted to other products. The rate of internal [14C}glycolate accumulation/min is plotted versus external concentration. Internal [Glycolate] (mM)/min 213— 1.5— 0.0 ‘0’ 0.0 510 10.0 15.0 External [Glycolate] (mM) Figure 3. I 20.0 .‘u.;«¢-'*\ ‘2‘: - ‘.' “ 20 stimulated growth of strain 137. Addition of 10 mM glycolate was found to be optimal for growth stimulation and to saturate metabolism (Figure 3). The growth stimulation by glycolate of Chlamydomonas reinhardtii strain 137 was light dependent; cells were unable to grow with glycolate as a carbon source in the dark. The metabolism of glycolate was greater in light than in the dark (Figure 4). The uptake and metabolism of glycolate was greater at lower pH (Figure 5) but less growth stimulation by glycolate occurred at low pH (14). Both growth stimulation by and metabolsim of glycolate was influenced by conditions of aeration during growth of algae. The most stimulation of growth by glycolate was observed in flasks that were fitted with a styrofoam stopper; aeration was limited by diffusion through the stopper. Some stimulation was observed in flasks aerated with atmospheric levels of CO and no stimulation was 2 observed in flasks bubbled with C02—enriched air. Consistent with this observation, air-grown cells metabolized more glycolate than COZ-grown cells (24). + Effect of NH4- on metabolism. Glycolate metabolism is dependent on the gvailability of NH3 for the transamination of glyoxylate to glycine. As reported (17) NH4Cl reduces the AOA stimulation of glycolate excretion. This is true also for methylamine. The decrease in glycolate excretion was observed if NH4Cl was added to the cells before the AOA but not if NH4Cl was added after the AOA. 21 Figure 4. Time Course of Glycolate Metabolism in Light and Dark. The metabolism of 5 mM 1-[14C1glycolate in the light (0) and dark (0) by 5% w/v suspension of COZ-grown Chlamydomonas reinhardtii strain 90 in 25 mM Hepes pH 7.5 was measured. Radioactivity in the cells is calculated and expressed as glycolate even though metabolism has occurred. Internal [Glycolate] (mM) 5.0—: 22 l r r T 1 10 20 :50 40 50 60 time (min) Figure 4. 23 Figure 5. The Effect of pH on Glycolate Metabolism. Amount of 14 C in cells due to uptake and metabolism of 5 mM 1-[14C]glycolate after 1 hr at different pHs by 5% w/v suspension of C02—grown Chlamydomonas reinhardtii strain 90 was measured. The buffer was 5 mM each Ches—Mes—Hepes adjusted to pH 4.5, 5.5, 6.5, 7.5 and 8.5 with NaOH. Radioactivity in the cells is calculated and expressed as glycolate even though metabolism has occurred. 24- 35A?— row no rnN 7 nu TI. 0 so r0 so no I A I s a I. 4” p p o. o. b o. o. no so no Po AU ,5 AU .5 oz 92 4: 1. AEEV 7356038 BEBE Figure 5. A‘."‘m_._ -\._. . C. ‘1 Discussion Glycolate synthesis Evidence for glycolate being produced by the oxygenase activity of Rubisco is the dependence of the amount of glycolate excreted on the different atmospheres. Aeration with N2 in the light reduced glycolate excretion. Because of 02 release during photosynthesis, it is very difficult to completely eliminate 02 from the cells. But with AOA the amount excreted did not increase as much as when cells were bubbled with air. Bubbling with 02 increased glycolate excretion even without AOA. Routinely cells were exposed to 1 mM bicarbonate; when bicarbonate was increased to 10 mM, glycolate excretion was reduced although it took 100 mM bicarbonate to almost eliminate glycolate excretion. This emphasizes how relatively inefficient Rubisco is for the carboxylation reaction relative to the oxygenation. Even with the inorganic concentrating mechanism, the internal inorganic carbon concentration is not as high as in C02-enriched air and not high enough to eliminate the oxygenase reaction of Rubisco. The two C02—concentrating mechanism mutants, pmp and pa, had higher glycolate excretion because of the inability to concentrate 002. Glycolate excretion in the mutants is higher than in wild—type because glycolate synthesis is increased while the metabolic capacity is the same. That the excreted glycolate was produced by the 26 oxygenase activity of Rubisco is supported by the absence of glycolate production by the mutant F-60 which lacks ribulose-S-P kinase. These cells were maintained on acetate for heterotrophic growth as they were unable to grow autotrophically; however, growth on acetate of wild—type cells did not affect the amount of glycolate excretion. Glycolate excretion as a result of the oxygenase activity would be expected to be reduced because of the reduced photosynthetic activity. The F-60 mutant can not synthesize ribulose-bis-P and consequently the supply of substrate for both carboxylase and oxygenase activities of Rubisco was eliminated. The C02 fixation of F-60 was only about 1% of the wild-type so that the reported excretion of 12% of the 14 total C fixed is not directly comparable in amount with the wild-type. Glycolate metabolism Conditions for growth stimulation by glycolate. Levels of glycolate dehydrogenase are higher in air—grown cells. Therefore there is higher metabolism of glycolate by air-grown cells (10, 24). The maximal stimulation at 10 mM glycolate is consistent with the activity of glycolate dehydrogenase and the normal flux of glycolate through the C2 cycle (10). The activity of glycolate dehydrogenase is considered limiting to the production of glycine for decarboxylation. Strain 137, having more glycolate dehydrogenase than strain 90, can liberate more C02 and unfit-.“m - ‘5 ~. 27 exhibit growth stimulation on glycolate (data not shown). Growth stimulation is observed only in the light and metabolism of glycolate is greater in light (10, 24, 25). This is expected if the refixation of CO from glycolate 2 metabolism by Rubisco is necessary for stimulation of growth. The difference between light and dark metabolism should be the amount of CO refixed by Rubisco. The 2 importance of CO fixation to glycolate metabolism and 2 growth stimulation is further shown by use of the inhibitors DCMU, HPMS and INH and the F-60 mutant of Chlamydomonas reinhardtii. The F-60 mutant lacks phosphoribulokinase, is incapable of autotrophic growth having 1% of wild type C02 fixation, and shows no growth stimulation on glycolate. 1400 from DCMU did not prevent the release of 2 14 1[ C]glycolate metabolism in Chlorella but it blocked electron flow preventing C0 fixation (15, 24). DCMU 2 decreased the assimilation of glycolate (26) and prevented the growth stimulation (14, 27). When another inhibitor, HPMS, which also inhibits glycolate dehydrogenase, was added at 10 mM, glycolate metabolism was decreased by 90% to less than dark levels of utilization (24) and growth on glycolate was prevented (27). All of the data support the conclusion that the refixation of photorespiratory C0 is the primary cause of 2 the observed growth stimulation of glycolate. Utilization of glycolate is a balance between competition of external C02 and photorespiratory C02 and metabolism of internal and 28 external glycolate. Addition of 002 competes with the photorespiratory CO for Rubisco and growth is not limited 2 by CO availability. 2 The availability of CO is influenced by pH. At higher 2 pH bicarbonate is the predominate species of inorganic carbon. The effect of pH on the amount of 00 available is 2 probably the explanation for the lack of growth stimulation on the plate medium. The TMP medium is 20 mM Tris, pH 7.3 compared to the minimal medium of 7 mM potassium phosphate, pH 6.8. The higher buffered pH serves to trap CO as 2 bicarbonate which the external carbonic anhydrase converts to 002. However addition of bicarbonate can also increase utilization of external glycolate. When bicarbonate was added with glycolate to COZ-grown cells, metabolism was increased to a level seen with air cells (figure 6). The metabolism of exogenous glycolate may decrease when the C2 cycle is already metabolizing internal glycolate at maximal capacity. When cells are starved for 002, they use photorespiratory CO to augment growth but external 2 glycolate can not augment the cell when it is saturated with the glycolate it is producing internally. The carbon atoms of glycolate that go on to glycine are important for cell maintenance; while the F—60 mutant did not grow on glycolate, it did not die. The F—60 had incorporation rates of glycolate that were half of the wild—type (14). This half is the C2 carbon of glycolate Figure 6. Effect of Added Bicarbonate on Glycolate Metabolism by COZ-grown Cells Compared to Air-grown Cells. The metabolism of 1-[14C1glycolate in 30 mM Hepes pH 7.5 by 5% w/v Chlamydomonas reinhardtii strain 90 grown with air (I) or C0 (0) was measured. Bicarbonate (1 mM) was 2 added every 5 min to one set of CO ~grown cells (0) during 2 the experiment. Radioactivity in the cells is calculated and expressed as glycolate even though metabolism has occurred. 