ABSTRACT AN EXAMINATION OF GIXCOLATE METABOLISM IN PLANTS By William J. Bruin Procedures for in C02 labeling of plant and algal metabolites in time periods of a few seconds were used to investigate the labeling pattern of glycolate, glycine and serine formed during photosynthesis. Glycolate was degraded enzymatically, glycine by ninhydrin, and serine by periodate. The presence of enzymes for some of the related reactions of glycolate metabolism was used as con- firmatory evidence for the metabolic sequence from glycolate to serine. After photosynthesis with 1“C02 by the unicellular green algae, Chlorella pyrenoidosa for 5 to 15 seconds, gly- colate and glycine were labeled predominantly in carbon—2, while serine was carboxyl labeled. By 30 seconds glycolate and glycine were uniformly labeled while serine remained somewhat carboxyl labeled. These results are different from those previously reported with higher plants which form uniformly labeled substrates of the glycolate pathway. ‘Metabolism of exogenous glycolate-l-luc by the alga, Scenedesmus, produced glycine-l-luc and serine-i-luc. Serine hydroxymethyltransferase activity was also detected in algal William J. Bruin extracts. These results establish that glycolate metabol- ism in algae is significant in Spite of the fact that algae exhibit only limited photoreSpiration and excrete part of the glycolate at certain stages of development. The results are best interpreted by the hypothesis that algae contain a metabolic pathway for conversion of glycolate to glycine and serine. In addition formation of oarboxyl labeled serine from 3-ph08phoglycerate during photosynthe— sis occurred to a large extent. Changes in the labeling pattern with time and in the presence of inhibitors of the glycolate pathway, a-hydroXy-Z-pyridinemethanesulfonate and isonicotinylhydrazide, are interpreted to suggest two dif- ferent sites or metabolic pools of these compounds in the cell. In plants with photorespiration, rapid glycolate biosynthesis and metabolism occurs, and the serine is uni- formly labeled. This was confirmed with bean leaves. Manganese-deficient bean leaves had a reduced rate of for- mation of glycolate, glycine and serine. This is a further implication for the involvement of manganese and photosyn- thetic electron tranSport in glycolate biosynthesis. Serine biosynthesis in three plants without C02- photoreSpiration was investigated during 14002 fixation. In these plants, sugar cane, corn and Amaranthus, serine had an unusually heavy labeling in the beta-carbon. After 15 seconds of photosynthesis by corn leaves 69% of the lb’C William J. Bruin label of serine was in the 03-carbon. These uneXpected results may be significant in the mechanism of COZ fixation and transcarboxylation during photosynthesis in this type of plant, which first fixes the COZ into the Cu-dicarboxylic acid cycle. In plants with photoreSpiration, some enZymes of the glycolate pathway are found in a subcellular microbody termed peroxisome. The intracellular location of serine hydroxymethyltransferase, the enzyme catalyzing the glycine to serine conversion, was examined. This reaction may be of Special importance for C02 release during photoreSpira- tion. The activity of this enzyme was assayed in both directions. Assays were run on fractions from Spinach leaves after separation by differential centrifugation and by isopycnic sucrose density gradient centrifugation. Serine hydroxymethyltransferase activity was located in the mito- chondrial fraction, and none was found in peroxisomes. Consequently glycolate metabolism and photoreSpiration in the peroxisomes must act in concert with this Specific step in the mitochondria. Three pathways or sequences for serine biosynthesis are suggested by the results. Uniformly labeled compounds of the glycolate pathway are formed in higher plants. Initially or momentarily this pathway in algae can produce Cz-labeled glycolate and glycine. Carboxyl labeled Serine from carboxyl labeled 3-phDSphoglycerate is a dominant William J. Bruin metabolic product in algae, but of little apparent signi- ficance in higher plants. C3-labe1ed serine is formed in plants which have a high initial rate of C02 fixation by the Cu-dicarboxylic acid cycle. The differences observed in metabolic pathways when using ll‘LCO 2 and when using labeled glycolate are interpreted to be indicative of com- partmentation of Specific reaction of the glycolate path— way. AN EXAMINATION OF GLYCOLATE METABOLISM IN PLANTS By William J. Bruin A THES IS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 C," (+"’-/ 7/ f ACKNOWLEDGMENTS I would like to eXpress appreciation to Dr. N. E. Tolbert for guidance during the course of this work and for initially providing the incentive which lead me to undertake graduate study in the field of biochemistry. I also thank Dr. Paul Kindel for valuable advice concern- ing the methodology of the glycine degradations and Mr. Charles Frolik (NSF Undergraduate Fellow) for assistance with part of the algal photosynthesis. The helpful assistance of Mr. Ed. Nelson and Mr. Doug Randall during