WWW. ———-— This is to eertifg that the thesis entitled E‘XNY-‘t‘m c3 0Vécnu‘t. Amt: Dunn-m} plm+osgpktucs b} QMMWQM A(&¢( presented by LULC— on'tn Clank} has been accepted towards fulfillment of the requirements for D Foci'ort-thf Mmdegree in——B—{°—C—heml.5fvj 77. gm Major professor ‘ 57¢ Date :0“ 517/ !7 67 0-169 PLACE ll RETURN BOX to runovothlo chockoutfrom your record. TOAVOID FINESmnonorbdmddoM MSU Is An Affirmative Action/Equal Opportunity Institution cmm.‘ sured l for ma‘ excreie the al“ amounts bout 4 also ex fied acf 8Cids e] ”'3 301‘ or EEEQrtaI acids n: niZed ce C6113 GI and glyc: mifiutes ( tone and ABSTRACT EXCRETION OF ORGANIC ACIDS DURING PHOTOSYNTHESIS BY SYNCHRONIZED ALGAE by Wei-Hsien Chang Synchronized cultures of Angigtrodesmus braunii'were grown dur- ing a 16-hr light and 8-hr dark regimen at 30° with a 1 to 4 dilution at the end of each dark period. The photosynthetic ability, as mea- sured by 1L‘COZ fixation, was the highest for young growing cells, low for mature cells, and lowest for dividing cells. The amount of 1LPG excreted during photosynthesis followed the same trend. The 1“ C-compounds excreted during photosynthesis changed during the algal life cycle. Young growing cells excreted glycolate in large amounts, but none of the other acids. Dividing cells excreted only about #% as much glycolate-IQC as young growing cells. Dividing cells also excreted mgggrtartrate, isocitric lactone, malate, and an unidenti- fied acid, U3, and occasionally some citrate and glycerate. Of the acids excreted by dividing cells, glycolate and mgggrtartrate were the major ones and they were excreted in comparable amounts. Excretion of ggggrtartrate when glycolate excretion decreased implied that the two acids might be metabolically and physiologically related. Kinetic studies on the excretion of these acids by the synchro- nized cells were done during 30 minutes of photosynthesis. Growing cells excreted glycolate. With dividing cells, mgggrtartrate, malate and glycerate were excreted in relative largest amounts within 1 or 2 minutes of photosynthesis, glycolate in 5 minutes, and isocitric lac- tone and U3 in 30 minutes. Wei-Hsien Chang Large-scale separation and purification of the excreted acids were carried out by repeated anion exchange chromatography on AGI- acetate columns with acetic acid gradient elution followed by formic acid gradient elution. The acids were eluted from the column with the acetic acid gradient in the order of glycolic, malic,‘mg§gy tartaric, isocitric and citric acids, and isocitric lactone; U3 was eluted with a subsequent formic acid gradient. The‘mgggrtartaric acid was identified by cochromatography on paper chromatograms and by gas-liquid cochromatography as the tri- methylsilyl derivative. Isocitric lactone was identified by cochro- matography on paper, and by hydrolyzing it to isocitrate. The iso- citrate was then identified by cochromatography and by converting it to glutamate by a reaction catalyzed by isocitric dehydrogenase and glutamic-aSpartic transaminase. Isocitric lactone was experimentally shown to be excreted as such by the dividing cells. 14C03, subsequent excre- After a period of photosynthesis in NaH tion of 1L‘C-labeled acids in the light and in the dark was analyzed both for the amount and for the components. In the light, excretion of glycolate and the other acids continued. In the dark, glycolate excretion was completely stopped, while the excretion of the other acids continued in even larger amounts. Upon addition of CMU during the light, the excretion pattern was similar to that in the dark. Aeration with oxygen during the dark prevented the excretion of iso- citric lactone. The distribution of 1“ C in meso-tartaric acid was determined. The carboxyl carbons were about n times as radioactive as the middle carbons. Since glycolate is known to be uniformly labeled, glycolate Wei-Hsien Chang could not be the precursor of carboxyl labeled‘mgggrtartrate, nor mgggr tartrate a direct precursor of glycolate. The biosynthesis of‘mgggr tartrate by these algae is unknown, but the carboxyl labeling pattern suggests that a carboxylation to form a Cu-precursor of the tartrate might exist. From the data, it was speculated that glycolate might be formed from a precursor common to both mpggrtartrate and glycolate. The reason for the Specific excretion of glycolate, mpggrtartrate and isocitric lactone is not known, except that all three acids may not be further metabolized by the algae. EXCRETION OF ORGANIC ACIDS DURING PHOTOSYNTHESIS BY SYNCHRONIZED ALGAE By Wei-Hsien Chang A.THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1 967 p L+ra:;.\\ (5,354; E‘ ACKNOWLEDGMENTS The author wishes to express his earnest appreciation to Professor N. E. Tolbert for his guidance, understanding, support, and encouragement throughout the course of this investigation and for counsel in the preparation of this théSis e ’ He is also grateful to Professor W. W. wells for his help in using the gas-liquid chromatograph. Dr. J. L. Hess and Dr. P. Kindel advised him during part of the investiga- tion. Mr. W. Bruin helped with the determination of specific activity, and Mrs. D. Randall helped in the prepa- ration of the thesis. Support from a National Science Foundation Grant No. GB #15“ to Dr. N. E. Tolbert is acknolwedged. ii Wu Tu my 3; i. r. :u T n . Wu. ”J I .hi Q?" 1" WWI“; P1 at Pt TABLE OF CONTENTS INTRODUCTION AND LITERATURE REVIEW . . . . . MATERIALS AND METHODS . . . . . . . . . . . Algae . . . . . . . . . . . . . . . . Synchronous culture of algae . . . . Chemicals . . . . . . . . . . . . . . Photosynthetic experiments . . . . . Paper chromatography and radioautography Anion exchange chromatography . . . . . . Gas-liquid chromatOgraphy . . . . . . . . Degradation of 1L’Cuorganic acids . . . . Determination of specific activities of carbon atoms Large-scale preparation of unknown acids excreted by A. braunii O O O O O O I O O O O O O O O O O C O O 0 RE ULTS AND DISC USS ION C O O O O O O O C O O C O O O C O O O O Photosynthetic CO -fixation and excretion by synchronized algal cells during their life cycle . . . . . . . . . . . Compounds excreted during photosynthesis Kinetic studies of the photosynthetic excretion . . . . Preparative separation and partial purification of the compoundsexcreted.................. Purification and identification of U1 as isocitric laCtoneeeeeeeeeeeeeeeeeeeeeeeee Purification and identification of 02 as meso-tartaric ECid O O O O O O O O O O O O O O O O O O O O O O O O 0 Purification and properties of U3 . . . . . . . . . . . iii Page 11 13 14 15 17 19 20 22 22 27 37 42 5o 56 62 SUI‘MR‘I BIBLIOSI TABLE OF CONTENTS (Continued) Page “C02 photosynthesis . . 66 Excretion of labeled acids after 1 Distribution of 1("LC-activity in meso-tartaric acid . . . 74 Discussion concerning metabolic relationships between mGSO-tartaric and EIYCOliC fields e e e e e e e e e e e e 78 Discussion concerning isocitric lactone excretion . . . . 83 SUMMARX O O O O O O O O O O O O O O O O O O O O O O O O O O O O 85 BImJImR-Apm O O O O O O O O 0 O O O O O O O O I O O O O O O O O 88 iv 9.' Talble .o‘ 10 11 Table No. 1 10 11 LIST OF TABLES A.Life Cycle of A, braunii Cells in A Synchronous Culture and Their Relationship to Photosynthetic ACtiVityeeeeeeeoeeeeeeeeeeeeee Amount and % Distribution of 140 in the Compands Excreted During 5-min. Photosynthesis in NaH1 by Synchronized Cells of A, braunii . . . . . .3 . . Amount and 1. Distribution of 1”c in the Compounds Excretfg During Different Periods of PhotOSynthesis in NaH CO3 by Synchronized Cells of A. braunii . . Retention Volumes for Organic Acids Excreted by A, braunii on an AGl Resin Column after pH- Gradient Elution . . . . . . . . . . . . . . . . . . Effect of Variations in Methods for Concentration of Supernate from A, braunii on the Amount of Radioactivity Recovered as Isocitric Lactone or Isocitric Plus Citric Acids . . . . . . . . . . . . Degradation of U2 (meso-tartaric acid) and U3 . . . Gas-Liquid Chromatography of TMS-Derivatives of TartariCACidsandUZeeeeeeeeeeeeeee Amounts of 1“'0 Excreted by 1L"C-ILabeled Cells of A, braunii under Different Conditions . . . . . . . Amount and % Distribution of 14C Afiong Compounds Excreted by A. braunii During NaH1 Fixation and Afterwards During Different Treatment; 0 e e e e e 0 Effects of Darkness, CMU and 0 on the Excretion of Acids by luC-Labeled Cells of _. braunii . . . . . . Specific Activity of the Carbon Atoms of meso- TamariCACid(Uz)eeeeeeeeeeeeeeeee Page 23 30 38 “7 55 61 63 67 7O 73 76 Figgre No. LIST OF FIGURES Changes in the Activity of Photosynthetic 14cc _ Fixation and 1[to-Excretion During the Life CycIe OfA.braunii................'.... Radioautograms of Supernates and Cell-Extracts After 5-min. Photosynthesis in NaHlucOB by Synchronized Cells of A, braunii . . . . . . . . . 1A C-Activity in the Compounds Excreted During 5-min. Photosynthesis in NaHlL‘CO3 by Synchronized cenSOfAebrauniieeeeeeeeeeeeeeee % Distribution of 14C Among the Comggunds Excreted During 5-min. Photosynthesis in NaH CO3 by synChronized Cells Of A. braunii e e e e e e e e e pH-Gradient Elution of Organic Acids Excreted by A0 braunii With an AG]. ReSin COlumn e e e e e e e 0 Paper Chromatographic Maps of Organic Acids Excreted by A, braunii Page 24 28 32 3h 48 INTRODUCTION AND LITERATURE REVIEW Excretion of organic compounds by algal cells has often been reported. Both the quantity and components excreted may vary for dif- ferent species, and for different environmental conditions. Allen (1) and Antia, et a1. (3) reported that from 10% to as high as 45% of the organic matter formed by algal cells was excreted. Recent reports (25, 30, 56), however, agree that the excretion of organic compounds by algal cells amounts to 2 to 6% of the total carbon fixed. According to Allen (1), the amount of excretion by Chlagydomonas species was parallel to the growth. However, working with other green algae, Forsberg, et a1. (25) claimed that organic compounds were excreted during the phase of declining relative growth rate and in the station- ary phase of growth. In fact they reported some uptake of external carbon compounds before excretion started. High light intensity (1, 23, 30) and nitrate as nitrogen-source (1) have been found to favor excretion. 