3O 100.0—I E I q air cells 0 80.0~ /"\ E E a v P"! 3 60.0— .9 O . '- 0 .. CO2 cells + HCO3 2.1 £2. __ 40.0-4 ' C C c L. (D .J 4.4 5 CD2 cells 20.0“ . . . , o .4 -/1 O O /o ~ I I T I I 0.0 10.0 20.0 30.0 40.0 50.0 External [Glycolate] (mM) Figure 6. 31 that goes on to serine. The product labeling patterns from [14C]glycolate are evidence for use of both the photorespiratory 002 and the carbons of glycine. When 14 1[ C]glycolate was added, label appeared in the same products as with 2[14C1glycolate but more label was found in compounds that would be labeled by addition of 14002 for CO2 fixation. The labeling pattern of glyceric acid support the use of photorespiratory CO2 from the C1 carbon of glycolate (28). Effect of NH4: on metabolism. When AOA was present before the ammonia was added, glycolate was excreted but not when ammonia was present before the AOA to prevent enzyme inhibition. Addition of methylamine during the experiment also decreased glycolate excretion. Addition of nitrogen facilitates glycolate metabolism. T ' -- .~ .4“; ‘ ...‘.".a‘-Lo<”“' 7 CHAPTER 3 THE EFFECT OF AMINOOXYACETATE ON CO2 FIXATION AND GLYCOLATE METABOLISM BY WHOLE LEAVES AND ISOLATED CHLOROPLASTS AS COMPARED TO CHLAMYDOMONAS REINHARDTII Introduction Aminooxyacetic acid (AOA) has been used to inhibit the activity of enzymes requiring pryridoxal phosphate (PLP) by forming a Schiff base with pyridoxal phosphate (29). The effects of AOA have been described for aminotransferases (29), phenylalanine lyase affecting phenyl propanoid compounds (30), 1-aminocyclopropanecarboxylate synthase affecting ethylene and polyamine synthesis and epinasty (31, 32), glycine decarboxylase (33), and nitrate reductase (34). The severe inhibition of C02 fixation by higher plants treated with micromolar concentrations of AOA has been reported (35). However, even 2 mM AOA did not inhibit CO2 fixation by algae, although glycolate excretion by Chlamydomonas (l7) and Chlorella (18) was increased. AOA inhibited glycolate metabolism in algae presumably by blocking the transamination of glyoxylate to glycine but it 32 ,1 *7 t—q——————_—u—_———u_flfl‘jg.rw— 33 is unknown if this effect in the algae were the basis for its severe inhibition of photosynthesis in higher plants. Jenkins et al (35) raised the question of effects of AOA on reactions in the chloroplasts. In an attempt to answer that wuestion, the effects of AOA on CO2 fixation and glycolate accumulation were compared in leaves, isolated chloroplasts and algae. Chloroplasts are capable of glycolate synthesis but not of its subsequent metabolism. Synthesis of glycolate occurs when Rubisco adds oxygen to ribulose-bis-P instead of C02. This reaction, known as the oxygenase activity of Rubisco, results in the formation of one molecule of phosphoglyceric acid and one molecule of phosphoglycolate. Phosphoglycolate is hydrolyzed by phosphoglycolate phosphatase in the chloroplaSt to glycolate which moves to the peroxisomes and mitochondria for further metabolism. An effect of AOA on chloroplast fixation might be due to an effect on 2 glycolate synthesis, possibly by affecting Rubisco or phosphoglycolate phosphatase. Methods and Materials Whole leaves Barley, corn and peas were grown in vermiculite and watered with half strength Hoagland solution. Tobacco was grown in soil under greenhouse conditions with 16 hr of supplemental fluorescent lighting. 34 Soybeans were grown in perlite in environmental chambers. Leaf sections of about 20 cm2 were cut from leaves of mature tobacco, soybean, pea and spinach plants. The first leaf on a young corn or barley plant was used for experiments. The basal and of the leaf was submerged in 1 ml of test solution in a 1.5 ml microfuge tube. Leaves were allowed to take up test solutions for 20 min under room light and temperature before they were placed in a sealed chamber at room temperature. Illumination from a slide projector (1000 uE/s/m2) was filtered through a solution of CuSO4. After a 5 min period of preillumination, [14CJNaHCOU3 was injected through a serum stopper into a dish of 2 N HCl to generate 330 ppm CO Leaves were quickly removed after 2. 5 min of CO2 fixation and placed in boiling 80% alcohol. An extraction in boiling 20% alcohol followed, the extracts were combined, and concentrated for counting and chromatography. Chlorophyll fluorescence of leaves was recorded on a Nicole osscilloscope connected to a fluorometer probe (36). Isolation of chloroplasts and measurement of $3992 fixation Chloroplasts were isolated from market spinach and intactness, chlorophyll and 002 fixation were determined (37). C02 fixation was determined in a buffer adjusted to pH 8 with NaOH containing 300 mM sorbitol, 10 mM bicine, 2mM EDTA, 1 mM MgCl 1 mM MnClZ, 50 mM sodium pyrophosphate, 14 2’ lmM ribose-S-phosphate and 0.5 mM ADP. [ C]NaHCO was 3 added to the chloroplast suspension in flattened test tubes 35 in a glass water bath at 2200. They were illuminated with light from a slide projector filtered through CuSO4. Measurement of light reactions Light—dependent pH changes by thylakoids of lysed chloroplasts were measured by a pH meter interfaced with an expanded scale recorder (38). Uncoupling of electron transport to photophosphorylation was measured by using the ferricyanide reduction assay for chloroplast intactness with the oxygen electrode (39). Ferricyanide and pyocyanine were used to accept electrons from the photosystems. Addition of NH4Cl uncoupled the production of ATP from the production of oxygen, increasing the rate of ferricyanide reduction. Glycolate excretion from chloroplasts The suspending medium was separated from the chloroplasts by centrifugation and applied to Dowex 1 columns in the acetate form to remove interfering compounds (40). Glycolate was eluted from the columns with HCl and determined by the Calkins assay (21). Chromatography Samples were chromatographed on Whatman No. 1 paper in two directions using phenol/water and butanol/proprionic acid/water (23). Papers were sprayed with 0.5 M NaHCO3 before they were completely dry to prevent volatilization of glycolic acid. Chromatograms were exposed to X—ray film and compound identificatioon was based upon a standard chromatographic map. Spots were cut out, eluted in water and radioactivity determined by liquid scintillation counting. Enzyme assays Rubisco carboxylase activity from To-‘hir av ',,—— , 36 14 chloroplasts was assayed as acid stable counts after CO2 fixation (37). Oxygenase activity was measured by the rate of 02 uptake with an oxygen electrode (41). Phosphoglycolate phosphatase activity was assayed by measuring phosphate liberated from phosphoglycolate incubated with lysed chloroplasts (42). Materials 14 C]NaHCO was obtained from Research Products [ 3 International, Corp. Results Effects of Aminooxyacetate on Photosynthesis by Whole Leaves Effects of AOA on CO2 fixation by leaves. CO2 fixation by whole leaves incubated with AOA for 20 min was severely inhibited. The 002 fixation by barley, corn, peas, tobacco and soybean leaves treated with varying AOA concentration is shown in Figure 7. Barley and corn were the most sensitive plants to AOA. Since corn is a C4 plant and because 03 and C plants have different CO compensation points which are 4 2 an indication of glycolate metabolism, the effect of AOA was tested on these plants at different 002 concentrations. At low CO2 levels there is more glycolate synthesis than at high 00 However, there was noo difference in inhibition of 2. 002 fixation by AOA relative to controls in either tobacco or corn leaves exposed to 100, 400,or 1000 ppm CO (Figure 2 37 Figure 7. Percent CO2 Fixation of Leaves Treated with Aminooxyacetate. The basal end of leaves were placed in tubes containing the AOA solution for 20 min before exposure to 330 ppm 14CO2 for 5 min. (Corn (I) Barley (.)' Peas (D) , Tobacco (O ), Soybean (A )) 38 100.01 I D 75.0 a .. 50.0—I €02 Fixation (V; Control) 25.0 0.0 2.0 4l0 6.0 [AOA] (mlvl) Figure 7. 39 Figure 8. Percent C02 Fixation of Leaves Treated with Aminooxyacetate at Different Levels of C02. Leaves of corn or tobacco were treated with 0.1 mM AOA for 20 min before exposure to 100, 400, or 1000 ppm 14CO2 for 5 min. 4O d/N/ \..>\.3x -- .Ee / / sews/Wes .1 , - _ -W «444,4..\A.4..4-..3\4...«.»«3qaxwu A/agaaaquqqa . .,_..,.)...3.,. ...._ ...\ \. \ //\< v. x ._. . xx. \3.\/..\ \ x _ . f: _..;.:,...,_..4,..m .g 3.... 3 K 33. .. .3..3_,_3.< 3.3 3. ...../.. 3. N3»...3.3..3....\.<<.NK . .4 Wlfl, flu I C All 100— Mu a M _ O .U PU. O 8 6 d- 04 90.3.3.8 NV 35:3: 030w C Q Q CLOQ ppm CO2 Figure 8. 