photosynthesis eXperiments with higher plants is appreci- ated. Finally I would like to acknowledge the encourage- ment and moral support of Dr. and Mrs. Tom Klaasen during the many trying periods of this work. Financial support was received in part from the National Institutes of Health and from a National Science Foundation grant GB #154. ii TABLE OF CONTENTS GENERAL INTRODUCTION 0 O O O I O O O O O O C O O PHOTOSYNTHESIS AND GLYCOLATE METABOLISM IN ALGAE Literature Review . . . . . . . . . . . . . Formation of Glycolate Glycine and in Vivo O O O O O O O O O O O O O I O Metabolism of Exogenous Glycolate . . . Use of Inhibitors . . . . . . . . . . . PhotoreSpiration in.Algae . . . . . . . Glycolate Oxidase in.Algae . . . . . . . Blue Light Effects . . . . . . . . . . . Summary of Glycolate Metabolism by Algae Materials and Methods . . . . . . . . . . . Glycolate Degradation . . . . . . . . . Serine Degradation . . . . . . . . . . . Glycine Degradation . . . . . . . . . . Specific Activity of BaCO3 . . . . . . . Self Absorption Curve for Ba003 CO Trap 0 o o o 0 Ca ibration of th Counter . . . . . Glycine Decarboxylation by Ninhydrin . . Growth of Chlorella pyrenoidosa . . . . Photosynthetic 14002 Fixation Procedures Separation of Amino Acid Fraction . . . Chromatography and Radioautography . . . iii Planchets Page 10 ll 12 13 14 14 .16 18 18 18 19 20 20 21 23 26 27 Metabolism of Exogenous Glycolate by Scenedesmus . . . . . . . . . . . . . . . . Serine Hydroxymethyltransferase in Chlorella . Results 0 O O O O I O O I O O O O O O 0 O O O O O Glycolate Degradation . . . . . . . . . . . . Serine Degradation . . . . . . . . . . . . . . Gly01ne Degradation O I O O O O O O O I O O 0 Theory for Planchet Counting . . . . . . . Calibration of Counter and Determination 0f Nk o o o o o o o o o o o o o o o 0 Degradation of Glycine-lac Standards . . . Degradation of Glycine From Photosynthesis by Chlorella O O O O O O O O O O O O O O Glycine and Serine Formation in Scenedesmus . Serine Hydroxymethyltransferase Activity in Chlore 11a 0 O O O O O O O O O O O O O O O 0 Discussion . . . . . . . . . . . . . . . . . . . SERINE FORMATION IN PLANTS WITHOUT PHOTORESPIRATION . Literature Review . . . . . . . . . . . . . . . . Serine From 002 Fixation in Higher Plants . . Prior EXperiments With Isolated Chloroplasts . Metabolism of Glycolate in Leaf Tissue . . . . Materials and Methods . . . . . . . . . . . . . . Photosynthetic J-l"*COZ Fixation . . . . . . . . Chromatography and Radioautography . . . . . . Serine Degradation . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . Total 1L’COZ Fixation and Radioactive Products. Serine Labeling in.PlantS With B-Carboxyla- tion I O O O O O O O O O O O O O O O O O 0 Discussion 0 O O O O O O O O O O O O I O O O O 0 iv Page 28 29 30 3o 32 3b 3H 3H 37 38 40 40 #2 48 48 48 49 51 53 53 55 55 55 55 59 61 Page GLXCOLATE BIOSYNTHESIS IN MANGANESE-DEFICIENT BEANS . . 6“ Introduction and Literature Review . . . . . . . . 6“ Materials and Methods . . . . . . . . . . . . . . 64 Manganese Deficiency . . . . . . . . . . . . . 6” Isotopic Procedures . . . . . . . . . . . . . . 65 Serine Degradation . . . . . . . . . . . . . . 66 Results . . . . . . . . . . . . . . . . . . . . . 66 Total 1“cog Fixation and Radioactive Products . 66 Serine Degradation . . . . . . . . . . . . . . 67 The Effects of Hydroxypyridinemethane sulfonate C O O O O O O O O O O O O O O O O 74 1313011881011 0 O O O O O O I C O O O O O O O O O O O 76 INTRACELLULAR LOCATION OF SERINE HYDROXYMETHYL- TRANSFERASE O O O O O O O O O O O O O O O O O O O O O o 78 Literature Review . . . . . . . . . . . . . . . . 78 Serine Hydroxymethyltransferase in Plants . . . 78 Glycine-Serine Interconversion in.Animals . . . 79 Serine Formation in Isolated ChlorOplastS . . . 81 Materials and Methods . . . . . . . . . . . . . . 83 Fractionation of Subcellular Particle by ISOpycnic Sucrose Density Gradient Centrifugation . . . . . . . . . . . . . . . 83 Preparation of Linear Sucrose Density Grad-lent O O O O O O O O 0 I O O O 0 O O O O 814’ Preparation of Mitochondria in Mannitol . . . . 85 Enzyme Assays . . . . . . . . . . . . . . . . . 86 Serine to Glycine Conversion . . . . . . . . 86 Glycine to Serine Conversion . . . . . . . . 87 V Preparation and determination of standard formaldehyde solution . . . 88 Enzymic incorporation of formaldehyde into serine . . . . . . . . . . . . 89 Cytochrome c Oxidase . . . . . . . . . . . 89 Glycine Decarboxylation . . . . . . . . . 90 Protein Determination . . . . . . . . . . . . 91 Results. 0 O O O O O O O O O O O O O O O O O O O 91 Distribution of Serine Hydroxymethyltrans- ferase Among Subcellular Particles . . . . 91 Serine Hydroxymethyltransferase Activity in Mannitol Prepared Mitochondria . . . . 97 Serine Hydroxymethyltransferase in PeI‘OX1someS. O O O O O O O O I O O O O O O 97 Serine Hydroxymethyltransferase in Chloroplasts . . . . . . . . . . . . . . . 99 Reversibility of the Enzyme . . . . . . . . . 99 Glycine Decarboxylation . . . . . . . . . . . 