'Various compounds have been found to be excreted by algae. Beside organic acids, which are the major excretion compounds, amino acids, other nitrogenous compounds, and various carbohydrates have been reported. Glycolic acid is the common compound which is excreted in relatively large amounts by all the algae. Except for glycolic acid, the types of other compounds excreted seem to be specific to the different algal species. Chlgmgdomongs (1) excreted some poly- saccharides, in addition to glycolic, oxalic and pyruvic acids, but 2 no nitrogenous compounds. Hellebust (30), who surveyed a large number of green algae found 9 to 38% of the total carbon excreted as glycolate, 0.2 to 5.9% as protein, and 2.8 to 10.3% as lipid, together with some amino acids and peptides. Excretion of nitrogenous compounds has also been analyzed. From 25 to #0% of total N-fixation was excreted according to Henriksson (32, 31), but 5 to 8% was reported by Magee and Burris (51) and Allen and Arnon (2). Henriksson (32) found that shaking of the culture and high nitrate could decrease the excretion of nitrogenous compounds. Accord- ing to Watanabe (95), nitrogen-fixing blue-green algae excreted only 3 amino acids, i.e., aSpartic and glutamic acids and alanine. Stewart (81) found many kinds of amino acids were excreted, among which alanine, threonine, leucine, glutamic acid and basic amino acids were the main ones. Other minor compounds reported in algal excretion have been glycerol (30), mannitol (30), some analogue of acetylcholine (93), dimethylsulfide or related sulfur compound (4), and a yellow, nitro- geneous compound, probably a protein-carotenoid complex (22). The studies mentioned above were, in most cases, done from an ecological viewpoint. Therefore, they were either run in the field or in the ocean under natural conditions, or carried out in a large scale for a long period of time. Few experiments were performed in a labora- tory with biochemical equipment and techniques. Glycolate excretion is the only subject that’has been extensively studied from a biochem- ical point of view, and with biochemical equipment and techniques. Rapid formation of glycolate by algae during photosynthesis was first reported by Benson and Calvin (8) in 1950, but it was not until 1956 that Tolbert and 2111 (86) first reported that the glycolate 3 formed by Chlorella was excreted into the medium. The glycolate excre- tion by algae has since been confirmed by many investigators, including Lewin (50) for Chlamdomonas, Pritchard, et al. (62) for Chlorella, Miller, et al. (55) for Chlorella, and Hess, et al. (35) for Scenedesmus. -One group with Fogg in England is investigating the ecological Significance of glycolate excretion. The amount of glycolate excretion during photosynthesis under normal conditions was reported by Tolbert and Zill (86) to be 3 to 10% of the total carbon fixed, or 3 to 8 mg glycolic acid per liter of algal suspension. However, it can be enhanced by low CO concentration (55, 62), high 02 partial pressure 2 (55, 86. 99), high pH (21, 57, 60), and high light intensity (62, 86). Light and manganese have been shown to be indispensable for glycolate excretion (35, 55, 62, 86). Although there have been reports that glycolate can be taken up by algal cells (21, 55, 99), especially when illuminated, Hess and Tolbert (34) have recently claimed that no sig- nificant uptake by algae of either glycolate or phosphoglycolate was observed. In spite of intensive studies and numerous speculations, the physiological significance of glycolate excretion by algae still remains unexplained. Hess and Tolbert (34) Speculated that glycolate might be an end product of carbon metabolism in algal cells because it was excreted but not taken up, and because of the absence in algae of glycolate oxidase which is necessary for its metabolism. The pathway of glycolate biosynthesis is unknown, although there are two hypothesis in the current literature. The most likely 14C—incorporation into products pathway was based on kinetic studies of of photosynthesis (98). This hypothesis postulates that glycolate may be derived from a photosynthetic product of the carbon reduction cycle. Ll, Since Schou, et al. (68) found that the glycolate molecule is uniformly labeled, the possible source of such a glycolate molecule would be the top two carbon atoms of a sugar phOSphate which are also uniformly labeled. Thus, Bassham (5) and others have proposed that glycolate arose from a sugar phOSphate with the participation of thiamine pyro- phosphate. Holzer's group isolated a glycolaldehyde-TPP intermediate, which was oxidized nonenzymatically to glycolyl-TPP, and then hydro- lyzed to free glycolate (2A, 36). Racker's group were also able to demonstrate glycolate formation from fructose-6-phosphate by chloro- plasts with ferricyanide, a strong oxidant (9). They also isolated a glycolaldehyde-phosphoketolaseintermediate(26). In analogy, Tolbert (85) has suggested that phosphoglycolate might be formed by the cleavage of a ketose diphOSphate molecule, probably through a TPP-CZ- ph03phate intermediate. However, none of these reactions have been shown to occur enzymatically or‘lgflgizg, Another hypothesis for glycolate biOSynthesis is a gg_ggyg,formation by direct condensation of two molecules of CO2 (82). From data of specific activity measure- ments, Zelitch (101) favored the £13 9233 synthesis. However, Hess and Tolbert (33) did not confirm Zelitch's results. A biochemical study of acid excretion by algal cells during photosynthesis could best be done with the use of a synchronized cul- ture. Shifts in metabolic pathways and change in enzyme activity of algal cells during their life cycle have been recognized (17, 39, #1). The most significant correlation between cellular activity of algae and the life cycle was observed for the photosynthetic activity. Thus, Sorokin (71, 72, 7A) and Kates and Jones (40) were able to show a rhythmic change of photosynthetic activity during the life cycle of . f ..r [living .5. . i \ F. ..\...rbi.u.r, L 5 Chlorellg or Chlgmydomonas species. Sorokin (73) further separated the small younger cells and the larger older cells by fractional cen- trifugation. He clearly showed that the small younger cells were invariably more active than the large older cells. He, therefore, concluded that the change in photosynthetic activity during the life cycle is characteristic of normal cell development. However, there are some reports (15, 75) in which no rhythmic change of photosynthe- tic activity was observed during the life cycle. If a minor phenomenon‘is taking place, or a minor component is being formed, simultaneously with a major one, the minor one may be obscured by the major one and tends to be overlooked. This is usually the case when a random algal culture is used in a biochemical study of some cellular activity. The activity of the rapidly growing young cells dominates. In this study, therefore, excretion of organic acids was measured, both quantitatively and qualitatively, during the life cycle of a synchronized algal culture. Ankistrodesmus braunii was used in this study because it was easily synchronized, its morpho- lOgical change during the life cycle could be clearly distinguished microscopically, and the cells were easily filtered through a Millipore filter. As reported in this thesis, the amount of photosynthetic excretion changed during the life cycle as did the photosynthetic activity, and the compounds excreted also changed. In part the results are a study of changes in glycolate excretion during the algal life cycle. Most of my effort was devoted to the identification of mpggr tartaric acid, which was the second most important acid excreted by the dividing cells of‘A, braunii, and of isocitric lactone which was also being excreted. MATERIALS AND METHODS Essa: The strains of algae used in these experiments were obtained from the "Culture Collection of Algae" at Indiana University, Bloomington, Ind. (80). Ankistrodesmus braunii (Naeg.) Collins (No. 2h5), Scenedesmus obliguus (Turp.) Knger (No. 393), and Scenedesmus guadricaudg (Turp.) Breb. (No. 77) were obtained on protease-agar slants and cultured on an inorganic salt medium with Hoagland's micronutrients (58); Chlmdomonas reinhardtii Dangeard, (-) strain (No. 90) was obtained on a soil-extract-agar slant and cultured on the high phOSphate medium described by Orth, et al. (60). Inocula were first taken from stock cultures on agar slants into 100 ml of the respective culture medium in a 250 ml Erlenmeyer flask for growth and multiplication over a period of a week to 10 days. This culture was then transferred to 1.5 l of fresh sterile culture medium in a 2.5 l "low'form" Fernbach flask fer further growth and multiplication. The continuous cultures were diluted 1 to 10 with fresh medium.about every 3 days before becoming dark green or brownish, and each culture was renewed from the respective stock culture every 2 to 3 months. The culture flasks were fitted with air inlet and outlet, both provided with a cotton filter. Aeration with 0.2% (v/v) CO2 in