41 8). Because corn and barley are grasses it is possible the monocot plants may be more sensitive to AOA than the dicot plants. Other compounds that inhibit glycolate metabolism were examined for an effect on C0 fixation by leaves. Pyridine 2 hydroxymethane sulfonate (HPMS) inhibits glycolate oxidase, aminoacetonitrile (AAN) inhibits glycine decarboxylase and aminotriazole inhibits catalase. None of these had an inhibitory effect on CD fixation comparable to AOA (Figure 2 9). The products of 002 fixation after treatment with AOA were chromatographed (Table 3). However due to great inhibition of CO2 fixation the amount of 140 incorporation into all of these products was low. With AOA there was an increase in glycolate, except in peas, as expected from the algal data. The increase in glycolate in corn was very pronounced since corn leaves normally have a small pool of 14 glycolate. Percent distribution of C into glycolate increased while glycine and serine decreased, sugar phosphates increased and sucrose decreased. The effect of AOA on 14C distribution among products of 002 fixation was somewhat similar in plants and algae and indicated a severe inhibition of glycolate metabolism. The major difference between plants and algae was the inhibition of total CO2 fixation Effects of AOA on light reactions in whole leaves. Although AOA inhibits enzymes by forming a Schiff base with 42 Figure 9. Percent CO2 Fixation of Control by Leaves Treated with Inhibitors of the 02 Cycle or Uncouplers of Electron Transport. Inhibitors were AAN (I), aminotriazole (X) and HPMS (A) and uncouplers of electron transport were methylamine (O) and NH4Cl (6)). Basal ends of leaves were submerged in test solution for 20 min before exposure to 14002 at 330 ppm for 5 min. The curve for AOA treatment (0) is shown for comparison. 002 Fixation (75 Control) 100.0 750-4 50.0~ 25.0- 0.0 43 AAN aminotriazole ‘methylamine _ HPMS O NHaCl AOA 0.0 i 2.0 410 630 8.0 10.0 [Inhibitor] (mM) Figure 9. 44 NM Ln 0 CONMLONNO’) v— mo smabmm E m: m.: m; a o.m m— mama mm4 so coaumxwm moo do mscsoo;m mcos< :o3u3nflgom3a 0:3 mmpmcomozdugmmsm mUHOm 03cmmgo mumaoosam mCHme + meaosam muficm 0:35m mmogozm poacoLm .m maoms 45 PLP, AOA could also function as a protonated amine at physiological pH. Other amine compounds such as methylamine and NH4Cl are known inhibitors of photosynthesis by uncoupling electron transport in the thylakoids. Methylamine and NH4Cl at the same concentrations as used for AOA did not greatly affect CO2 fixation in whole leaves indicating that effects on electron transport were not the cause of ADA inhibition of 002 fixation. To further examine a possible inhibitory effect of AOA on electron transport, chlorophyll fluorescence of whole leaves treated with AOA was measured. Results showed no change in initial fluorescence, but there was a slight difference at longer (40 sec) times Figure 10). Effects of Aminooxyacetate on Photosynthesis in Isolated Chloroplasts Effects of ADA on light reactions in chloroplasts. Compounds which act as electron transport uncouplers increase the rate of 02 evolution and will affect proton pumping across thylakoid membranes by decreasing the pH gradient necessary for the production of ATP. The addition of AOA to lysed chloroplasts increased ferricyanide reduction as measured by 02 evolution almost as well as the standard uncoupler NH4Cl (Figure 11). AOA decreased the change in pH with the electron acceptor pyocyanine but not as dramatically as NH4Cl (Figure 12). Effects of AOA on CO2 fixation by isolated 46 Figgure 10. Chlorophyll Fluorescence Transients of Whole Leaves. Corn or tobacco leaves were treated with 0.1 mM AOA for 20 min prior to measuring relative fluorescence. Fluorescence of nontreated leaves is shown for comparison. A. Relative fluorescence of corn. B. Relative fluorescence of tobacco. 47 1 mM AOA N 40 sec -——- 0.1 mM AOA ' ontrol 40 sec Figure 10. 48 Figure 11. Effect of Aminooxyacetate on Ferricyanide Reduction by Lysed Chloroplasts. Oxygen evolution by lysed chloroplasts is shown; arrows mark addition of 10 mM AOA. Response with 5 mM NH Cl is added for comparison. 4 02 Evolution (change in uM) 49 100 751’ U" O l 25" 10 mM AOA / “t i i i 0 l 3 5 time (min) Figure 11. 50 Figure 12. Light-dependent pH Change by Lysed Chloroplasts. With 0.1 mM pyocyanine as the electron acceptor, the change in medium pH induced by lysed chloroplasts in 0.5 mM bicine pH 6 was measured. Arrows indicate light on or off. control (I); 5 mM NH4Cl (’0): 10 mM AOA (D) Change in pH Si Q—-Off 0007 '. 0006'”- 0.05m 0.04m 0.03ii + 10 mM AOA 0.02mJ Control 0001-”- , / 5 IBM NHACl 0.00~» ‘——*’J TOD. n g ; r : O l 2 time (min) Figure 12. 52 chloroplasts. The severe inhibition of CO2 fixation in leaves could be due to a direct effect on 002 fixation in the chloroplasts. To consider this possibility, the effect of AOA on 002 fixation by isolated chloroplasts was examined. When 0.1 to 10 mM AOA was present in varying concentrations in chloroplast suspensions, 002 fixation was increased and the increase was greater with higher levels of AOA (Figure 13). When the bicarbonate concentration was varied, AOA had a greater effect at the higher bicarbonate concentrations (Figure 14). With 10 mM AOA there was a two-fold increase in fixation at 10 mM bicarbonate and no increase at 2 mM bicarbonate. With 1 mM AOA there was only a 20% increase in fixation at 10 mM bicarbonate. The stimulation of 002 fixation by AOA was not as pronounced at pH 6 where rates of fixation are lower than at pH 8 (data not shown). AOA stimulation of CO2 fixation by chloroplasts was not due to an effect on Rubisco (Table 4). Isolated chloroplasts were lysed and both the carboxylase and oxygenase activities were assayed in the presence of AOA. The activity of phosphoglycolate phosphatase was not altered by AOA (data not shown). Effects of AOA on glycolate excretion from chloroplasts. Since AOA increased glycolate excretion from Chlamydomonas, AOA might increase glycolate excretion from chloroplasts. AOA did not increase glycolate excretion from isolated chloroplasts (data not shown). 53 Figure 13. Time Course of CO2 Fixation by Isolated Chloroplasts with Aminooxyacetate. Chloroplasts were given 10 mM sodium bicarbonate in the presence of varying concentrations of AOA. Buffere is bicine pH 8 containing sorbitol and salts as given in Methods and Materials. (OmM AOA (A), 0.1 mM (0), 1 mM ([3) and 10 mM (I)) 54 24uO-1 10 mM AOA 2013a i E i 0 1 mM AOA Cn 1613~ E \ L 0.1 mM AOA .0 (D 25 La 12.0~J N C) o 2 8.0—1 0 mM AOA o E 2L 4134 0'0 i F l I i C) 2 4- £3 8 10 finm Unm) Figure 13. 55 Figure 14. Effect of Aminooxyacetate on the Time Course of CO2 Fixation by Isolated Chloroplasts with Bicarbonate Concentration. Chloroplasts were given 10 mM AOA at 1 and 10 mM sodium bicarbonate. Buffer is bicine pH 8 containing sorbitol and salts as given in Methods and Materials. (1 mM bicarbonate (A ) , 1 mM Bicarbonate + AOA (o ), 10 MM bicarbonate (E1) and 10 mM Bicarbonate + AOA (I )) 56 SAD—i 10 mM HCO3- + AOA 4J3— IE L) CD E _ \ 3.0_ 10 mM HCO3 13 . Q) 25 L1. (\1 8 2.0.. / _ 1 mM Hco3 + AOA 12 O E :k 1 mM HCO - 1.0- 3 CLC) rt i' 'r T’ i O .2 4’ 6 8 ‘10 time (min) Figure 14. 57 Table u. Effect of Aminooxyacetate Carboxylase/Oxygenase Activity Activation Assay Conditions Conditions Standard Standard + 1 mM AOA Standard + 1 mM AOA Standard+ no HCO§, no MgC12 Standard + 1 mM AOA Standard+ Standard activation conditions were 100 mM Bicine, 20 mM MgC12, on Purified Ribulose-P2 Carboxylase Activity umole C02/min/mg i i 0 0 .20 .06 .20 .724 .601 Oxygenase Activity umole OZ/min/mg 0.110 0.0968 0.115 0.0523 0.0445 10 mM NaHCO§, 1 mM DTT and 0.2 mH EDTA. Enzyme was incubated for 10~3O min at 30°C at pH 8.2 Standard assay mix includes 100 mM Bicine pH 8.2, 20 mM MgC12, 10 mM NaHCOE, 5 mM ribulose-PZ, 1 mM DTT and 0.2 mM EDTA. + Concentration of AOA was an uM carryover from activation. 58 Effects of Aminooxyacetate on CO Fixation by Algae 2 14002 fixation was not altered by the presence of 2 mM AOA with either air- or In a previous report total COz-grown Chlamydomonas reinhardtii(17). AOA blocked glycolate metabolism and glycolate was excreted into the medium by the algae. The distribution of label among photosynthetic products was altered in a manner similar to that of higher plants. Discussion Effects of Aminooxyacetate on C0 Fixation by Leaves 2 Effects of AOA on light reactions of whole leaves. AOA did not inhibit CO2 fixation by isolated chloroplasts or by algae; however, AOA severely inhibited CO fixation by whole 2 leaves. AOA does not appear to act as an inhibitor to electron flow since initial chlorophyll fluorescence was not altered. At the longer times (40 seconds), a change in fluorescence probably reflected alterations in carbon metabolism. The amine properties of AOA were probably not inhibiting 002 fixation as inhibition was not observed with similar concentrations of methylamine and NH4C1. Effects of AOA on 002 fixation by whole leaves. AOA is an inhibitor of pyridoxal phosphate requiring enzymes, especially of alanine and aspartate aminotransferases (43). 