101 Discu8310n O O I O O O O O O O 0 O 0 O O O O O 0 102 BIBLIOGRAPHY. O O O O O O O O O O O O O O O O O O O O 105 vi LIST OF TABLES Table Page 14c Distribution in Glycolate Pathway Inter- b mediates . . . . . . . . . . . . . . . . . . . 2 Distribution of 14C in Compounds From Short- Time Photosynthesis . . . . . . . . . . . . . Culture Medium for Chlorella . . . . . . . . . . 22 4 Degradation of Specifically Labeled Commercial GlYCOlate O I O I I I I I I I I I O I I I I I 31 5 Percent Distribution of Label in Photosynthetic GlyCOlate I I I I I I I I I I I I I I I I I I 31 6 Degradation of Specifically Labeled Commercial serine O O O O O C O O C C C C C I I C O O O O 33 7 Percent Distribution of Label in.Photosynthetic serine I I I I I I I I I I I I I I I I I I I I 33 8 Degradation of Specifically Labeled Commercial Gly01ne I I I I I I I I I I I I I I I I I I I 39 9 Percent Distribution of Label in.Photosynthetic GlyC1ne I I I I I I I I I I I I I I I I I I I 39 10 Percent Distribution of Radioactfivity in Glycine C . and Serine From Glycolate-l-1 41 11 Serine Hydroxymethyltransferase Activity in Chlorella I I I I I I I I I I I I I I I I I I 41 12 Labeling Pattern of Serine From Higher Plants . . 5O 13 Products of Photosynthetic lucoz Fixation in Plants With B-Carboxylation . . . . . . . . . 56 1h Labeling Pattern of Serine From B-Carboxylation P lants C O C O I C C O I C I I O I I O I I O O 60 ‘15 Percent Distribution ofluc Among Products of 1 002 Fixation by Manganese-Deficient Bean Leaves (Deficiency Obtained by Lime Treat- ment) 0 o o o o o o o o o o o o o o o o o o o 68 vii Table Page 16 Percent Distribution of lac Among Products of 02 Fixation by Manganese-Deficient Bean Leaves (Deficiency Obtained by Withholding Manganese) . . . . . . . . . . . . . . . . . . 69 17 Relative Amounts of Glycine and Serine Formed During Photosynthesis by Bean Leaves at 6" . . 70 18 Distribution of 14c in 6" Serine From Beans . . . 72 19 Metabolites of the Glycolate Pathway Formed During Photosynthesis . . . . . . . . . . . . 73 20 Effect of Hydroxypyridinemethanesulfonate Treat- ment on Photosynthetic Products by Manganese- Deficient Leaves . . . . . . . . . . . . . . . 75 21 Distribution of EnZymes in.Particulate Fractions. 92 22 EnZyme Activity in Mitochondria Prepared in MannitOl I I I I I I I I I I I I I I I I I I I 98 viii LIST OF FIGURES Figure Page 1 Carbon Flow for the Initial Reaction of the Glycolate Pathway . . . . . . . . . . . . . . 3 2 1”C02 Fixation Apparatus for Algae . . . . . . . 2“ Self-Absorption Curve for BaC03 Planchets . . . 35 4 Enzyme Distribution on a Linear Sucrose Grad.ient I I I I I I I I I I I I I I I I I I 96 5 Enzymic Incorporation of Formaldehyde into Serine I I I I I I I I I I I I I I I I I I I 100 ix FMN NAD NADH OHPMS PAIP POP OP PPO LIST OF ABBREVIATIONS flavin mononucleotide nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide a-hydroxy-Z-pyridinemethanesulfonate pyridoxal phOSphate 1,4—bis[2-(5-phenyloxazolylil-benzene 2,5-diphenyloxazole GENERAL INTRODUCTION The investigation of glycolate biosynthesis and metabolism in photosynthetic tissue is a currently active and rapidly moving area of plant biochemistry. During the course of my thesis work knowledge in several large fields of interest has been significantly increased including the comparison of glycolate metabolism in algae vs. higher plants, the relationship of glycolate metabolism to photo- reSpiration and the localization of glycolate oxidation in the peroxisome. Consequently, what initially appeared to be a single problem has developed into several areas of work of sufficient magnitude to be considered separately. Work begun before some of the current trends were evident will be discussed in light of our present understanding, and in general distinctions will be maintained between unicellular green algae, higher plants with photoreSpiration and higher plants without photoreSpiration. The separate sections of the thesis are indicated in the Table of Con- tents. It is hOped that this thesis will serve to clarify some diSputed points of glycolate metabolism in a variety of plants. PHOTOSYNTHESIS AND GLYCOLATE METABOLISM IN ALGAE Although glycolate has long been recognized as an early product of photosynthesis, its biOSynthetic route still remains obscure. The subsequent metabolism of gly- colate, however, has been examined extensively. That part of the glycolate pathway, described by Tolbert (78), which is pertinent to this thesis is presented in.Figure 1. The sequence proceeds from glycolate to glycerate. Literature Review Formation of Glycolate, Glycine and Serine _i_._1;1 z.i_v2 . Hess and Tolbert (25) reported that in unicellular green algae the glycolate pathway probably did not operate as indicated in Figure 1 Since they could obtain little evidence for a glycolate oxidase, and no metabolism of 1LLC labeled glycolate was observed in crude algal extracts. 