air was maintained by passing air from an oil-free compressor through a cotton filter and distilled water, and then mixing the air with 7 appropriate volumes of C02 which was monitored by bubbling through distilled water. The flasks were placed on a reciprocating shaker (Eberbach Corp., Ann Arbor, Mich.) of about 60 excursions per min., thus providing a gentle,but thorough agitation of the 1.5 1 medium in the large flasks. The shaker was held in a controlled environ- ment chamber ("Sherer Controlled Environment Lab," Model CEL 37-14; Sherer-Gillett 00., Marshall, Mich.) kept at 15°. Continuous light from cool white super high fluorescent bulbs (General Electric, F72T12-Cwel500) on the top of the chamber provided a light intensity at the level of the flasks of 1,200 ft-c. These conditions main- tained a temperature in the culture medium of 200 as monitored by a thermometer. Sygchronous culture of algae: For synchronization of the algal cultures, Tamiya's method (84)of'Wprogrammed" light-dark regimen with periodic dilution was slightly modified according to Wanka's light-dark time schedule (9“). Initially a dense, random culture of algae was diluted one to one with fresh medium.and kept growing under continuous light for another day or two. By this method, which is similar to that reported by Stange, et al. (79), a partially synchronized culture was obtained, in which most of the algal cells were resting at a certain stage of growth and only a few dividing cells could be observed microscopically. The algal cell population was then counted with a "Levy and.Levy-Hausser counting chamber" in which each of the smallest square units was equivalent to 2.5 x 10"7 ml. The algal cells were diluted with fresh medium to a papulation of A x 106 cells/m1, or 1 cell/smallest square unit. 8 One liter of the diluted algal culture in a low form Fernbach flask was placed on a reciprocating shaker held in another controlled environment chamber. Illumination in this chamber was set for cycles of lfi—hr light of the same intensity and of 8-hr dark. The tempera- ture during the light period of the chamber was kept at 250 in the chamber, which was sufficient to maintain a temperature in the cul- ture medium of 30°. During the dark period the temperature was kept at 300 by means of additional heat from darkened tungsten lamps which were connected to a thermostat. Since the diluted algal cells were already partially synchronized in a certain growth stage, they were put into the growth chamber at about the middle of the light period, so that the remaining period of illumination would bring them up to mature cells which would divide during the following dark period. At the end of the dark period the population of daughter cells was counted again and the "division number" was calculated. In most cases, the division number for Ankistrodesmus braunii was between 3 and h and that for Scenedesmus obli uus, Scenedesmus uadric uda, or Chlgmydomongs reinhardtii was between 6 and 8. The daughter cells were diluted at the end of the dark period again to 4 x 106 cells/ml, or 1 cell/smallest square unit, and the next cycle of light and dark was started. Two generations of such a light-dark-dilution cycle were in most cases enough to bring a partially Synchronized culture to complete synchronization. The algal cells were examined micro- scopically and classified, as described in Results and Discussion. Chemicals: All of the chemicals used in this work were standard reagent grade. Authentic compounds for chromatography were calcium, gluconate, 9 fumaric, maleic, and malonic acids as obtained from Nutritional Bio- chemicals Corp. (Cleveland, Ohio); dihydroxymaleic and dihydroxya tartaric acids from Aldrich Chemical Co. (Milwaukee, Wisc.); DL- isocitric lactone, (+) tartaric; and.mg§gytartaric acids from Calbiochem (Los Angeles, Calif.): oxalic acid from J. T. Baker Chemical Co. (Phillipsburg, N. J.); tartronic acid from General Bio- chemicals (Chagrin Falls, Ohio): and mesoxalic acid prepared by N.anmn. Organic solvents used for paper chromatography were n-butyl alcohol, ethyl acetate, and 88% phenol (without preservative for chromatographic purposes) from Mallinckrodt Chemical works (New York, N. L); formic acid from J. T. Baker Chemical Co.; and propionic acid from Eastman Organic Chemicals. Enzymes used in this work were acid phosphatase, glutamic oxalacetic transaminase, and isocitric dehydro- genase from Sigma Chemical Co. (St. Louis, Missouri); alkaline phos- phatase from.Nutritional Biochemicals Corp; and phosphoglycolate phosphatase from Donald Anderson. Bio-Rad Laboratories (Richmond, Calif.) supplied anion exchange resin AGl x 8 (100-200 mesh, Cl-form) and cation exchange resin AG5OW x 8 (100-200 mesh, H-form). Chemicals used for gas-liquid chromatography were pyridine from Eastman Organic Chemicals; hexamethyldisilazane from.Peninsular Chemresearch (Gainesville, Florida); trimethylchlorosilane from General Electric Co., Silicone Products Dept. (Waterford, N. Y.): and OV-i and Gas~Chrom Z from Applied Science Laboratories (State College, Pa.). Those used in degradation experiments were mercuric chloride and sodium meta periodate from.Eisher Scientific Co. (Fair Lawn, N. J.); potassium.persulfate from Allied Chemical (Morristown, 10 N. J.); DL-serine from Nutritional Biochemicals Corp., silver nitrate from D. F. Goldsmith Chemical & Metal Corp. (Chicago, 111.), barium hydroxide and barium chloride from J. T. Baker Chemical Co. Chemicals used for liquid scintillation counting were p-dioxane and phenethyl- amine from Eastman Organic Chemicals, naphthalene and xylene from J. T. Baker Chemical Co., toluene from Mallinckrodt Chemical Works, absolute ethyl alcohol from Commercial Solvents Corp. (Terre Haute, Ind.); and d-NPO (d-naphthylphenyloxazole), POPOP (1,14-bix-2-(5- phenyloxazoly1)benzene) and FPO (2,5-diphenyloxazole) from Packard Instrument Co. (Downers Grove, 111.). Other chemicals used were: TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) from Calibiochem, manganous chloride from J. T. Baker Chemical Co., NAIF from Sigma Chemical Co., DL-aspartic acid from Nutritional Biochem- icals Corp., and CMU (3-(p-chlorophenyl)-1,1-dimethylurea) from E. I. DuPont DeNemours & Co., Wilmington, Del. 14CO received 3 from either Oak Ridge National Laboratories (Oak Ridge, Tenn.) or Radioactive compounds used in this work were Ba New England Nuclear Corp. (Boston, Mass.), orthophOSphoric-BZP acid from Tracerlab (Waltham, Mass.), benzoic-iuC acid from Packard Instrument Co., oxalic-U-MC acid from Volk Radio Chemical Co. (Chicago, Ill.): and calcium salts of glycolic-i-mC, glycolic-2-1%, and phosphoglycolic-MC acids from Orlando Research Inc. (Orlando, Florida). Solutions of NanCO3 for photosynthetic experiments were prepared by generating 14CO2 gas from Ball’CO3 by the addition of lactic acid in one arm of an evacuated, Y-shaped apparatus, and capturing the 14002 gas in a calculated amount of an NaOH solution in the other arm of the apparatus. The specific activity of the 11 NaHmCO3 was generally 50%, and the final solution always contained 1 pc 1L‘C/Z pan Photosypthetic egperiments: The normal photosynthetic experiments were similar to those of Bassham and Calvin (6). The algal cells at a designed stage of their life cycle were centrifuged from the medium at 1,000 x g for 5 min. The packed cells were washed once by resuspending in a small volume (usually less than 10 ml) of distilled water in a graduated centrifuge tube and centrifuged again at 1,000 x g for 5 min. The packed volume was recorded and the cells were then resuspended in a volume of 0.001 M phosPhate buffer at pH 6.0, so that a final suspension of 1% (v/v) algal cells was obtained. Small scale (5 to 20 m1) and medium.scale (20 to 80 ml) photo- synthetic experiments were performed in a "lollipop" (13) of the correSponding volume. White light of 3,000 ft-c was obtained from two 300-watt KEN-RAD reflector flood lamps, positioned perpendicular to the plane of the lollipop, one on each side. The lollipop was placed in a water bath maintained at 20°. Five-liter diphtheria toxin culture bottles, filled.with distilled water, were placed between the lamps and the water bath in order to absorb excess heat from the lamps. After 5 min. preillumination and aeration by a stream of air, 50 P1 of NaHluCO3 solution per 10 ml of algal suSpension was injected at zero time and the lollipop was closed and briskly shaken in the light path. The NaHIuCO3 solution contained 1 Pc 114C per 2 P1, and the percent inc varied between 25 and 50%. Aeration of an Ankistrodesmus 12 suspension was not possible because severe foaming forced the cells to flood out of the lollipop, but instead, the lollipop was occasionally shaken in the light path in order to keep the cells from settling. When the photosynthetic experiments were to run longer than 5 min., another aliquot of the NaHlnCO3 solution was added either at zero time or after 5 min. of photosynthesis. At various time intervals as designed, 2 to 4-ml aliquots of the suspension after shaking were taken with a pipette and quickly filtered through a Millipore filter (AAWP-025) with suction. The cells on the filter were immediately rinsed under continuous suction with 1 m1 of distilled water. The entire process of filtration and washing was usually completed within 15 sec. The filtrate and the washing were combined in a graduated test tube, acidified with 1 or 2 drops of glacial acetic acid, aerated for 10 min. with 12002 gas, made up to a certain volume by adding water, and then thoroughly mixed. An aliquot of less than 0.5 ml of this solution, called "supernate," was counted in 10 ml of Kinard's liquid scintillator (44) with a Packard Tri-carb Liquid.8¢ilifllation Spectrometer, Model 3310. This liquid scintillator, suitable for counting aqueous solutions, was made by dissolving 0.1 gcx-NPO, 10 g PPO, and 160 g naphthalene in a mixture of 770 m1 xylene, 770 ml p-dioxane, and 462 ml absolute 1LPG-activity excreted was calculated as ethyl alcohol. The total cpm/ml algal suspension. The remaining portion of the supernate was evaporated to dryness with a shaking evaporator (Buchler's Rotary Evapo-Mix) at 35-380 under reduced pressure, and the residue was analyzed by paper chromatography and radioautography. The washed cells on the filter were immediately transferred 13 into a beaker containing about 5 m1 of boiling 80% methanol, the extract filtered through a Millipore filter, and the broken cells washed twice with 2 to 3 ml of boiling 80% methanol. The filtrate and the washing were combined in a graduated test tube, acidified with glacial acetic acid, aerated with 12002, made up to a certain volume (usually 10 ml) and thoroughly mixed. A 0.1 or 0.2-m1 aliquot of the solution, called "cell-extract," was counted in 10 ml of’Kinard's liquid scintillator. The 1LLC-activity in the cell- extract was also calculated as cpm/ml algal suspension, and the total inc-fixation was then calculated by summation of the 1“(L-activity excreted and in the cell-extract. It is obvious, therefore, that this figure of total 1L’s-fixation does not include the fixation into compounds insoluble in 80% methanol, such as some proteins or poly- saccharides. An adequate aliquot of the cell-extract was evaporated to dryness and analyzed.by paper radioautography. as described for the supernate. Paper chromatOgggphz ggd radioAutography: Two-dimensional paper chromatography was carried out according to the procedures described by Benson, et al. (7). The solvent sys. tems were waterbsaturated phenol (made by mixing 4 vol. of 88% phenol with 1 vol. of water) for the first direction and butanol-propionic acidawater (freshly made by mixing equal volumes of butanol-water (1,246 m1:84 m1) and propionic acid-water (620 ml:790 ml) for the second direction. In many cases the second solvent system was modi- fied, according to Hartley and Lawson (28), by adding 0.1 ml of 6 N NaOH to 100 m1 of the solvent mixture. This modification gave preferential separation of particular compounds and better visualiza- 14 tion on paper of acid Spots of lower Rf values by bromcresol green spray (0.04% solution in 95% ethanol). ,Another solvent system, which was used for one-dimensional separation of various organic acids, consisted of butanol-ethyl acetate-formic acid (1:1:1, by vol.) as reported by Schramm (69). ‘Whatman No. 1 chromatographic paper was used throughout these experiments. After drying overnight in the air, the approximate glycolic acid area on the chromatograms was sprayed with 0.1 MNaZCO3 solution in 50% ethanol in order to prevent sublimation of the free acid. Cochromatography on paper was performed by mixing the radio- active sample with a nonradioactive, authentic acid in a test tube, evaporating the mixture to dryness, and spotting the residue on the origin as usual. Radioautograms were made by exposing the dried paper chromato- grams to "Kodak Medical X-ray Film, Blue Brand" from Eastman Co., Rochester, N. Y. After an appropriate period (3 days to 2 weeks) of exposure, the films were developed to locate radioactive Spots on the chromatograms. The radioactive areas were then counted with a thin window (DuPont Mylar film) gas flow'countergusing a Nuclear Chicago Sealer, Model 161A. Helium, passing through ethanol which was cooled with an ice bath, provided the gas for the counting chamber. Anion exchange chromatogrgphy: Analyses of organic acids by anion exchange chromatography were performed by a modification of the method described by Palmer (61). The anion exchange resin, AG1 x 8, Cl-form, was converted to the acetate form and packed to about 8 cm high in a cylinder of 0.4 cm inner diameter and 12 cm in length. The total volume of the resin 15 bed was about 1 ml, which was approximately equivalent to an exchange capacity of 1.4 milliequivalent. After thorough washing with deionized water a radioactive sample solution was introduced onto the column, which was again washed with deionized water until no radioactivity could be detected by liquid scintillation counting in the effluent. Fractional elution of the organic acids from the resin column was effected by the use of a pH-gradient provided by a simple apparatus described by Palmer (61). The gradient elution was initiated by introducing 3 N acetic acid from a reservoir flask into a mixing flask containing 200 ml of deionized ‘water. A constant pressure of 3 pounds per square inch was applied to the reservoir flask in order to maintain a flow rate of 1-2 ml/min., and the effluent was collected in 4-ml fractions with a Gilson fraction collector. After 500 m1 of 3 N acetic acid had been used, the reser- voir was refilled with 3 N formic acid, leaving the acetic acid solu- tion in the mixing flask, and the gradient elution was continued. The peaks of radioactive acids in the effluent were first approximately located by counting 0.1 or 0.2-m1 aliquots of every other tube in 10 ml of Kinard's liquid scintillator, and then precisely located by counting every tube around each peak. The radioactive compounds contained in each peak were collected by evaporating the combined solution to dryness with a shaking evaporator, and were then analyzed by paper radioautography. Ggs-liguid chrgmatogrgphg: Gas-liquid chromatOgraphy of organic acids as their trimethyl- silyl (TMS) derivatives was performed by the method of Sweeley, et al. (83). An F‘& M Scientific 402 high efficiency gas chromatograph 16 (Hewlett-Packard, F‘& M Scientific Division, Avondale, Pennsylvania) equipped with a 3% 0V-1 column (6 ft x 0.25 inches) coated on Gas- Chrom Z, 80-100 mesh, was used. The carrier gas, Argon, was passed through the column at an optimal flow rate of approximately 50-60 ml/min., and the effluent gas was Split with a device into two streams, one leading to a hydrogen flame ionizing detector and the other to a collection port (18), which enabled the collection of fractions of the TMS derivatives by inserting a long-tip, disposable pipette. A trimethylsilylating reagent (96) made by mixing in order 1.7 ml anhydrous pyridine, 0.2 ml hexamethyldisilazane, and 0.1 ml tri- methylchlorosilane, was used for trimethylsilylation of organic acids. One-tenth ml of the fresh reagent was added to 0.1-1.0 mg of authentic organic acids, or 1,000-100,000 cpm of unknown organic acids which had been collected from an anion exchange column and dried overnight in a centrifuge tube over KOH pellets in a vacuum desicator. The mixh ture was shaken vigorously for about 30 sec. and allowed to stand for 5 min. or longer at room temperature, and then a 1 to 301pl aliquot was injected with a microsyringe into the column for chromatography. The chromatography was ran either by linear temperature-programming of SO/min. starting at 100°, or under isothermal conditions at a desired temperature for better resolution (83). 'While peaks of TMS derivatives in the effluent were located by an automatic recorder connected to the hydrogen flame ionization detector, the major portion of the radioactive derivatives was collected at the collection port in fractions. The radioactivity from each collection was dissolved in 10 ml of Kinard's liquid scintillator and counted. 17 Cochromatography was accomplished by mixing a radioactive acid sample with a nonradioactive, authentic acid in a centrifuge tube, drying, trimethylsilylating, and then chromatographing the TES derivatives. Fractional collection of the radioactive TMS derivative was programmed in accordance with the appearance of the peaks due to the nonradioactive derivatives recorded on a recording chart. Degrgdgtion of 1“Cuogganic gcids: lac in the molecules of 1“Caorganic acids, The distribution of purified either by paper chromatography or by anion exchange chroma- tography, was determined by a modified technique (1a) of Sakami's procedures (66) for the degradation of serine, which gave 1 mole each of 002, HCOOH, and HCHO on periodate oxidation at room temperature. The degradation vessel was a 50 ml, 3-necked, pearbshaped flask fitted with a separatory funnel for adding reagents, a condenser with an attached C02 trap, and an inlet tube for aeration. The degradation followed three separate steps as outlined below. The first step was cleavage of serine into 1 mole each of C02, HCOOH, and HCHD by periodate oxidation at room temperature. To the flask were added 1,000-10,000 cpm of the 1“Cuorganic acid in less than 1 ml water, 0.2 mmole (21 mg) of nonradioactive DL-serine in 1 ml water as a carrier, and 2 ml of 0.5 M phosphate buffer at pH 5.8. Then, to the closed system, 0.75 mmole (160 mg) of NaIO in 3 ml water 4 was introduced through the funnel and the system was aerated at room temperature for an hour. Most of the excess NaIO“ was consumed by reaction with additional 0.2 mmole of carrier serine during another hour of aeration at room temperature. The second step was oxidation of HCOOH to C0 by boiling with 2 18 HgClZ. After adding 3 ml of 1.5 M phosphate buffer at pH 2.5 to the flask, the System was closed and 1 g of HgClZ, dissolved in 5 ml of hot water, was introduced through the funnel. The system was then aerated for an hour while boiling gently. The last step was the oxidation of HCHO, as well as all other carbon compounds, to CO2 by boiling with KZSZOB. After adding 1 ml of 5% AgNO3 solution to the flask, the system was closed and 1.5 g of K dissolved in 5 ml 232°8' of hot water, was introduced through the funnel. The system was first aerated for 45 min. with gentle boiling, and then aeration continued 30 min. longer with vigorous boiling. In some cases, for the determination of the 1“C-activity in ECHO formed by periodate oxidation, the second step was modified by eliminating the second addition of 0.2 mmole carrier serine to decomp pose the excess NaIO“. Instead7the closed system was heated in the presence of the excess NaIO“ which oxidized both HCOOH and HCHO to C02. The increase in 14COZ-yield in this step over that in the normal HgClz-oxidation step was considered as the 1“bu-activity due to HiuCHO. 