59 Therefore it would be expected that production of 14C labeled amino acids during 14CO2 fixation would decrease and organic acid levels increase. The percentage of 14C in glycine, serine and alanine did decrease and the percentage in glycolate increased. The oxidation product of glycolate is glyoxylate which is aminated to make glycine and glycine is further metabolized to serine. This amination step was apparently inhibited by AOA as expected. Jenkins et al (35) reported similar results in 14C product distribution when barley and maize leaves were treated with AOA. They observed that corn leaves accumulated more [14C]glycolate than barley leaves and that barley leaves were more sensitive to AOA than corn leaves at much lower concentrations of AOA than used in this report. The changes in distribution of photosynthetic products with AOA treatment is consistent with inhibition of aminotransferases. AOA has been shown to inhibit utilization of glucose due to inhibition of the aminotransferases (44). It has been reported that with inhibition of aminotransferases, the exchange of reducing equivalents between mitochondria and cytosol via the malate-aspartate shuttle is reduced. This leads to a decrease in levels of intermediates in the TCA cycle and to lowered ATP levels (45). Accumulation of glycolate itself during photosynthetic CO2 fixation may not be the cause of inhibition. Jenkins et al (35) reported barley to be more sensitive to AOA than 60 corn yet the percentage of counts accumulated in glycolate was less than in corn. The C2 cycle inhibitor HPMS causes glycolate to accumulate but did not cause inhibition of 002 fixation. Also there was no change in toxicity at higher CO2 levels which should reduce glycolate synthesis. It is possible that accumulation of glycolate can contribute to the toxicity of AOA in combination with inhibition of aminotransferases involved in other metabolism. Glycolate is oxidized to glyoxylate which if not converted to glycine is futher oxidized to oxalate and hydrogen peroxide or to 002 and formate. Oliver (46) reported that in the absence of amino acid donors, more CO2 was released by the pathway of direct oxidation of glyoxylate than from the pathway of glycine decarboxylation. Inhibition of the aminotransferases would simulate a depletion of amino acid donors. Different species have different activities of formate dehydrogenase (47) and thus some may be more sensitive to increases in formate levels. Formate has been shown to inhibit 002 fixation in isolated chloroplasts (37). Alternatively, glyoxylate itself could react to form adducts with sulfhydryl groups as discussed by Hamilton (48). Effects of Aminooxyacetate on Photosynthesis in Isolated Chloroplasts Effects of AOA on light reactions. Since AOA increased 02 evolution in the ferricyanide assay and decreased the 61 light-dependent rise in pH, AOA appears to have acted as an uncoupler of electron transport. It is not clear how compounds like AOA, NH Cl and methylamine could function as 4 uncouplers of electron transport and still stimulate CO2 fixation by isolated chloroplasts. It is possible that AOA may function to some extent as an uncoupler but this may not be its primary effect. In some cases uncouplers may not affect CO2 fixation if the dark reactions are limiting in photosynthesis; partial inhibition of the light reactions would still provide enough ATP and NADPH for CO fixation. 2 The lack of an effect by AOA on the light reactions in isolated chloroplasts is consistent with the lack of change in initial in leaf fluorescence. Effects of AOA on 002 fixation by chloroplasts. The inhibition of CO2 fixation by leaves is not due to an effect on CO2 fixation within the chloroplast or to an effect on the enzymes Rubisco and phosphoglycolate phosphatase. AOA did not affect activity of either of these enzymes in extracts and did not inhibit 002 fixation by isolated chloroplasts. It would seem that AOA inhibition of 002 fixation was effecting changes occurring in other cell compartments. The response of isolated chloroplasts to AOA was more similar to the algae than leaves to AOA treatment. The stimulatory effect of AOA on C0 fixation by 2 isolated chloroplasts is postulated to be by its action as a base. A similar increase in CO2 fixation was observed when chloroplasts were incubated with 10 mM NH4C1 and 62 methylamine. The umoles fixed at the 2 min time point of treated chloroplasts was greater in magnitude than controls but if subsequent rates were plotted, the slope was the same in treated and control chloroplasts (data not shown). Typically there is a lag in CO fixation during which the 2 levels of the reductive cycle intermediates increase. Ribose—S-P and ADP were added to the buffer system to shorten this lag. During photosynthesis changes in pH of the stroma of the chloroplast are believed to occur with the stroma pH rising to 8. Enser and Heber (48) discuss the effect of acids on the pH gradient between stroma and thylakoid; acids inhibit the alkalinization of the stroma and inhibit the sugar bisphosphatases for the regeneration of the 03 cycle intermediates. In contrast to acids, bases permeable to the membrane raise the stroma pH sooner thus shortening the lag. This case has been reported for NH4Cl by Benedetti et al (50) and Tilbury et al (51). Benedetti et al (50) also observed a stimulation of CO2 fixation at higher concentrations of bicarbonate as wasobserved with AOA. Both NH4Cl and methylamine are known to be uncouplers of electron transport but in this case their mode of action is postulated to be by assisting in the alkalinization of the stroma of the chloroplast. Glycolate excretion by chloroplasts. The lack of an effect on glycolate excretion by chloroplasts is consistent with the lack of effect of AOA on Rubisco and phosphoglycolate phosphatase activity. The effect of AOA is 63 implied to be on glycolate metabolism in the peroxisomes and/or mitochndria. Effect of Aminooxyacetate on CO2 Fixation by Algae An explanation for the resistance of algae to initial toxic effects of AOA during the time (up to 3 hours) of the experiment has been that the algae were able to excrete glycolate into the medium. In higher plants glycolate must remain in the cell. Perhaps glyoxylate accumulation does contribute substantially to the short-term toxicity of AOA treatment before the full effects of disruption of metabolism due to the inhibition of PLP—requiring enzymes are developed. CHAPTER 4 THE MECHANISM OF TRANSPORT 0F GLYCOLIC ACID BY CHLAMYDOMONAS REINHARDTII Introduction The amount of glycolate excretion has been described and the conditions favoring excretion determined (16). However, little is known about the actual transport process of glycolate. The saturation of glycolate uptake has been claimed (15, 16) but as pointed out, metabolism has often confused the process of transport. Published data on glycolate uptake by algae have described the process over long periods of time (hours) and as has been shown in Chapter 2, these long time experiments were measuring glycolate metabolism rather than the kinetics of uptake. Questions related to the excretion of glycolate are why and how does it occur. Why it occurs is related to the balance between synthesis and metabolism; excretion occurs when synthesis exceeds metabolism. The question still remains why the cell would excrete rather than sequester molecules of fixed carbon. This is related to the 64 65 specificity of excretion for glycolate. Glycolate is excreted by the alga Chlamydomonas reinhardtii but D-lactate will accumulate under anaerobic conditions and will remain in the cell to be metabolized when the cell becomes aerobic again (52). Without understanding why the cell excretes glycolate, the question of how it is excreted has been addressed. Glycolate, because of its pKa of 3.8, exists primarily as an anion at physiological pH and it is generally believed that anions are not permeable across the cell membrane. Therefore, it is postulated that there exists a protein transporter for glycolate which may be essential to cell survival and must be present constitutively. Chlamydomonas reinhardtii are capable of heterotrophic growth in the dark with acetate as the carbon source. Based on the structural similarities