14C in Furthermore the intramolecular distribution of several intermediates of the pathway formed from 1LACOZ dur- ing photosynthesis seemed to support their hypothesis in that the labeling patterns of glycolate, glycine and serine were inconsistent with the proposed scheme. This data is shown in Table 1. Several other reports of 140 labeling patterns in 2 amassed opsaooaao as» to soaaooom adapHsH one not scam.soaaoo .H enemas. mommoa mommoa mommoa _ _ _ mooo + muoaummz onus mouoanm _ ommhommsmaps _ ommsowoaomSmmv . moooo Iosaaq moooo onshoomawla moooo A: mmmaommsmap IHmSpoamxoaoms mafihom N m momm No N m2 mos“. 9.62. < mo mos _ «wmmnowmzoap _ tammmcaxo _ moooo uosass moooo cacaooaao moooo If? 0“. (fl (N Table 1 14C Distribution in Glycolate Pathway Intermediates (From Ref. 25) Time (Sec) 5 7 10 12 20 6O I. Glycolic Acid c-1 27+ 39* 35X 30° 42° 43x 48° 47* C-2 73 61 65 7O 58 57 52 53 II. Glycine C-i sox 49° c-2 50 51 III. Serine C-1 78x 80X 69° c-2 6 4 7 0-3 16 16 24 x - Chlorella pyrenoidosa (chick) + - Chlorella pyrenoidosa (Warburg) o - Chlamydomonas reinhardtii 5 compounds of the glycolate pathway from algal photosynthesis also appeared. These results were from short term photosyn- thetic experiments with 14002. Zak and Nichiporovich (90) did a time study of the labeling patterns of 3-phoSphoglyceric acid, alanine, serine and glycine in Chlorella pyrenoidosa. As shown in Table 2 the first three compounds contained about 80% of the radioactivity in the carboxyl carbon at times up to 19 seconds. Glycine, however, diSplayed a uniquely skewed distribution being initially carboxyl labeled but becoming 0-2 labeled. PasserauEEIEI. (55) reported similar data for Chlorella vulgaris. After a 60 second period of photosynthesis alanine and serine were still heavily carboxyl labeled while glycine appeared uni- formly labeled (see Table 2). Of these three compounds glycine had the highest Specific activity, which approached that of glycolic acid. The distribution of label within the glycolate itself was not reported, probably because it did not accumulate. These results are particularly different from those with higher plants where serine has been found to be uniformly labeled (24, 60). Thus evidence for the conversion of glycolate to glycine in algae from C02 fixation studies is not well established, and the glycine to serine reaction does not seem to be contributing significantly to the total serine pool. These apparent discrepancies between the prOposed reactions from the higher plant investigations and the Table 2 Distribution of 1LAC in Compounds From Short-Time Photosynthesis M Time (Sec) 4 6 9 13 19 1. Chlorella rancidosa (From Ref. 90) Glycine C-1 100 73.7 49.0 38.2 0.2 - 26.3 51.0 61.8 Serine C-1 100 90.1 82.3 80.9 0-2 - - - 8.5 C-3 - 9.9 17.7 10.6 3-P-Glycerate C-1 100 99 87.0 73. C-2 - 0.4 6.4 12.7 C-3 - 0.6 6.6 14.1 Alanine C-1 100 83.8 75.8 79.8 C-2 - 7.2 14.8 9.6 C-3 - 9.0 9.4 10.6 2. Chlorella vulgaris (From Ref. 55) Glycine Serine Alanine C-1 46.3 C-1 66.0 C-1 69.5 C-2 53.7 C-2 + C-3 34.0 C-2 + C-3 30.5 7 observed labeling pattern with algae may indicate the occur- rence of two metabolic pools for glycine such as those pro- posed by Bassham.g§.gl. (68). These workers have shown that there were two amino acid pools in Chlorella; one was very small and was rapidly labeled within seconds with 1["002 to a constant Specific activity, while the other pool was large and acquired label from 1”€02 more Slowly over a period of minutes. The rapidly labeled pool may have repre- sented a photosynthetic compartmentalization within the cell while the slowly labeled pool may have been indicative of the larger glycine pool for the bulk of protein synthesis. Metabolism of Exogenous Glycolate In general eXperiments where glycoLate was fed to algae have been unsuccessful because of the apparent inabil- ity of these organisms to take up this compound. An.ear1y report on glycolate metabolism (65) indicated that in Scenedesmus glycolate was converted to nearly equal amounts of glycine and serine in the dark; and in the light the radioactivity from glycolate appeared in a wide range of compounds. Glycolate-2-1“c produced glycerate-2,3-1”c while glycolate-i-luc was converted to predominantly car- boxyl labeled glycerate. The glycolate uptake was conducted at pH 2.8, although the authors claimed this did not affect normal photosynthesis. Recently, E. B. Nelson.gtla;.(52) were able to observe glycolate uptake by synchronous cultures of Scenedesmus ‘1) 't. i_ b.’ 8 obliguus at one stage of the growth cycle. One metabolic product of radioactive glycolate taken up was glycine (un- published). Indirect evidence for the conversion of glycolate to glycine in algae has been reported by Whittingham and co- workers (43). In Chlorella glucose-i-luc provided carbons to form glycine-Z-luc. This is consistent with a cleavage of the tOp two carbons to form glycolate-Z-luc, which in turn would be converted to glycine—Z-luC. Use of Inhibitors Metabolic inhibitors have also been employed as another method of elucidating biosynthetic pathways. In the case of the glycolate pathway there have been several reports on the accumulation of intermediates when Specific inhibitors have been used. a-Hydroxy—Z—pyridinemethane- sulfonate (OHPMS) caused the accumulation of glycolate in higher plants while glycine formation was greatly reduced (24). This inhibitor blocked the metabolic sequence at the Site of glycolate oxidase. In algae, however, there was no significant accumulation of glycolate in the presence of OHPMS in eXperiments by Hess and Tolbert (80), even though the net rate of COZ fixation was greatly stimulated by this inhibitor at pH 7.6. The percentage of 14c in alanine, aSpartic and glutamic acids was reduced more than that in glycine and serine. The amount of a—ketoglutarate and pyruvate was increased as if the inhibitor were preventing 9 normal aminotransferase reactions. Gimmler gt_§l. (21) using synchronous cultures of Ankistrodesmus braunii found a five-fold increase in the excretion of glycolate in the presence of OHPMS with no increase on the total 1L"C02 fixation. The amount of stim- ulation of glycolate excretion by the inhibitor varied with the stage of growth of the life cycle. Eight hours after the beginning of the light phase there was no stimu- lation of total fixation, but at most stages of the life cycle the OHPMS treated cells did fix a somewhat larger quantity of 1”002. Thus the usefulness of this inhibitor for algal studies would seem to depend on the stage of growth of the culture. An inhibitor of aminotransferase reactions, iso- nicotinylhydrazide, was used in Chlorella by Whittingham (59). A marked increase in the formation of glycolate and glycine during photosynthesis was Observed. In Scenedesmus azaserine also caused a significant accumulation of glyco- late during photosynthesis while the overall COZ fixation was stimulated (1). Scenedesmus cells suSpended in 6% ethanol also channeled the majority of the radioactivity 14 from CO2 into glycolate and glycine (40). Unfortunately the site of action of these inhibitors is not Specific enough to allow very definite conclusions to be drawn about glycine and serine formation. But the results seem consistent with those from the higher plant 'C.’ (.7 ' (I) .' 10 and with the function of a glycolate pathway in algae. PhotoreSpiration in Algae Another factor to consider in metabolic studies of glycolate is its role in photoreSpiration. Zelitch has shown that the C02 evolved during photoreSpiration came from the carboxyl group of glycolic acid (96). The role of glycolate in photoreSpiration raises the possibility that the metabolism of glycolate may be different in the light than in the dark. The only striking difference observed previously was that glycolate was metabolized to sucrose in the light whereas in the dark the sequence went to glycerate and then to a lesser extent into compounds of the citric acid cycle (78, 86). PhotoreSpiration has also been observed in some algae; however, the phenomenon appears to vary with genera (8). For example, Chlorella and Scenedesmus exhibited a low COZ compensation point ( <3 ppm) in.21% oxygen, while Nitella, a multicellular green alga, had a higher 002 com- pensation point typical of land plants With photoreSpiration. In.Ankistrodesmus no light stimulated net oxygen uptake was observed (9). On the other hand Hoch‘gtlgl. (27) did report a light dependent 02 uptake in Scenedesmus which seemed to be inhibited by dichlorOphenyldimethylurea. Nitella was found to have an active glycolate oxidase, and glycolate stimulated COZ evolution in the dark at 21% oxygen (16). Likewise Acetabularia appeanato be an alga like Nitella 11 which has glycolate oxidase and glycolate metabolism more Similar to higher plants (5). Thus it is probably unjusti- fiable to Speak of "algae" as a biochemically uniform group and to eXpect all genera of green algae to metabolize glycolate in the same way. Glycolate Oxidase in.Algae Closely related to the phenomenon of photoreSpiration is the occurrence of glycolate oxidase. Although Hess and Tolbert (25) did not observe a typical glycolate oxidase in either Chlamydomonas or Chlorella grown in C02 enriched air, Zelitch and Day (97) were able to detect an enzyme which catalyzed a dye-linked oxidation of glycolate in air-grown cultures of Chlamydomonas. Nelson and Tolbert (51) have confirmed this finding and have suggested that glycolate metabolism and excretion by algae are regulated by the availability of 002 in the growth medium. Both Nelson and Tolbert (50) and Gimmler 33 El. (21) have observed that the rate of glycolate excretion in synchronous cultures of algae changes during stages of their life cycle. If excre- tion of glycolate is related to a lack of further metabo- lism via a glycolate oxidase, then these data support the idea that glycolate metabolism varies with the life cycle. Thus in algae whether the glycolate pathway is operative or not may depend on the environmental conditions and the stage of growth. Recently Merrett and Goulding (47) have shown a light 12 dependent assimilation of acetate by autotrophically grown Chlorella. The main products were glycolate, glycerate and serine; and from kinetic studies it was apparent that glycerate was formed from glycolate. This particular strain of Chlorella had an active "glycolate oxidase" as assayed by formation of the glyoxylate phenylhydrazone (41). Because of this the