1“Cu-Activity not recovered after this step was attributed to carbon atoms in molecules other than C0 HCOOH, and HCHD. 2’ During each step of the degradation, CO evolved was carried 2 by aeration through the condenser and introduced into a U-tube with a sintered glass filter fused at the middle. The U-tube was filled with 5 ml of’COz-trapping solution (100) which was prepared as follows. Five grams of PPO and 100 mg of POPOP were dissolved in a solution of 270 ml redistilled phenethylamine (b.p. 65-670 at 6 mm Hg pressure) and 270 ml absolute methanol, and then diluted to 1 liter with toluene and stored in the dark. After each step of the 19 degradation procedure the trapping solution was carefully transferred to a counting vial, and the trap was rinsed twice with 5 ml of scin- tillation fluid (100), made by dissolving 5 g PPO and 100 mg POPOP in 1 liter of toluene. The final volume of the counting solution in the vial was approximately 15 ml. The sum of radioactivity in cpm obtained through the three steps of degradation was calculated as 100%, and the % distribution of 140 in C0 HCOOH, HCHO, and other 29 carbon compounds was calculated on this basis. Determination of spegific activities of carbon atoms: Determination of the specific activity of carboxyl (i.e., 1 and 4) and the middle (i.e., 2 and 3) carbon atoms of mgggrtartaric acid molecules was performed essentially according to the method of Van Slyke, et a1. (89). The first step of the degradation by perio- date oxidation was the same as that described for serine degradation, except that 0.2 mmole (33.6 mg) of nonradioactive gasp-tartaric acid was used in place of serine as the carrier. An important difference was that the closed system had been flushed with N gas for 15 min. 2 before periodate was introduced into the flask, and instead of aeration, the N2 gas was continuously passed through during the entire process. The decomposition of excess NaIO“ by the second addition of nonradioactive carrier was omitted. The second step of the degrada- tion by HgClZ-oxidation was the same except that N gas was continu- 2 ously passed through the closed system. The CD2 evolved in each step was trapped in a known volume and concentration of a Ba(0H)2-BaCl2 solution (88). The recovery of C02 in each step of the degradation was calculated by back-titrating the Ba(0_H)2-_BaCl2 trap with a standardized HCl solution. Planchets were then prepared from the 20 BaCO3 suspensions and, after drying to a constant weight, the radio- activities were determined with a Nuclear Chicago low background gas flow counter, Model C 115. The specific activity of each carbon was calculated by the following equation: A Ni x mg C in sample + mg—C in blank FNk mg C in sample where A gives specific activity in mpc./mg C; N8 is observed sample count; F, "infinite thickness" factor, determined from a self- absorption curve (65): and Nk’ the counter constant, determined from a BaCO3 sample produced by a total combustion of standard benzoic- 1“C acid. Large-scale prgparation of unknown acids excreted by As braunii: Fully mature or dividing cells of‘é, braunii were collected by centrifugation from a synchronized culture between the 3rd and the 4th hour of the dark period. They were washed and resuspended in an enough volume of 0.001 M.phosphate buffer to give a final concentra- tion of 0.5-0.8% algal cells. A photosynthetic experiment in NaHluCO 3 was run with 80 ml of the suspension in a medium-size lollipop, and a large-scale experiment using air but no 1“(002 was carried out in parallel in a "high form" Fernbach flask containing 200-400 ml of the suspension. The flask was placed between two diphtheria toxin culture bottles filled with water for absorption of heat, and illum- ination by two 300-watt KEN-RAD lamps, one from each side, was con- tinued for 30 min. while the suspension was slowly aerated with air and occasionally shaken. 21 After 30 min. the supernates were collected separately by con- trifugation, then filtered through a Millipore filter and stored at -18°. A 2 to 4-ml portion of the radioactive supernate was evaporated to dryness and analyzed by paper radioautography. In case the 1LPG-- activity in the unknown acids matched or exceeded that in glycolic acid, the remaining portion of the radioactive supernate was combined with the larger volume of the nonradioactive supernate, and the mix. ture was evaporated to a small volume with a rotary evaporator (Buchler's Flash Evaporator). The concentrated supernate was then passed through a column of cation exchange resin, AG50 in H-form, and the column was thoroughly washed with deionized water. The eluate and the washing were combined in a test tube and again evaporated to a small volume with a shaking evaporator. The concentrate was neutra- lized and introduced onto a column of anion exchange resin, A81 in acetate form, and then chromatographic separation of the acids was carried out by pH—gradient elution as described before. Radioactive acids in each peak were analyzed by paper radioautography, and peaks containing the unknown acids were saved for further purification by repeated anion exchange chromatography. RESULTS AND DISCUSSION Photosynthetic COB-fixation and eycretion by synchronized algal cells during their life gycle: The life cycle of A, braunii cells, kept synchronous by a 16-hr light and 8-hr dark regimen with 1 to 4 dilution at the end of each dark period, is depicted in Table 1. Along the life cycle, the algal cells were collected at different stages of development, and 5-min. photOSynthesis and excretion experiments were carried out as described in methods. The changes in photosynthetic activity, as measured by 1“Cog-fixation and 1“'Cuexcretion for 5 min., are shown in Figure 1 and also summarized in Table 1. The photosynthetic ability, measured by 1“C0 -fixation and 2 14 expressed as C-activity fixed per unit volume of cell suSpension, followed a distinct trend: growing cells were more active than mature cells, and mature cells more active than dividing cells. 14 Since the amount of COZ-fixation at the peak of the activity, 1.6., at 6 and 10-hr stages in the light period, exceeded 90% of the NaHluCO added, the real photosynthetic ability at these stages was 3 probably even higher than the values in Figure 1. A similar trend for change in photosynthetic ability as measured either by 0 2 evolution, C02-fixation, or relative quantum efficiency during the life cycle of synchronized algal cells has been reported by Sorokin and his coworkers for Chlorellg species (71, 72, 74), and by Kates and Jones for Chlamydomongs geinhardtii (no). Sorokin (73). with 22 23 TABLE 1 A Life Cycle of A, braunii Cells in A Synchronous Culture and Their Relationship to Photosynthetic Activity 4 Stage of Morphological Observation Photosynthetic Life Cycle Activity* Pictorial Description Drawing Light, 0 hr 6 j Daughter cells; Low, not growing increasing Light, 2 hr fl fl Fast growing cells Higher, increasing Light, 6 hr é? 67 Growing cells; Highest, cannot divide still if put in dark increasing Light, 10 hr Premature cells: Highest, can hardly divide started if put in dark decreasing Light, 14 hr Mature cells: Low, ready to divide decreasing Light, 16 hr (Dark, 0 hr) starting to divide decreasing Dividing cells: Lower, daughter cells still not yet released decreasing fl 5’ fl Fully mature cells; Lower. i i Dark, 4 hr Dividing cells; Lowest starting to release daughter cells fl a a 2 m. 5 i % Dark, 6 hr ;;9 Dividing cells; Low, still releasing increasing daughter cells a 0 Dark, 8 hr 00 ’0 Daughter cells; Low, (Light. 0 hr) 0 0 not growing increasing ‘— *As measured both by 1“C0 -fixation and inc-excretion. (For details see Figure .) 24 685 0 H33 93 co eouoaowo 3a 03a & .codmcoamsm HHoo RH HE\.CHE m CH venomous hud>apowo mo octoponHHoo “m .Au: 0H .pgmev mHHoo wadsouw mo pommuxosHHoo “Q .p:o>Hom psooom one Scam poppfiso was momz pump pmooxo .m we came one no .Aas : .xampv mHHoo mcapfi>ap Seam mpmcaogom am .An: OH .psmwflv mHHoo mcwsoam Eonm camshomsm “d 2::an .« co ofioo eooaooogoém an m8 moz 5.. oaoofiiooooé .cfisnm nopm< mpoanuKMIHHoo pas mopmcnommm mo mssmmopomoaosm N MMDGHM A . . GLYCOLATE 'ls'a- é '- GLYCOLATE BUTANOL- PROPIONIC ACID - WATER - NAOH -——) . ORIGIN . «a '<— PHENOL-WATER D GLYCOLATE GLYCERATE ,_ MALATE ALAN'NE GLUTAMATE ASPARTATE GLYCINE SE‘RINE P-PYRUVATE e a ‘ PHOSPHATE ESTERS ORKNN \) .'.“ MALATE CITRATE U, aq.’ U2 U3 BUTANOL- PROPIONIC ACID - WATER - NAOH——) ORIGIN a ALANINE I], h n \‘J C GLYCOLATE BUTANOL - PROHONIC ACID - WATER ’ ORIGIN e‘ I GLYCOLATE GLYCERATE MALATE 'I GLUTAMATE CITRATE ASPARTATE SE RINE ~. P-PYRUVATE PHOSPHATE ES TE RS _ w:- a... x ‘ e ORIGIN F] 30 TABLE 2 Amount and % Distribution of :4C in the Compounds Excreted During 5-min. Photosynthesis in NaH1 CO by Synchronized Cells of‘A. braunii. Values are cpm x 103/ml 1% cell suspension; in parentheses are the % of the total 140 excreted. —_ a Compound Excreted Stage of Glycol- Isocitric meso- Life Cycle ate - Malate Lactone Tartrate U3 Total (U1) 012) Light, 0 hr 38.9 1.7 0.2 20.7 0.5 62.0 L- (62.7) (2.7) (0.3) (33.5) (0.8) Light, 2 hr 235.4 0.3 0 9.3 0 245.0 (96.1) (0.1) (3.8) Light, 6 hr 735.1 0 0 5.9 0 741.0 (99.2) (0.8) Light, 10 hr 1,044.0 O 0 0 0 1,044.0 (100.0) Light, 14 hr 343.3 0.3 0 11.4 0 355.0 (96.7) (0.1) (3.2) Light, 16 hr 80.5 1.0 0 19.0 0.5 101.0 (Dark, 0 hr) (79.7) (1.0) (18.8) (0.5) Dark, 2 hr 28.3 0.8 0.1 18.5 0.3 48.0 (59 0) (1 6) (0.2) (38.6) (0 6) Dark, 4 hr 10.9 1.1 0.6 14.2 2.2 29.0 (37 8) (3 8) (2.0) (48 9) (7.5) Dark, 6 hr 23.6 1.5 0.5 20.9 1.5 48.0 (49.1) (3.2) (1.0) (43.5) (3.2) Dark, 8 hr 42.8 2.1 0.5 25.3 1.3 72.0 (Light. 