between glycolate and acetate, an hypothesis for the glycolate transporter is that it will use monocarboxylic acids in general. Transport systems may be classified as active, requiring energy to concentrate a molecule, or as passive. Facilitated diffusion may be described as a passive process. The substrate molecule is not accumulated and energy is required only in the maintenance of the cell rather than for the transport process itself (53). To demonstrate the existence of a facilitated diffusion system, certain criteria must be met (54). The system should saturate with substrate, have time and temperature dependencies different 66 from simple diffusion, and be subject to inhibition by protein modifying agents and to competition by substrate analogs. As Stein (54) states, some of the criteria are more important than others and even then they may not be exclusive, eg. a system may saturate only at extremely high, inaccessible concentrations and this may actually be an advantage to the cell in the transport of molecules which are carbon sources. There is little literature on the tranport of monocarboxylic acids in plants. Some information is available on organic acid uptake in plants but most reports are concerned with the transport of dicarboxylate anions into mitochondria, chloroplasts, and tonoplasts. Little has been written about monocarboxylate acids, partly because it has been felt that they diffused as the undissociated form across the membrane. However, the effective concentration of the protonated species may not be great enough to prbvide sufficient transport by diffusion (55). Raven (56) reports that acetate is moved by diffusion in the cyanobacteria but notes that the movement of glycolate and lactate are not as readily categorized. It is not clear that the transport of acetate is by diffusion. Moore and Wilson (57) found acetate transport into plant mitochondria was either proton symport or hydroxyl antiport in contrast to acetate transport into membrane vesicles from an alkaliphilic Bacillus sp. which was found to be dependent on a sodium gradient with sodium as the co-transport species (58). 67 Ihlenfeldt and Gibson (59) state that the acetate is rapidly metabolized in cyanobacteria and that it is difficult to separate transport from subsequent metabolism. In the older literature on glycolate transport, time points of hours were used and the saturation described (15, 16) is more likely that of metabolism rather than transport. Glycolate transport from the chloroplast was concluded to be by diffusion until experimental time points less than 10 seconds were measured (11). Then a protein transport molecule was indicated. A lactate transport system that will not use pyruvate and that is different from the dicarboxylate system is induced in diatoms grown with lactate (60). The transport of lactate and glycolate has been extensively described for erythrocytes (61). Movement of these acids is accomplished by three components: diffusion, a nonspecific carrier and a specific carrier. By use of specific inhibitors, the proportion moved by each component was determined. Pyruvate will compete with lactate for both the specific and nonspecific carriers (62). The transport of pyruvate into mitochondria has been much more controversial (63, 64). At concentrations above 5 mM, there may be a diffusion component to pyruvate transport, but at lower concentrations pyruvate is transported by a system separate from the dicarboxylic and tricarboxylic acid transporters. The system will exchange pyruvate with acetoacetate but not with acetate. The 68 existence of a pyruvate monocarboxylic acid transporter on the cell membrane has been suggested (55). The transport of hexoses and amino acids has been examined in the green alga Chlorella by Komor (65, 66) and found to be electrogenic, usually as proton symport. In contrast, monocarboxylate acid uptake, at least in mitochondria and erythrocytes, appears to be as an acid uniport or as an acid anion—hydroxyl antiport. In either case neutrality is maintained and the external pH is increased. The distinction between movement with a proton or in exchange for a hydroxyl can be made kinetically (67). Glycolate exchange across the chloroplast membrane can occur either with a proton or in exchange for glycerate in keeping with the flow of carbon through the photorespiratory cycle (68). By comparing glycolate uptake by Chlamydomonas reinhardtii with the same process by chloroplasts as a photosynthetic organelle and by erythrocytes as a whole cell, it was anticipated that knowledge of the mechanism of glycolate excretion could be gained. Studies of uptake of glycolic acid by the algae is complicated by transport across organelle membranes as well as across the plasma membrane. No attempt has been made to distinguish between these processes. The uptake rather than the efflux of glycolate was measured and the assumption was made that efflux and influx occur by the same process. To measure efflux, the algae would have to be preloaded with 69 [14C]glycolate and there was concern that metabolism of this glycolate would occur before efflux could be measured. Methods and Materials Growth of algae Chlamydomonas reinhardtii strain 90 was grown in 1 l of the minimal medium of Sueoka (19) in 3 l Fernbach flasks at room temperature with shaking, constant illumination and aeration with C02-enriched air. Cells were harvested by centrifugation and washed once with water before resuspension in buffer. Chlorophyll was determined by the method of Arnon (22). Erythrocytes Erythrocytes were obtained by venous puncture into a vacutainer and blood was allowed to clot on glass beads. After centrifugation, cells were resuspended in 6.5 mM sodium phosphate buffer pH 7.4 containing 4.5 mM glucose and 140 mM NaCl to a hematocrit of 50% and kept at 4°C (69). Protein was determined by a modified Folin-Lowry procedure (70). Glycolate uptake Uptake was measured using the silicone oil centrifugation method as modified by Howitz and McCarty (71). Short time measurements of about 2 seconds were accomplished by pelleting cells in an upper layer of 200 ul through a 50 ul layer containing [14C]glycolate in buffer containing 10% Percoll into 20 ul 4N NaOH. In longeer time points, [14C]glycolate was added to the 200 ul of cells 70 before centrifugation. The cells, [14C]glycolate and NaOH layers were separated from each other by layers of AR 20 and AR 200 silicone oils mixed 2:1 and 1:1, respectively. Label in the cell pellet was determined by liquid scintillation counting. Internal volume was determined by incubation with 3H20 and [14C]sorbitol and calculations of inteernal concentration was made using the Siloil program written by Dr. H. David Husic. Linear regression was performed using the Datalab program by EMF Software. Materials [14 C]sorbitol were from NEM. Percoll was from Pharmacia and C]glycolate was obtained from ICN and 3H20 and [14 Wacker silicone oils AR 20 and AR 200 were a gift from SWS Silicones Corp., Adrian, MI. Results Saturation When internal concentration (uptake) was plotted against external concentration, saturation was not observed with Chlamydomonas or erythrocytes (Figure 15). Glycolate was not accumulated in the cells but reached an equilibrium between internal and external concentrations. The uptake of acetate, formate and D~lactate by Chlamydomonas also came to equilibrium rapidly (Figure 16). The internal concentration was dependent on the cell (as measured by chlorophyll) 71 Figure 15. Glycolate Uptake by Chlamydomonas reinhardtii or Erythrocytes. Control (I) or 10 mM NEM—treated (0) human erythrocytes (40% hematocrit) or C02-grown algae (300 ug chl/ml) (C)) were centrifuged through a buffer layer of 6.5 mM sodium phosphate pH 7.4 containing 4.5 mM glucose and 140 mM NaCl and [14C]glycolate at 40C for 2 sec. 72 1513- //p , Chlamydomonas 1213- /"\ :2 E V F"! g 9.0-« 2 C) 0 .25 CD l—_—J o __ 6A3— E L. :9 ’ erythrocytes E 3JD~ erythrocytes + NEM CLO "- 1. ' I T 7 Oil 213 413 6A) 813 External [Glycolate] (mM) Figure i5. 73 Figure 16. Uptake of Monocarboxylic Acids by Chlamydomonas. COZ-grown algae (300 ug chl/ml) were centrifuged through a buffer layer of 20 mM Hepes pH 7 containing l4C—labeled acid at 4°C for 2 sec. (glycolate (O), D~lactate (I), acetate (¥~),formate (0 )) Internal Concentration (mM) 74 15-- o glycolate lactate lO-- I acetate formate 5-- a x o / 0.. of4 Us 410 10:0 External Concentration (mM) Figure 16. 