glycolate formed both from acetate and from CO2 was not excreted, but instead was metabolized further. These authors suggested that the metabolism followed the sequence: glycolate-——-—9 glycerate-—-——9 serine. This strain of Chlorella appears quite unique, however, in that it also has the ability to take up exogenous glyco- late and to grow heterotrOphically on this substrate. Radio- activity from glycolate-i-luc rapidly moved into C02, glycine, serine, glycerate and citrate (M. J. Merrett, personal com- munication). Thus in the case of exogenous glycolate a typical glycolate pathway appears to be Operating, while glycolate formed in the cell may follow a different metabolic route. Blue Light Effects Hess and Tolbert (26) have studied the effect of blue light on glycolate biosynthesis in algae. Chlamydomonas reinhardtii grown in blue light for several generations incorporated 30-36% of the total soluble 140 fixed into glycolate; however, the amount of glycine and serine did not 13 increase. In cells grown in red light only 3% of the soluble radioactivity occurred as glycolate. These results differed somewhat from those of eXperiments conducted with plant materials grown in white light which were then eXposed to photosynthetic conditions in red or blue light. In such cases amino acid production in general was increased in blue light as measured by total protein formation (39, 85). Cayle and Emerson (11) examined photosynthesis in Chlorella during illumination with red or blue light. These workers saw almost three times greater Specific activity in the amino acids formed in blue light than those formed in red. Examination of the labeling pattern of the glycine formed showed that in blue light photosynthesis at 30 seconds the glycine was predominantly a-labeled (C1:C2 ; 22:79), whereas in red light the distribution was more uniform (42:58). These results have never been confirmed. Also using Chlorella, Becker 22.3l- (3) found no excretion of glycolic acid into the medium during blue light photosynthesis, while normal glycolate excretion was observed with red and white light. They suggested the gly- colate was being further metabolized to glycine and serine in blue light rather than being excreted. Summary of Glycolate Metabolism by Algae In summary, in some cases reported concerning algal photosynthesis the glycine labeling from 1[+002 does not reflect that in glycolate, and in all instances cited serine 14 does not appear to come exclusively from glycine in these unicellular organisms. At the times when algae do not excrete glycolate, perhaps this compound is being further metabolized by oxidation to glyoxylate and subsequent transamination to glycine. Blue light and low C02 may stimulate this route. In addition algae without photoreS— piration may Show a different pathway of glycolate metabol- iSm than those organisms which are able to photoreSpire glycolate-i-luC to 1“C02 via the glycolate pathway. Materials and Methods Glycolate Degradation The enzymic procedure of Zelitch (95) was used with some modification. A crude glycolate oxidase preparation was made from Spinach leaves by grinding the leaves with two volumes of water, adjusting to pH 5.4, and centrifuging to remove debris. Saturated (NH4)2304 in 0.001 M EDTA, pH 7.5 was added with stirring to 0.21 saturation. The protein which precipitated in this step was discarded. Then solid (NH4)2804 was added until 0.43 saturation was reached. This protein fraction was collected by centrifugation and suSpended in a volume of 0.43 saturated (NH4)2804 equal to 1/20 the original leaf homogenate volume. This crude gly- colate oxidase preparation was stable several months when stored frozen. Tris buffer, 0.08 M, pH 8.7 was chosen Since Richardson and Tolbert (61) have shown that in 15 phoSphate buffer glyoxylate, the first product of the enZymic oxidation, can be oxidized further to oxalate by the action of glycolate oxidase, whereas this secondary oxidation is inhibited in Tris buffer at pH 8.3. In the subsequent degra- dation procedure, oxalate would be oxidized to two C02 mole- cules by ceric sulfate giving erroneous degradation results. Other materials used included 0.01 M_sodium glycolate, 6 x 10'“ M flavin mononucleotide (FMN) adjusted to pH 8.7, and 0.2 N ceric ammonium sulfate in.2 N H230“. The C02 trapping solution (89) contained 27 m1 redistilled phenethlyamine, 27 ml methanol, 500 mg PPO (2,5-diphenyloxazole), and 10 mg POPOP (1,4 bis [2—(5-phenyloxazolyl)]benzene) made up to a volume of 100 ml with toluene. The solution for rinsing the trap contained 5 g PPO and 100 mg POPOP in one liter of toluene. Degradations were carried out in 50 ml three-neck flasks each equipped with a condensor, to which the C02 trap was attached, an air inlet tube, and a dropping funnel. For the enzymic oxidation of glycolate 8O umoles buffer, 10 umoles carrier glycolate and radioactive sample were added to the flask in a volume of 2-3 ml. The 002 trap was filled with 5.0 m1 phenethylamine solution and attached. Then 0.1 m1 of the crude glycolate oxidase suSpension dissolved in 1.0 ml 6 x 10"” y FMN