0 hr) (59.4) (2.9) (0.7) (35.1) (1.9) 31 excreted compounds and elucidation of the physiological conditions responsible for their excretion have been a major effort to be reported in this thesis. In subsequent sections will be given results for the identification of U1 as isocitric lactone and of U as meso—tartrate. 2 U3 remains unidentified. The radioactivity of all the Spots on a radioautogram of a supernate was added to make 100%, and the % distribution of radioactiv- ity among the compounds excreted was computed on this basis (Table 2). The actual radioactivity in cpm of each compound excreted by 1 ml cell suspension was then calculated from the % distribution and the respective amount of total 14C-excretion as shown in Figure 1. It is obvious from the data shown in Table 2 that maximum amount of glycolate was excreted by the rapidly growing cells and that mature or dividing cells excreted much less glycolate. Maximum amounts of the other five compounds were excreted by mature or dividing cells, or in other words, the excretion of the other five compounds was approximately in a reciprocal relationship to glycolate excretion. This relationship is also shown in Figure 3 by plotting along the life cycle the change in the 1[JO-activity in glycolate excreted, together with that of total excretion. Thus total 1“Cuactivity in the other five compounds is represented by the difference of the two curves, i.e., the shaded area in the Figure. The two different patterns of photosynthetic excretion are further clarified in Figure 4 by plotting along the life cycle the change in % distribution of 1“C among the compounds excreted. Figure 4 emphasizes that the curve for glycolate excretion is complementary to that for‘mgggptartrate excretion. Excretion of‘U followed the 3 same trend as that for meso-tartrate, but the results were less clear, 32 N .m: es. s .H D .03de .opmaocham mo soapomoxm “some popasm .cofimcommom HHoo ma dismo 5“ 2930988 opmHooanHu OIIO .SOHmcommam HHoo RH HE\Emo CH cowponoxo Hooch UIIU flmmmmmm .«.oo aaaoo oosasossosam so moosfimez on onoososaoooosd .sasim mosses ooooooam aosooasoo one on aoasaoossosfl m mmDmHm 33 el foo [Iv 3:55 as; O. :33 — O_ gOI x wdo 34 .AHDV onopoeH OHavHoomH CH .opeams :H . 5 CH .ANDV opmapaspuomos sH .opoHoohHw CH :oaponokosodH R I I so ohoxol coHpoaoxolu: HR Drvlfl coa ohoxol .o eon a A‘r.naV :oHpvoKonoeH R TO “assess .< oo eHHoo ooeasoesosam as no oqfimez on oasesosaoososm .cfissm wcHaea ooponoxm mpcsomaou esp woos : mmDon co soapsoaeoasa a 35 \ $.85 oEP sauteohnomos 22330 2e... Om UO!IeJoxa-0,. °/. 00. 36 perhaps because of less formation during 5-min. photosynthesis. Rates of photosynthetic 1“Cog-fixation and 1uC-excretion with synchronized cultures of Scenedesmus obliguus, Scenedesmus uadricauda, and Chlamydomonas reinhardtii were examined only qualitatively. Analy- ses of the supernates revealed that glycolate excretion was maximum by growing cells and minimum by dividing cells. However, glycolate was the only major compound excreted by these algae throughout their life 4“ cycle, and no formation in appreciable amounts of the unknown compounds by mature or dividing cells was observed. Since the synchronous algal cultures were not strictly sterile, and some bacterial cells were sometime observed microscopically, there existed the possibility that the bacterial growth might have been favored during the dark period, and that the bacteria might have changed the amount of glycolate excreted by mature or dividing algal cells in relation to the unknown compounds. This possibility, however, was eliminated by two facts. First, mature or dividing cells of A, braunii fed either glycolate-luC or phosphoglycolate-lac in the light or dark failed to metabolize the radioactive compounds. Secondly, mature or dividing algal cells of other Species grown under the same circumstances and conditions did not excrete significant amounts of 1"co . It 3 was therefore concluded that these compounds in the supernates were the unknown compounds during 5-min. photosynthesis in NaH formed and excreted by the algal cells, that glycolate was the major excretion product of all the algae, and that excretion and accumula- tion of large amounts of the other compounds were characteristic of mature or dividing cells of A, braunii. However, conditions for detecting the excretion of the unknown compounds by the other algae 37 have not been carefully studied. Kinetic studies of the photosynthetic excretion: Kinetic studies were performed to examine further the mode of formation and excretion of those compounds during photosynthesis, and to establish the best conditions for isolation of enough of the unknown compounds for identification. During the course of the standard photo- Hpia “31‘ synthetic experiments in NanCO3 with A, braunii cells at different developmental stages (as described in Table 3). 2-ml portions of the cell suspension were removed after 1, 2, 5, 10 and 30 min. The super- nates were separated with Millipore filters and analyzed.both for total 1L‘C-activity by scintillation counting and for radioactive com- ponents by paper radioautography. The % distribution of lac among those compounds excreted at each stage of development was computed from the respective radioautogram, as described before, and then the actual 1L‘C-activity in cpm of each compound per ml cell suspension was calculated (Table 3). In almost all cases, the amount of an excreted compound increased with the increasing period of photosynthesis. The rate of increase in amount, however, varied from one compound to the other, and thus there occurred different values for the % distribution of the excreted com— pounds during the course of photosynthesis. .In most cases the percent of lac excreted as malate, glycerate, and mgggptartrate was the highest (as underlined in Table 3) within 1 or 2 min. of photosynthesis, while the total amount of excretion was still low. Isocitric lactone and U3, 14 in contrast, contained a consistently increasing percent of the C excreted with increasing length of photosynthesis, implying that they 38 TABLE 3 Amount and % Distribution of 1“c in the COTpounds Excreted During Different Periods of Photosynthesis in NaH CO by Synchronized Cells of‘A.‘braunii. Values are cpmhx 103/ml 1% cell suspension; in C parentheses are the % of the total excreted. Stage of Compound Excreted Algae and Glycol- Glyc- Isocitric meso- 4 Length of ate Malate erate Lactone Tartrate U Total in Synthesis (U1) (02) 3 2 Dark, 0 hr 1 min. 0.58 0.21 0.14 0 0.32 0 1.25 (46.8) (16. (11,4) (25.3) L 2 min. 1.36 0.26 0.22 0 0.81 0 2.65 (51.4) (9.8) (8.1) (ll-Z) 5 min. 22.40 0.39 0.36 0.14 4.30 0.16 27.75 (9.9.22) (1.4) (1.3) (0.5) (15.5) (0.6) 10 min. 50.63 0.85 0.92 0.92 9.80 7.88 71.00 (71.3) (1.2) (1.3) (1.3) (13.8) (11.1) 30 min. 71.38 3.78 2.70 69.76 33.53 359.60 540.75 (13.2) (0.7) (0.5) (12,2) (6.2) (66. ..... 7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Dark, 3 hr 1 min. 0 0.29 0 0 0.86 0 1.15 (2 .0 (25.0) 2 min. 0 0.32 0.19 0 1.73 0.16 2.40 (13.3) (8.3) (72.0) (6.7) 5 min. 0.30 0.38 0.21 0.07 4.14 0.85 5.95 (L1) (6.3) (3.6) (1.1) (69.6) (14.3) 10 min. 0.58 0.46 0.23 0.30 9.54 2.69 13.80 (4.2) (3.3) (1.7) (2.2) (69.1) (19.5) 30 min. 1.83 1.67 0.76 18.00 63.29 66.95 152.50 (1.2) (1.1) (0.5) (11.8) (41.5) (42.2) 39 TABLE 3 (Continued) Stage of Compound Excreted Algae and Glycol- Glyc- Isocitric meso- Length of ate Malate erate Lactone Tartrate U Total Synthesis (01) (U2) 3 Dark, 5 hr 1 min. 0 0.31 0 0 0.99 0 1.30 (24.1) (75.9) 2 min. 0.10 0.35 0 0 2.60 0 3.05 (3.3) (11.4) (82.3) 5 min. 8.92 1.37 0 0 8.21 0 18.50 (48.2) (7.4) (44.4) 10 min. 27.72 2.03 0 0.24 16.51 0.80 47.30 (58.6) (4.3) (0.5) (34.9) (1.7) 30 min. 143.21 4.52 0.59 7.07 27.90 13.16 196.45 (2222) (2.3) (0.3) (356) (14.2) (612) ..... J------------..---..-........----.....-.. Dark 8 hr (LightI 0 11%) 1 min. 2.31 0.40 0 0 2.35 0.14 5.20 (44.5) (Lg) (511.2) (2.7) 2 min. 6.50 0.46 0 0 5.12 0.37 12.45 (52.2) (3.7) (41.1) (3.0) 5 min. 43.85 1.92 0 0.07 22.89 2.57 71.30 (61.5) (2.7) (0.1) (32.1) (3.6) 10 min. 106.19 3.82 0 0.95 41.55 6.69 159.20 (66.7) (2.4) (0.6) (26.1) (4.2) 30 min. 524.40 12.77 0 16.32 68.12 87.99 709.60 (2L2) (1.8) (2.3) (9.6) (12.4) 40 TABLE 3 (Continued) Stage of Compound Excreted Algae and Glycol- Glyc- Isocitric meso- Length of ate Malate erate Tartrate 03 Total Synthesis (U2) Light, 2 h; 1 min. 3.12 O 0 0.03 0 3.15 (98.9) (1.1) 2 min. 16.79 0.02 0 0.29 0 17.10 (98.2) (0.1) (1.2) 5 min. 173.84 0.35 0 1.76 0 175.95 (98.8) (0.2) (1.0) 10 min. 304.72 0.31 0 2.46 0.31 307.80 (99.0) (0.1) (0.8) (0.1) 30 min.1,)41.85 1.05 0 4.22 7.38 1,054.50 (98.8) (0.1) (0.4) (0.2) Light, 8 hr 1 min. 5.80 0.26 0 0.29 0 6.35 (91.4) (4.1 (4.5) 2 min. 229.69 0.23 0 0.23 0 230.15 (99.8) (0.1) (0.1) 5 min. 976.00 0 0 1.95 0 977.95 (99.8) (0.2) 10 min.1,323.17 0 o 3.98 0 1,327.15 '(99.7) (0.3) 30 nin.:3463.45 3.50 7.00 20.95 3.50 3,498.40 (99.0) (0.1) (0.2) (0.6) (0.1) f..