75 concentration (Figure 17). Concentration dependence was not done with erythrocytes. With the algae, the total glycolate (nmoles) inside the cells increased with increasing cell volume (figure 17A) but the concentration (mM) decreased with increasing cell volume (Figure 17B). The number of nmoles taken up increased from 4 to 18 with an increase from 0.5% to 5% cell suspension. With the dilute 0.5% cell suspension, there was about 12 mM internal [14C]glycolate at 12 mM external [14C]glycolate. When the cell suspension was increased tenfold to 5%, the internal concentration was only 1 mM at 12 mM external [14C]glycolate. Time Course and Temperature and pH Dependencies The uptake of glycolate was very rapid, reaching equilibrium within the time of the shortest technically possible time point (about 2 seconds) at 40C The uptake of glycolate was more rapid than the uptake of benzoic acid by diffusion (Figure 18). No difference in glycolate uptake at 4°C and 22°C was detectable. Uptake of benzoic acid was more rapid at 22°C than at 4°C as expected for a diffusion process (Figure 18). There was no difference in glycolate uptake in the light or in the dark. The uptake of glycolate and benzoic acid at different external pH was compared (Figure 19). Benzoic acid uptake was strongly dependent on pH but glycolate showed a plateau over the same range despite similar pKa values. 76 Figure 17. Uptake of Glycolate by Different Concentrations of Cell Suspension of Chlamydomonas. COZ—grown algae were resuspended to 5% (A), 2.5% (B) and 0.5% (0) w/v and centrifuged through a buffer layer of 25 mM Hepes pH 7 containing [14C]glycolate at 4°C for 2 sec. A. Label in cell pellet is expressed as nmoles of [14C]glycolate. B. Label in cell pellet is expressed as concentration (mM) of [14C]glycolate. Internal Glycolate, nmoles Internal [Glycolate] (mM) 18.0-1 15.0— 12.0- 9.0~ 6.0~ 3.0~ 0.0 77 2.5 Z 0.0 18.0— 15.0~ 12.0- 9.0a 5.0-1 3.0-4 oo, oo 1 ‘ ' 1 ' ' l r ‘ F 3.0 6.0 9.0 12.3 External [Glycolate] (mM) 0.57. f T fl lio l . ' 1 ‘ ' l 3.0 6.0 9.0 External [Glycolate] (mM) Figure l7. 78 Figure 18. Time Course of Glycolate Uptake. COZ-grown Chlamydomonas (50 ug chl/ml) cells were incubated with 1 mM [14C]glycolate and with 0.82 uM [14C]benzoate in 20 mM Na citrate pH 3, 4, 5 or 20 mM Mes pH 6 for times indicated. The time course shown is for glycolate in 20 mM Hepes pH 7 but the time course was the same over the pH range. Internal concentration of benzoate varied with external pH as shown; uptake at pH 5 and 6 was loo low to be shownm on this scale. Uptake of glycolate was the same at 4°C and 2200 but benzoate reached equilibrium quicker at 22°C than at 40C. The benzoate curve at pH 4 at 22°C is shown at 10 times the internal concentration for ease in comparison. (glycolate (o ), benzoate, pH 3 ()4), benzoate, pH 4 (I), benzoate, pH 4, 22°C (C>)) 0.5-- Q ‘3 Internal Concentration (mM) 0.1-. 79 0.3» 0.2» 0 ~30 5 O glycolate ~ 0 4 o x ° “.0 3 benzoate, pH 3 q ‘02 benzoate, pH 4.22‘ *0 - .01 ' benzoate, pH 4 4 so so Iéo Time (s) Fl'QUre l8. Internal Concentration (mM) 0'0 80 Figure 19. Uptake of Glycolate as a Function of External pH. COZ-grown Chlamydomonas (50 ug chl/ml) were centrifuged through 0.5 (O) and 5 uM (O) [14C]glycolate or 0.82 uM [l4CJbenzoate (u) at 4°C in 20 mM Na citrate pH 3, 4 and 5, 20 mM Mes pH 6, 20 mM Hepes pH 7 and 20 mM Epps pH 8. 81 20.00-— 16.00- A S benzoate I v C: O 1:3 12.00-3 C L. .4.) C (1) O E Q 8.004 '5 C L. B o 5 uM glycolate C o _ 4.009 o oi o 0.5 uM glycolate 0.00 - ; ‘\; 7' pH Figure l9. t T r 1 r T 1 1 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 82 Inhibition Glycolate uptake was inhibited by treatment of Chlamydomonas cells with 10 mM NEM or with 1 mM KCN for 10 min at 4°C (Figure 20). The inhibition by NEM was observed only at cell concentrations greater than 200 ug chl/ml; KCN treatment was tried only at high cell concentration. Inhibition by NEM was greater than with KCN. Erythrocyte uptake of glycolate was severely inhibited by NEM treatment (Figure 15). Decreased glycolate uptake by competition wtih structural analogs such as acetate, lactate and glycerate was not observed. Stimulation Addition of 0.2 ng valinomycin/ml and 100 mM KCl to the glycolate layer increased uptake (Figure 21A). The stimulation was optimal with 60 mM KCl with 0.2 ng valinomycin/ml present (Figure 218). Stimulation occurred with high KCl (100 mM) alone present in the glycolate layer but ionophore alone decreased uptake (Figure 22). As with the inhibition by NEM, the stimulation by valinomycin/KCl was dependent on cell concentration. The uptake of glycolate by cells at different chlorophyll concentrations treated with valinomycin, KCl, valinomycin and KCl and NEM was compared (Figure 23). The percent change from control at each chlorophyll concentration is given above the bar. 83 Figure 20. Inhibition of Glycolate Uptake. COZ-grown Chlamydomonas (300 ug chl/ml) were pretreated with water (0), 10 mM NEM (-) or 1 mM KCN (o) for 10 min at 4°C before centrifuging through [14C]glycolate in 20 mM Hepes pH 7 at 4°C. 84 control . 24.0—1 /i\ 20134 E E V '75“ 1613— +4 2 O 3. E 12.0- o B E o NEM a) 8.0- ' E; KCN 4.0+ ° ° 0.0+ , r 1—5 F T 0.0 5.0 10.0 15.0 20.0 25.0 External [Glycolate] (mM) Figure 20. 85 Figure 21. Stimulation of Uptake by Valinomycin/KCl. A. Addition of 0.2 ng valinomycin/ml and 100 mM KCl to the [14C]glycolate-20 mM Hepes pH 7 layer increased uptake at 400 by C02—grown Chlamydomonas (300 ug chl/ml). B. Stimulation is dependent on potassium concentration with 0.2 ng valinomydin/m; and 0.5 mM [14C]glycolate at 4°C in 20 mM Hepes~NaOH pH 7 3; Internal [Glycolate] (mM) 86 30.00 1 valinomycin/KCl 25.00— 20.00— 15.00— controlo 10.00— ' 5.00— 0-00 1 1 1 D 0.00 2.00 4.00 6.00 8.00 External [Glycolate] (mM) 0.80— 050- ° 0.40- Internal [Glycolate] (mM) 0.20- 0~00 1 1 1 r *1 0.00 20.00 40.00 60.00 80.00 100.00 External [KCl] (mM) Figure 21. 87 HUM + mpozmocofi ”m “Homz + muonaoa0a ”H um sapwoaamum no “Cauomaoc “:Hofipw>mmn “m “Houucoo no .czosm ma HOMucoo no poached mm 0x039: .oov um HUM no Homz SE om mafia muonmoaofl so HE\mponaocoH a: «.0 mcHGMMucoo no m mm 30 mmmwm1mmz XE om CH mmeoo>HmnovHH ZE m.w~ mo nm>mH m nmnouzp pmmSMflnpcmo who: AaE\H£o as oomv macoaopwecho czoum1moo .meuaD wumHoo>ao CO mufimm paw mohozmocoH mango mo uomwwm .NN ansmflm ESL 621 203 V61 9'96 121 [V1 2'29 001 88 0 2.0-i 1 0a (ww) [epics/([9] lDUJelUI 01) Figure 22. 89 mamnuo may we mxmuas onoamo mag 03 303 004 am zmz :5 OH nuaz quEummpumna 3m “HUM SE om 0cm Ha\aao>aoafifim> m: N.o .3 “HUM :5 om .m uua\:ao>ao:fiam> ma m.o “N ”Honuaoo ”a .mCOAHMpquocoo HH>SmouoH£o accumuufiv cu pmpcmamdmmn mmaoeop>EmHno axopm1moo >9 00v up w :9 mammm1mmz 25 ON cw mumfioo>HmH0¢HH 28 m Mo excums .coflumuucmocoo Ha>nmouoH£o so 0xMHQ: «o GawymHSEaum no coflyanwncH mo mocmpcmmma .mm mpsmflm 90 ID 86111114,, 0, 912111111». 3 9'1717 1—N 8 001 111m. " 5°93 G’m 119 KXXXXXXXXXXXXXXEF. 0, L89 KERDQDQDHEKEMFn 3 6'61 1., 2 001 1111-3 "3 9'08 11., 681 We, 0. 002 WLH 3 2'21 111%... 211 001 mu N All mrm 0 06 881. c, Z82 oumumamu~n 3 17 86 XXX}... '8 001 111111,. N 0°58 '.IZ'I 1'18 811 6'28 001 3 <5. 0 N .— 0' (mm) [910100109] [balaiul Figure 23. pg chl/ml 91 Discussion Saturation Because lactate transport in erythrocytes has been reported to saturate (62) and because lactate and glycolate are transported by the same carrier in erythrocytes, glycolate uptake in Chlamydomonas was compared to erythrocytes. Under the experimental conditions used here, glycolate uptake by algae or erythrocytes did not saturate with respect to external concentration, even up to 50 mM. Hubbard (72) reported linear uptake of glycolate by erythrocyte ghosts up to 1 mM. In the literature cited, transport into the erythrocytes was measured by incubation followed by centrifuging and washing of the cells. If the equilibration of the acids across the membrane is as rapid as indicated by the silicone centrifugation method, much exchange of glycolate would have occurred during the time of centrifuging and washing of the cells, giving rise to misleading results. In fact, because of the rapid equilibration shown in the time course, saturation with respect to substrate can not be demonstrated. The existence of the chloroplast transporter was not demonstrated until uptake times of less than 10 seconds were used (11). At 10 seconds, the time course of uptake has come to equilibrium in the chloroplasts. The uptake of glycolate by 92 Chlamydomonas is more rapid than uptake by chloroplasts of higher plants. Even at the time of the shortest time point, equilibrium is reached. Discussion of saturation for glycolate uptake by Chlamydomonas has no meaning at equilibrium conditions. The equilibrium concentration dependence on cell concentration as shown in Figures 17 and 23 implies that there may be many sites of this transporter. Stein (54) estimates 0.7 to 13 x 106 sites of the glucose transporter on the erythrocyte membrane. The transport process