was added through the funnel to initi- ate the reaction. The solution was stirred continuously with a magnetic stirrer, and the reaction was allowed to 16 proceed for two hours at room temperature with slow aeration. Next the glyoxylate produced by the enZymic reaction was oxidized to 002 from the carboxyl group and formats from the d-carbon by addition of 0.5 ml ceric sulfate. Any per- oxide oxidation of the glyoxylate during the enzymic reac- tion would have produced the same products from the same car- bon atoms. However, there was probably sufficient catalase in the crude enzyme preparation to destroy hydrogen peroxide as rapidly as it was formed. The reaction was complete in three hours at room temperature. Aeration was increased during the last half hour to assure complete removal of 1”002 from the flask. Although the formate could be oxidized to 002 in a subsequent step by boiling the solution with HgClg, in most cases the percent distribution was calculated by difference based on total radioactivity added and 1L’COZ released. The phenethylamine solution was transferred to a scintillation vial and the trap was rinsed with 10.0 ml of the toluene solution. The radioactivity was then counted with.a.Packard Tri-Carb Scintillation Spectrometer, Model 3310. Control degradations with Specific labeled glycolate samples were routinely included. Serine Degradation A method described by Chang and Tolbert was used (13). All carbons were recovered as 14C02 in the phenethylamine trapping solution described above. 17 The materials used were: DL-serine; 0.5 M sodium phOSphate, pH 5.8; 3.0 M sodium phoSphate, pH 2.5; reagent grade NaIOu. HgClZ, and K28208 (potassium persulfate); and a 5.0% aqueous solution of AgNOB. It is essential to use sodium phOSphate buffer Since potassium periodate is quite insoluble. For recovery of the carboxyl carbon 0.2 mmoles serine, radioactive sample, and 2.0 ml 0.5 M phOSphate were added to the flask. The C02 trap was attached and 160 mg NaIOu dissolved in three ml water were added through the funnel. The solution was stirred for one hour at room tem- perature. The carboxyl carbon of serine was oxidized to C02, the a-carbon to formate, and the B-carbon to formalde- hyde by this reaction. Another 0.2 mmole serine was added to destroy the excess periodate. After one hour 1.5 ml of the pH 2.5 phOSphate was added, and the flask was aerated for 30 minutes. The C02 trap was changed and the d-carbon, as formate, was oxidized to C02 by boiling the solution for one hour with 1.0 g HgClg dissolved in 5.0 ml hot water; the system was aerated continously. The trap was changed again and 1.5 g pctassium persulfate dissolved in 5 ml water was added. When the solution began to boil, 1.0 ml AgNO3 as a catalyst was added slowly to avoid a sudden uncontrolled reaction. This oxidation of the B-carbon to C02 was complete in about 1 hour. The system was aerated during the last half hour. 18 Glycine Degradation The ninhydrin decarboxylation described by Chang and Tolbert (13) was used. However, by empirical determin- ation it was found that the reaction yields varied and were never completely quantitative. Therefore a determination based on specific activity was worked out using procedures described by Van Slyke 22.3l' (83). Specific Activity of BaCO3 1) Self-Absorption Curve for BaC03 Planchets A radioactive sample of BaCO3 was prepared by adding 1.0 no NaHluCOB in 10 mmoles NaHCO3 to a solution contain- ing an excess amount of BaClz. The precipitate was formed into disks on fritted glass planchets by pouring the sus- pension into a precision bore glass tube clamped over the planchet and drawing off the solution byinction through the fritted glass. The precipitate was washed three times with about 7 ml water. The disks were then dried one hour at 110°. For a single disk the authors recommend using 0.5-2.0 mg carbon which correSponds to 8.2-32.8 mg BaCO3. Two sets of twelve disks of varying weight were prepared and counted on a Nuclear Chicago Low Background, Gas Flow Counter, Model C115 at a voltage setting of 1400 V. The data relating thickness and observable counts per minute (cpm) were used for establishing the self-absorption curve. l9 2) C02 Trap The trapping system consisted of three glass tubes connected in series to an aerating apparatus. The tubes were detachable for filling and for removal of BaC03. Nitrogen gas was used as the carrier for sweeping 14C02 out of the degradation flask into the C02 trapping system where the gases were bubbled through the trapping solution. The first tube contained concentrated H2304 as a scrubbing agent. For trapping C02 the next two tubes contained 2.50 ml standardized Ba(OH)2, approximately 0.3‘N, containing 12 g BaClZ'ZHZO per 100 ml solution. This reagent was stored under a COZ-free atmOSphere in a reservoir which was attached to a self-filling burette for diSpensing. Openings at the top of the reservoir and of the burette for maintaining atmOSpheric pressure were protected with tubes of Ascarite to eliminate atmOSpheric C02. At the beginning of the degradation the entire apparatus was flushed with nitrogen before the Ba(OH)2-BaC12 solution was added to the traps. When the C02 evolving reaction was complete, the traps were removed and the apparatus was quickly rinsed with 002 free distilled water. The BaCO3 suSpension was immediately titrated to a phenolphthalein end point with standardized HCl. Disks were then prepared as described above, weighed and counted in the gas flow counter. 