- 41 are later products of carbon metabolism. The most complicated and probably of most interest were the patterns for the percent distribution of 1“C in glycolate during the course of photosynthesis. When the algal cells were rapidly growing and excreting glycolate in a large amount, i.e., at the stages of 2 and 8-hr of the light period (Table 3), glycolate contributed more than 98% of the total excretion throughout the 30-min. photosynthetic course. At the stages of 0 and 3-hr of the dark period, when the algal cells had just started dividing, the maximum percent of 1”C excreted in glycolate appeared only after 5-min. photosynthesis. This fact, together with the observation of the earlier appearance of the maximum percent of the 1LIC-excretion in mpggytartrate and that the excretion of glycolate and mgggrtartrate were complementary to each other (Figure 4), implies a possibility that‘mgggrtartrate might be a metabolic precursor of glycolate, or that the two excreted acids might have a common photosynthetic precursor. During the early stages (0 and 3 hr) of the dark period, the maximum percent of 14C excreted as glycolate occurred after 5 min. of photosynthesis. During the later stages (5 and 8 hr) of the dark period, however, it occurred after 10 to 30 min. of photosynthesis. This shift could be due to changes in the algal cells from one stage to the other even during the photosynthetic period, and it could also indicate that the daughter cells were recovering in the dark their capability of normal photosynthesis that the young, growing cells possess. From the above results, the best condition for the formation of the largest amounts of the three unknown compounds (U1, U2 and U3) was the use of mature or dividing cells of A, braunii at the early stages 42 (0 to 4 hours) of the dark period. Photosynthesis in NaHluCO3 should continue at least for 30 min. Thus, highest yields of the unknown 14 C-compounds with the least accompanying glycolate-inc could be obtained with A, braunii cells from a well synchronized culture at an early stage of the dark period. From many experiments with well Syn- chronized cultures kept for many generations, the percent yield of excretion. In other words, even when using the best synchronized 14 cultures at the right time, one to two-thirds of the total C 14 the unknown C-compounds ranged from 30 to 70% of the total CZE"..1"I ‘ _ excreted was in glycolate-iuC. Occasionally, however, the use of a Fill... less synchronized culture obtained by dark incubation for one or two days of a dense culture of premature cells followed by dilution and illumination, as described by Stange, et a1. (79), resulted in much higher percent yields, sometimes nearly 100%, of the unknown compounds. It seemed, therefore, that newly synchronized cultures gave a higher percent yield of the unknown compounds. Although the reason is not known, it seems reasonable to assume that changes in percent C- distribution among the excreted compounds during the algal life cycle were determined by metabolic shifts, which are internally regulated in connection with differentiation. Preparative separation and partial purification of the compounds excreted: The procedures described in Methods for large—scale preparation of the unknown organic acids were followed. Photosynthesis, either in NaHluCO3 or in the air for 30 min., were carried out with mature or dividing cells of A, braunii at an early stage (3 to 4 hr) of the dark period. The supernate was separated and analyzed by paper chromatog- 43 raphy and radioautography. Because the separation of a large volume of supernate by centrifugation followed by filtration was a slow process, more compounds, in addition to those mentioned before, such as succin- ate, lactate, aspartate, glutamate, serine, glycine and sucrose, tended to leak out of the cells and appeared in the supernates. Only super- nates with the highest percent of 11+C (more than 40%) in the unknown compounds were saved and used for further separation and purification. ‘When the concentrated supernate was passed through a cation exchange column packed with AG5OW resin in H-form, the eluate showed by paper chromatography no loss of any of the unknown compounds, indicating that none of them were positively charged. Any amino acids that might have been present in the supernate were removed by this procedure. When the eluate was subsequently passed through an anion exchange column of A01 resin in acetate form, the eluate contained according to paper chromatographic analysis only sugars, in most cases sucrose. Glycolate and all of the unknown compounds were retained on the column. It was clear, therefore, that all of the unknown compounds bore only negative charges. The A01 column, binding the three unknown organic acids as well as some known organic acids, was then subjected to pH-gradient elution as described in Methods. The radioactive peaks were located by scin- tillation counting, and their contents and identity were checked by paper chromatography and radioautography. The pattern of separation of those known and unknown organic acids by two-step pH-gradient elu- tion with acetic acid followed by formic acid gradient, is ideally depicted in Figure 5, A and B. Minor components are shown as smaller peaks in the Figure. .xneHu mosses one on asasaesos one. oases. one no as com ease sends me: oHos OHEhom z m .4 pcodoonm ho HE oom msfipsao poems “COHpsHo ponneam oHoe OHEpom Am .nopcz HE ooN opsH noxfis was pace 0Hpooo zm ”cOHpsHo psoHoopm pHoa 0Hp00< A4 .I :EdHoo :Hmem Ho< so ands Handmpn .< ho poochoxm moHo¢ OHCsmno mo :oapdflm psofipmhoimm m mmDon 45 .2 8m 08 00. I H H m n: m: 8:5 2285 23 258.1 a .5 00¢ 00m CON . . 00. ll|< _ <_ _ _ _ _ 3:28.. ottoom: .3 3569...: _.3 _ _ __ _ _ 2.22 2 _ _ 05626: I 2 2 __ = . . t ms z .8 8.576» -ommEv N3 9.0030 8:5. 2286 22 osooq 3 Rig/(110v 3w GMIDIGH Mgnuov Dbl OMIDIGH 46 Since the pH-gradients were not perfectly reproducible with the use of such a simple apparatus, retention volumes of the organic acids varied from one experiment to the other, eSpecially for those acids with larger retention volumes. Nevertheless, the order of emergence of the acid peaks remained the same, provided that the samples were treated in the same way before chromatography. The range of variation of retention volumes of the acids, tOgether with their mean values which were used to compose Figure 5, are listed in Table 4. Although some characteristics of these acid peaks are shown in this Table, more details about the unknown acids will be given later in the sections concerning their purification and identification. Since some of the acid peaks were so close to each other, separa- tion and purification of one component from the others usually required repeated passage through the anion exchange columns. After each step of column chromatOgraphic purification, the identity and purity of each peak were checked by paper radioautography. A fast, one-dimensional separation with the butanol-ethyl acetate-formic acid system was found satisfactory for this purpose. The approximate location of the acid spots on such a chromatogram is depicted in Figure 6, together with a typical two-dimensional chromatogram, in which location of some other compounds of interest is added for reference. Samples of any radioactive compounds, pure in terms of radio- activity, could be obtained by eluting a Spot from a paper radioauto- gram. These samples, however, were not chemically pure, and when they were chromatographed again on paper and Sprayed with a bromcresol green solution, several yellow Spots not in coincidence with the radioactive spot were visualized. The contaminates were probably from the paper TABLE 4 Retention Volumes for Organic Acids Excreted by'A, braunii on an AGl Resin Column after pH—gradient Elution Organic Acid Retention Volume Range Mean Value Glycolic Succinic Malic 02 (mppprTartaric) U 1 a 2 "* U2 U1 (Isocitric lactone) U1' (Isocitric) * Acetic acid gradient elution 20 - 44 ml 32 ml 44 - 48 46 64 - 72 68 72 - 124 96 28 - 52 40 124 - 208 160 260 - 480 370 140 - 240 190 Formic acid gradient elution 44 - 104 74 120 - 280 200 *More detailed classification and description will be given in sections concerning their purification and identification. 48 senses 3.8an .393 8Q 2084 3595 .8 was: oHcmmamop ..aa .< encepfio m .opeauaoomH soapohowm maonpo D masseuse .e:opoeq opeaez opeaoozHu Hence psosaaomxm tomes afihpfioomH popoaoxm messedsoo .COfipsnaApmdp R new one momenpcoawm ca mosHeb “scamsoamzm HHoo RH mo HE H mam.:ds oco ca Uopoaoxo :HLEQO one mozdu> .mpcoEpaoae acceoQMfiQ madman mopesnopm< use soapexdm m mqm ___> ___> (Eq . 6) HZOH fHO HC-OH H -OH COOH COOH COOH COOH We have considered this scheme for‘pppprtartaric acid formation, because the biosynthesis and excretion of mppprtartaric acid and glycolate had a reverse relationship and because the excretion of these two acids along with isocitric lactone was unique. The first enzyme of this pathway is glycolate oxidase, the second step has 80 not been investigated and the third step could be similar to equation 5. Rather from the extensive work of Kornberg's group (05) and Vennes- land's group (06) with bacteria, the oxaloglycolate was decarboxylated to hydroxypyruvate which was then reduced to glyceric acid. No evi- dence has been published for the scheme in plants or algae, and in fact, Tolbert's group has cited extensive data to indicate that this scheme does not function for glycerate formation from glycolate in plants (85). The pathwgy for ppppytartaric acid biosynthesis as shown in equation 6 could not explain the formation of carboxyl labeled t? material. Glycolate molecules have always been found to be uniformly labeled in the shortest periods of 1[+002 fixation (12, 33, 30, 60, 68). So the pppprtartrate molecule formed from glycolate Should consequently be uniformly labeled. Furthermore, Hess and Tolbert (30) have found that algae were devoid of glycolate gxidase, which is reSponsible for the conversion of glycolate to glyoxylate. Thus, alternative path- ways for‘pgpprtartaric acid synthesis must be considered. Oxaloglyco- late might be formed, in analogy to the reactions catalyzed by pyruvate synthetase (19) andeQrketOglutarate synthetase (11), by ferredoxin-dependent carboxylation of tartronyl CoA, as proposed in equation 7. SCoA COOH cso (i=0 110-011 + co2 + Fdred —_> HCI"0H + CoASH + Fdox (Eq. 7) ('30011 COOH Tartronyl CoA 0xa10glycolate 81 No evidence exists for this enzyme. Another possible pathway for the formation of carboxyl labeled ppgprtartaric acid is analogous to the reaction catalyzed by malic enzyme (59), which would result in the reductive carboxylation of hydroxypyruvate (Eq. 8). goon coon 4. 