is rapid enough that equilibrium between internal and external [14 C]glycolate is achieved. The total number of nanomoles of glycolate taken up increased with greater cell density from 4 to 18 (Figure 17A), but the number of nanomoles did not increase in proportion to the number of cells (tenfold increase) and thus each cell has fewer nanomoles in it. The concentration decreased from 12 mM to 1 mM with a tenfold increase in cell concentration. The data imply that competition exists among sites for substrate molecules and this concept is elaborated further below. Time Course and Temperature and pH Dependencies The lack of time and temperature dependencies of glycolate uptake can be used to verify that metabolism was not involved in the measurement of the transport process. A metabolic process should vary with time and temperature. Glycolate metabolism at 4°C has not been measured but 93 glycolate uptake and metabolism at 22°C has been shown (10) to increase with time and to be light dependent as well. A transport process should vary also with temperature but because glycolate uptake is so rapid, this dependence has not been observed. This makes the difference in equilibration times between transport of glycolate and diffusion of benzoic acid more pronounced. The uptake of glycolate is much more rapid than the temperature-dependent diffusion of benzoic acid. Although the saturation of glycolate uptake by Chlamydomonas was not similar to that reported for erythrocytes, the pH dependence was similar. In erythrocytes glycolate uptake was about the same over the pH range 6 to 8 (73); for Chlamydomonas the pH range was broader, including pH 3 to 9. This lack of pH dependence is an argument for the existence of a transporter. Determination of intracellular pH by permeant weak acids depends on the trapping of the dissociated anion in the cell compartment. Since the concentration of the undissociated acid will vary logarithmically with external pH, the amount of trapped anion will also vary logarithmically. If glycolate crossed the membrane only as the undissociated acid, the uptake pattern should be similar to benzoic acid. Even at low pH (3 and 4) where some diffusion would be expected, internal glycolate came to the same equilibrium value as at higher pH. This argues that the amount of internal glycolate is controlled and any glycolate that may 94 accumulate due to diffusion is excreted until equilibrium is reached. Inhibition Some inhibition of glycolate uptake by KCN may not a specific inhibition of the transport process. With reduced energy production the membrane potential of the cell is not maintained in the same manner. The uptake of glycolate is not believed to be dependent on the membrane potential directly because glycolate is not accumulated in the cell. The dependence of glycolate uptake on the membrane potential will be discussed below. The inhibition of uptake by NEM only at high cell concentrations may be related to the postulated high number of transporter sites per cell. If there is competition among many sites for substrate molecules, then decreasing the number of sites for transport will decrease transport but will also decrease the competition. If the number of substrate molecules is kept constant and the number of sites decreased, then the number of substrates per site is increased and the competition between sites is decreased. Inhibition of some sites under these conditions may not decrease uptake because of the greater availability of substrate molecules. In glycolate uptake by Chlamydomonas, the rate of uptake is not limiting. At high numbers of sites, there is greater transport. If half the sites are inhibited, transport is halved. If the number of sites is 95 reduced by one—sixth in the presence of the same number of substrate molecules, transport would be expected to be reduced by one-sixth. The total amount of transport was reduced but the internal concentration increased (Figure 17). Transport had been limited by the number of substrate but upon dilution of the cells this limitation has been decreased. Now even if half the sites are inhibited, there are sufficient substrate molecules available that no reduction in uptake is observed because rate of transport is not limiting. Other possible explanations for the lower internal concentration with higher cell density exist. One is that 2 seconds is not enough time to allow complete equilibration but preliminary evidence indicates that the time course is not different between cells at 50 ug chl/ml and 300 ug chl/ml. Another explanation is that the incomplete equilibration is a result of reduced access of substrate to transport sites due to cell aggregation. The cell count increases about a hundred fold from approximately 106 cells/ml at 50 ug chl/ml to 108 at 300 ug chl/ml. High cell density may induce cell clumping and the effective surface area would be decreased. A third explanation is substrate depletion. If the transporter exists at 104 sites/cell and there are 108 cells/ml, then there are 2 x 1011 sites in 200 ul of cell suspension. This number of transporters is 16 exposed to 0.05 umoles or 3 x 10 molecules substrate in 50 ul of 1 mM glycolate solution. As the cells centrifuge 96 through the glycolate solution, cells at the leading edge of the band of cells are exposed to a higher substrate concentration than the trailing edge which is exposed only to substrate not already taken up. The final internal concentration measured in the cell pellet fraction is an average of internal concentrations. Not all substrate is taken up, however, because radioactivitiy is detected in the supernatant fraction and the radioactivity is higher than the in the pellet fraction. This observation implies that substrate depletion is not complete and may not be entirely the explanation for incomplete equilibration. Aggregation of cells may contribute to the incomplete equilibration. Stimulation This concept of limited availability of substrate, in this case protons, will serve to explain the stimulation of uptake by the ionophores and salts or by salts alone. If glycolate uptake requires a proton, the transport will be dependent on proton availability. A change in this availability is more noticable under circumstances of competition among sites for substrate, particularly when the concentration of glycolate anion is high relative to H+. This availability will be determined at least to some extent by external pH and by action of the plasma membrane ATPase. The scheme in Figure 24 was made to help visualize the proton exchange across the plasma membrane. Inhibition of such an ATPase activity by KCN will inhibit glycolate 97 Figure 24. Model Proposed for the Stimulation of Glycolate Uptake by High KCl. Addition of external KCl stimulates uptake of K+ into the cell. A proton is extruded by the plasma membrane ATPase in exchange for K+ to preserve electrical neutrality. Since H+ is required as co-transporter for glycolate uptake, increased H+ availability stimulates glycolate uptake. See also Figure 24. 98 l+ ATPase .i 1<+ , Na+ + H >- A- x A- with salt H+ +- PH 5 pH 7 H + H+ H H+ H+ H+ H+ AT i¥+< Page Hfz ATPase + H > H+ > - >. A > A \ _ A- A with ionophore F1°gure 24. 99 transport. Alteration of proton availability can also be achieved by treatment with ionophores or by high KCl. Valinomycin facilitates potassium exchange across the membrane. Uptake of potassium causes proton extrusion to maintain electrical neutrality in Chlorella (74). If proton extrusion is accelerated by potassium and glycolate uptake is dependent on proton availability, then glycolate uptake will be increased. The ratte of the transport process is not limiting. The extrusion of protons from the cell increases the proton concentration above that which is due to external pH. The stimulation by valinomycin/KCI was greater at higher glycolate concentration implying that glycolate uptake was limited by the availability of co-transport species. This increase can be achieved by the addition of potassium alone as was observed. Nonspecific ionophores, such as beavericin and nonactin, will accomplish the same end; protons will be extruded in response to increased cellular concentration of sodium or potassium. In the absence of cation, protons will be exchanged to eliminate the pH differential across the membrane. The ionophores alone have different effects at different pH. At pH 7 the ionophores tended to decrease glycolate uptake but tended to increase uptake at pH 5. At pH 5, the concentration of protons is greater on the exterior than in the cell. In the presence of an ionophore, protons will enter the cell and be extruded to maintain cell pH. CHAPTER 5 THE SELECTION BY FLUOROACETATE OF MUTANTS 0F CHLAMYDOMONAS REINHARDTII DEFICIENT IN ACETATE METABOLISM Introduction Glycolate stimulated the growth of Clamydomonas reinhardtii (14) and acetate can be used for heterotrophic growth. Because of the similarity in structure of glycolate and acetate and the involvement of glyoxylate and acetate in the glyoxylate in the glyoxylate cycle, it was thought that the glyoxylate cycle (Figure 25) may be involved in the metabolism of glycolate. In addition, the hypothesis had been made that glycolate and acetate were taken up by the same monocarboxylic acid transporter. The hypothesis that there is a single transporter and the observation of growth stimulation by glycolate was used as a basis for screening for mutants in transport and in acetate metabolism. Fluoroacetate is a toxic compound that blocks the TCA cycle by condensing with oxaloacetate to form fluorocitrate which inhibits aconitase (75). Fluoroacetate has been used to select for mutants defective in acetate utilization. 