0:0 + #00 + NADPH ——3 nc-0n + NADP . 8 mt ' 2 <7... | (Eq ) cnzon Hi-OH *coon The proposal by Marco (52, 53) that glycolate is the precursor of meso-tartrate in plants is rendered impossible for the algae by the results from experiments on the excretion of labeled acids after 14c02.rixation. As Shown in Tables 9 and 10, the for- mation and excretion of glycolate were completely inhibited in the photosynthetic dark, but‘pggprtartrate Synthesis in the dark was increased. This fact rather implies that‘ppgprtartrate might be a precursor of glyco- late. In such case, the reaction sequence in equation 6 might be reversed. Mppprtartrate would be first dehydrogenated to oxaloglyco- late, which, according to Kun and Hernandez (08), is unstable and either rapidly Splits into two molecules of glyoxylate or decarboxyu lates to hydroxypyruvate. The glyoxylate would then be reduced to glycolate by glyoxylate reductase which is present in algae. However, this pathway of glycolate formation from ppgprtartrate is unlikely for two reasons. First, the amount of increase in 14C-activity inlmgggr tartrate in the dark was too small in comparison to the large amount of 1“Ca-glycolate formation which was shut off. Secondly, carboxyl labeled mesa-tartrate will give rise to carboxyl Labeled glycolate, 82 rather than uniformly labeled glycolate, which has always been observed. Mention might also be made that the CO condensation hypothesis for 2 glycolate biosynthesis proposed by Stiller (82) and Zelitch (101) could neither be eliminated nor be substantiated by the labeling patterns in ppgpytartrate. A more likely metabolic relationship between ppgprtartrate and glycolate is that they are both derived from a common precursor X , rfifiE and that the pathway A, leading to glycolate,is light-dependent, while I pathway B, leading to mpgprtartrate, occurs in the dark. The relative ; activity of the two pathways could be regulated by changes which occur Glycolate (uniformly labeled) [Y] Pathway A: light-dependent 5' 7‘ [X] ‘\\\\::thway B: light-independent mesa-Tartrate (carboxyl labeled) during cell differentiation. Thus, pathway B would be more active Photosynthesis \ 002 , or dark fixation when the cells are dividing, while pathway A is predominant when the cells are growing. When pathway A is shut off in the dark, the for- mation of mggprtartrate can be increased, but not necessarily in direct proportion toreduced.glycolate biosynthesis. For this scheme to confirm with the labeling data, it is necessary to assume the existence of intermediate X which should not be uniformly labeled. However, in pathway A between }( and glycolate, there should exist a 10 symmetric intermediate Y , so that the C label becomes randomized in glycolate. The physiological significance of the excretion of meso-tartrate 83 is not known, just as the Significance of glycolate excretion remains unexplained. While glycolate was considered by Hess and Tolbert (30) as an end product of algal carbon metabolism, Vickery and Palmer (92) made a similar speculation concerning the formation of (+)-tartrate in tobacco leaves. In ana10gy, glycolate and mggprtartrate may also be considered aS end products of carbon metabolism.by growing and dividing cells, respectively, of A, braunii. Experimental data of 77‘ this study suggest that the formation and excretion of glycolate and mpgpptartrate may be related to each other, and that glycolate excretion must be somehow related to the photosynthetic processes. g.. No relationship could be established. Feeding radioactive meso- tartrate (U2) to A. braunii cells at various stages of development resulted in negligible uptake by the cells, and no conversion of the radioactive compound was observed by paper chromatography and radio- autography. Discussion concerning isocitric lactone excretion: The reason why the algae excrete the lactone rather than the free isocitric acid is unknown. In comparison with the excretion of glycolate and Eggpgtartrate, isocitric lactone may be excreted because it cannot be further metabolized. Isocitric lactone, though occuring in preparations of isocitric acid, has not previously been thought to be a naturally occuring compound or to be synthesized by plants. The specific excretion of the lactone suggests that an enzymic lactonization reaction may exist which might be associated with the algal membrane. The significance of this work on the lactone and these Speculations could be extended by further biological surveys to establish the uniqueness of this compound. 80 A metabolic relationship between isocitric lactone and glycolate biosyithesis exists in the glyoxylate cycle. In this cycle, isocitric acid.is Splitto succinate and glyoxylate by isocitritase. The glyoxy- late could be reduced to glycolate by glyoxylate reductase. Both of these enzymes have been reported in various algae, although no study has been made of them in A, braunii. In the absence of glycolate synthesis and excretion in the dark, isocitrate might accumulate and be converted to the lactone which could be excreted. Much further work would be necessary to establish this hypothesis. This pathway for the synthesis of most of the glycolate by algae has not been favored from kinetic experiments, from 114,002 labeling experiments, 1"LC-acetate feeding experiments (50). However, it is pos— and from sible that the glyoxylate cycle could account for the fermation of isocitric lactone which could be a minor reaction relative to the large amount of glycolate formation by some other route. SUMMARY Synchronized cultures of Ankistrodesmus braunii were grown during a 16-hr light and 8-hr dark regimen at 30° with a 1 to 0 dilution at the end of each dark period. The photosynthetic ability, ”C02 fixation, was the highest for young growing cells, low for mature cells, and lowest for dividing cells. The as measured by 1 amount of inc excreted during photosynthesis followed the same trend. The inc compounds excreted during photosynthesis changed during the algal life cycle. Young growing cells excreted glycolate in large amounts, but none of the other acids. Dividing cells excreted only about 0% as much glycolate-lac as young growing cells. Dividing cells also excreted pppprtartrate, isocitric lactone, malate, and an unidentified acid, U , and occasionally some citrate and glycerate. 3 Of the acids excreted by dividing cells, glycolate and‘ppgprtartrate were the major ones and they were excreted in comparable amounts. Excretion of ppgprtartrate when glycolate excretion decreased implied that the two acids might be metabolically and physiologically related. Kinetic studies on the excretion of these acids by the synchro— nized cells were done during 30 minutes of photosynthesis. Growing cells excreted glycolate. ‘With dividing cells, pggprtartrate, malate and glycerate were excreted in relative largest amounts within 1 or 2 minutes of photOSynthesis, glycolate in 5 minutes, and isocitric lactone and U3 in 30 minutes. Large-scale separation and purification of the excreted acids were carried out by repeated anion exchange chromatography on A01- 85 86 acetate columns with acetic acid gradient elution followed by formic acid gradient elution. The acids were eluted from the column with the acetic acid gradient in the order of glycolic, malic,[pp§prtartaric, isocitric and citric acids, and isocitric lactone; U3 was eluted with a subsequent formic acid gradient. The ppgprtartaric acid was identified by cochromatography on paper chromatograms and by gas-liquid cochromatography as the trimethyl- Ema silyl derivative. Isocitric lactone was identified by cochromatography on paper, and by hydrolyzing it to isocitrate. The isocitrate was then identified by cochromatography and by converting it to glutamate by a reaction catalyzed by isocitric dehydrogenase and glutamic-aspartic t“ transaminase. Isocitric lactone was experimentally Shown to be excre- ted as such by the dividing cells. After a period of photosynthesis in NaHluCO 10 , subsequent excre- 3 C-labeled acids in the light and in the dark was analyzed tion of both for the amount and for the components. In light, excretion of glycolate and the other acids continued. In dark, glycolate excrea tion was completely stopped, while the excretion of the other acids continued in even larger amounts. Upon addition of CMU during the light, the excretion pattern was Similar to that in the dark. Aera- tion with oxygen during the dark prevented the excretion of isocitric lactone. The distribution of 140 in ppgprtartaric acid was determined. The carboxyl carbons were about 0 times as radioactive as the middle carbons. Since glycolate is known to be uniformly labeled, glycolate could not be the precursor of carboxyl labeled meso-tartrate, nor pppprtartrate a direct precursor of glycolate. 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