100 101 Figure 25. The Glyoxylate Cycle. The reactions for the utilization of acetate are shown. The reactions from the C2 Cycle for the oxidation of glycolate to glyoxylate and the transamination of glyoxylate to glycine have been added. Also added is the diversion of isocitrate by isocitrate dehydrogenase to the TCA cycle. Malate is shown in two locations to indicate its role in continuation of the cycle and in metabolism to oxaloacetate and aspartate to be used as carbon skeletons for growth. The inhibition by fluoroacetate is shown in the conversion of citrate to isocitrate. (from Reference (82)) 102 Glyoxylate Cycle CHJCOOH Acetate ADP ‘M‘+¢ ATP CH3CO.SCoA Acetyl-CoA HS A Co H20 HOOCCOCH2COOH Oxaloacetate’ CHICOOH ' NADH + H . C(OH)COOH NAD' CHlCOOH Citrate HOOCCH(OH)CH2COOH Malate 1.. (brag-Drlsocicnce: NAD oxidoreductnc . HOOCCH=CHCOOH l 1 .........‘2’2‘.‘£3§Z‘Z.22'3.‘. HOHCCOOH Fumarate 1.1...41 CHCOOH _ FADHI CHzCOOH FAD T.C.A. Cycle "“"me HOOCCH2CH2COOH Succinate (hreo-Ds-lsocitrate :lyoxylue lyase r/ lsocurue lyase 1"“ OHCCOOH *3 HINCH2.COOH / Glyoxylate GlyCIne COOH \ H20 H(|:OH L-malate glyoxyla.e-lyase ( . (C133.'.Z°§Ilii.'3§.’ \* HSCoA Glyco ate 4 LB.) 11 HOOCCH(OH)CH2COOH Malate NADt NADH + Ht HOOCCOCHICOOH Oxaloacetate HOOCCH1CH(NH2)COOH L-Aspartate Figure 25. 103 Fluoroacetate inhibited the uptake and metabolism of acetate to CO2 by Anabeana flos-aquae (24, 76). McKenny and Melton (77) isolated mutants of Azotobacter vinelandii deficient in acetate kinase or phosphotransacetylase. Rothstein (78) selected mutants of Clostridium thermosaccharolyticum unable to produce acetate as a fermentation product. Harford and Weitzman (79) isolated mutants of g. ggli_K12 and Acinetobacter calcoaciticus lacking citrate synthase by selection on fluoroacetate. A mutant of Chlamydomonas dysosmos which was unable to use acetate and therefore an obligate autotroph was found to be lacking isocitrate lyase (80). Fluoroacetate can select for mutants in respiration (81). It was suggested that growth on fluoroacetate could also be used to select for mutants in the glyoxylate cycle (Figure 25) or for mutants in a permeability system for acetate. In addition, Harford and Weitzman (79) and Smith and Lequerica (83) stated that resistance to fluoroacetate may be due to an inability to take up fluoroacetate, i.e. transport mutants. One of the Azotobacter mutants isolated by McKenny and Melton (77) was reported to lack acetate uptake. The assumption implied in these suggestions is that uptake of acetate is by a process other than diffusion, i.e. that there is a transport system for acetate and that this transporter will also use fluoroacetate. In order to select for glyoxylate cycle mutants, it is assumed that fluoroacetate in addition to blocking the TCA cycle, will 104 function like acetate to induce the enzymes unique to the glyoxylate cycle, isocitrate lyase and malate synthase. Methods and Materials Mutagenesis Cells of Chlamydomonas reinhardtii strain 137 were treated with nitrosoquanidine (84). Cells from liquid culture were centrifuged and washed in 50 mM K-citrate pH 5.5. Nitrosoguanidine (10 ug/ml) was added and cells were incubated in the dark for 30 min. Cells were washed twice with minimal media before plating. Cell survival was found to be between 5 and 15%. The mutagenesis was done twice with the only difference being the order of the screening procedures. After the first mutagenesis, cells were plated onto minimal medium (19) containing 10 mM fluoroacetate and placed in the light. Colonies growing on fluoroacetate were replica plated onto minimal medium and minimal medium with glycolate to compare for growth stimulation. After the second mutagenesis, cells were plated onto minimal medium and colonies growing on the minimal medium were picked and replica plated onto minimal medium and minimal medium containing 20 mM glycolate. Those colonies failing to show enhanced growth on glycolate under conditions of an atmosphere of reduced CO2 were then transferred to plates of fluoroacetate medium. Only colonies showing both fluoroacetate resistance and lack of glycolate stimulation of growth were kept for analysis. 105 Colonies were plated on tris-acetate-phosphate (TAP) (20) in the dark to verify their inability to use acetate heterotrophically. Growth of mutant cultures for analysis Mutants were air-grown in 200 ml of TAP medium in 500 ml flasks with aeration and with continuous light (100 uE/s/mz) on an Eberbach shaker. Preparation of cell extracts Cells were harvested by centrifugation at 1000 g for 5 min, washed with water and resuspended at the ratio 1 gm cells per 10 ml homogenizing buffer containing 165 mM Tricine pH 7.5, 10 mM KCl, 10 mM MgC12, 10 mM EDTA, 10 mM DTT (85) and kept on ice. Cells were broken in 2 passes through a Yeda press at 1000 psi for 2 min. After centrifugation at 27,000g for 20 min, the supernatant fraction was assayed. Protein determination Protein was determined by a modification of the Folin-Lowry method (70). BSA was used as the standard. Chlorophyll was determined for the cell suspension by the method of Arnon (22). Enzyme assays Isocitrate lyase activity in the supernatant after cell breakage was measured by the method of Cooper and Beevers (85). The reaction was initiated with isocitrate and production of the glyoxylate—phenylhydrazone was monitored at 324 nm. Malate synthase activity in the supernatant was measured in vials containing 25 mM Tris pH 8, 3 mM MgClz, 2 mM acetyl CoA at 30°C. The reaction was initated by 106 addition of [14C]glyoxylate to 10mM final. The reaction was terminated by the addition of H202 to oxidize unreacted [14C]glyoxylate to 14CO2 and HCl to liberate the 14CO2 from the vials. Glycolate excretion Cells were diluted from the original 10% suspension in homogenizing buffer to 50 ug chlorophyll/ml using 3 mM potassium phosphate buffer pH 7. 14 [ C]NaHC03 (1 mM) was added every 15 min to the illuminated cell suspension and the amount of radioactivity excreted and the total amount of fixed radioactivity determined (17). Glycolate and acetate uptake The uptake of acid by cells (50 ug chl/ml) exposed to 0.1 and 1 mM Na 14C]glycolate or 0.1 and 1 mM Na [14C]acetate in 20 mM Na [ Hepes pH 7 was measured using the silicone oil gradient centrifugation method described by Howitz and McCarty for chloroplasts (71). Cells were pelleted through a layer containing the labeled acid at 4°C in the dark. The time of exposure of cells to [14C]glycolate or [14C]acetate was about 2 seconds. Chromatoggaphy Fractions of cell pellet and cell supernatant from the glycolate excretion experiments were concentrated and applied to Whatman 1 paper for two dimension chromatography (23). Papers were exposed to X—ray film aand identification of spots was made from a standard reference map. 107 Results Glycolate and acetate uptake. The measurement of excretion may reflect glycolate metabolism rather than changes in the transport of glycolate. Therefore, short timee uptake of [14C]glycolate was measured (Table 5). Because these mutants were unable to heterotrophically grow on acetate, uptake af acetate was measured in the same manner as glycolate but on a few of the mutants randomly selected based on the levels of isocitrate lyase and malate synthase activities. Most mutants took up glycolate to equilibrium just as the wild-type did. There were some (TWF 8, 12, 16, 17, 18, 23, 27, 29, 39, 41, 42, 44, 45, 46 and 48) that had about half the expected uptake. One mutant, TWF 28, had greatly reduced glycolate uptake and had reduced acetate uptake also (Table 5). Reduced uptake does not correlate with the class of enzyme activity. 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