20 3) Calibration of the Counter Total oxidation of a benzoic-140 acid sample was per- formed by the Van Slyke wet combustion method (83) to obtain a BaCO3 sample of known Specific activity. The materials used were: benzoic-luc acid with Specific activity of 5760 dpm/mg from.Packard; reagent grade KIO3: combustion fluid made of 5.0 g Cr03, 16.7 ml concentrated H3P04 (Specific gravity 1.7): and 33.3 ml fuming H2804. It was necessary to heat the fluid to dissolve the Cr03. A 10.0 mg sample of benzoic-14c acid dried to con- stant weight over P205 was transferred to the same three- neck flask system described before together with 300 mg K103 and a few boiling chips. The apparatus was flushed with nitrogen for 15 minutes; then the two traps were filled with 2.50 ml Ba(0H)2—BaC12 solution. Oxidation was initiated by addition of 5.0 ml combustion fluid. The mixture was brought to a boil and maintained there for three minutes or until iodine vapor became apparent. The heat was removed and after slight cooling, the system was aerated for at least 15 minutes. The C02 traps were then.removed and titrated to the phenolphthalein end point. This BaCO3 was used to prepare disks of known Specific activity. Glycine Decarboxylation by Ninhydrin The following materials were used: 0.1 M citrate, pH 4.7: 0.050 M glycine; radioactive glycine-i-luc and -2—14C; crystalline ninhydrin. Using the apparatus 21 described before, 1.0 ml buffer, 2.0 ml carrier glycine, and the radioactive sample were pipetted into the flask. The mixture was then brought to a boil while flushing with nitrogen. The traps were filled with the Ba(OH)2-BaClZ solution, and the reaction was initiated by adding 107 mg ninhydrin dissolved in 2.0 ml water. The reaction proceeded for ten minutes; then the heat was removed, and aeration was continued for at least another 15 minutes. The traps were then removed and neutralized as described previously. A determination was made using all reagents except glycine in order to evaluate the amount of 12C02 introduced from the reagents. Growth of Chlorella pyrenoidosa, Warburg The algae were originally obtained from Dr. M. Stiller and stored on agar slants. They were grown in a Warburg's K medium as modified by Stiller (72), which is shown in Table 3. The culture was prepared by adding EDTA to distilled water, then the salts, and finally the micro- nutrients. The medium was diSpensed into the culture flasks for sterilization by autoclaving. The algae were grown in two-liter Ehrenbach flasks in 1.2 1 medium on a reciprocating shaker in a controlled environment growth chamber at 22—230 with at least 1000 ft-candles of continuous light. Temperature in the medium was about 25°. The cultures were aerated with compressed air enriched with approximately 0.2% C02. 22 Table 3 Culture Medium for Chlorella pyrenoidosa, Warburg Component Stock Solution (g/l) ml Stock/l Medium Mg804°7H20 250 20 KH2P04 100 25 NaCl 100 20 KN03 100 20 Ca(N03)2 100 5 EDI‘A 3O 1 . 0 Micronutrients 0.8 Fe804'7H20 5 F3(N03)3'9H20 10 HBBO3 3 MnSOu°4H20 2 ZnSOn’7H20 0.22 Cuson'SHZO 0.08 (NH4)6M07024‘4H20 0.02 NaVO3 0.125 Dissolve micronutrients in 0.05 N H280“ 23 Photosynthetic l”C02 Fixation.Procedures Two to four day old cultures of the algae were har- vested by centrifuging at 1000 x g for 10 min at 4°. The medium was decanted and the packed cells were resuSpended in a few milliliters of distilled water. The suSpension was transferred to a 12 ml graduated conical centrifuge tube and again centrifuged 10 min at 1000 x g. The volume of the packed cells was then.read; for photosynthesis experiments the cells were suSpended in a volume of 0.001 M phOSphate, pH 6.5 to give a 1 or 2% suSpension. The buffer contained 0.01 M isonicotinylhydrazide in cases where the inhibitor was used; the cells were allowed to stand 45 min with aeration to assure uptake of the inhib- itor. The short time photosynthesis eXperiments were carried out in two ways. In one case a "lollipop" appara- tus was fitted with a reservoir for the NaH14C03 solution (Figure 2). The principle of this apparatus was a rapid mixing of an algal suSpension with a NaHCO3 solution as they passed into a line flowing by gravity into a killing solution of methanol. The time of photosyntheSis was con- sidered to be that time required for the algae to flow from the three-way stopcock, where the radioactive bicar- bonate was mixed in, to the end of the dropping tube. This time was standardized by dyes in blank runs. This tube was illuminated with two 300 watt Kenrad reflector NoH'4c03 Amce 24 Air O [i 900 0 UL \@_Flood E Lamp Three-Way Stopcock \ FIOOd / Lamp F:=#————-CMmp — Methanol F__—_ Figure 2. 14co2 Fixation Apparatus for Algae .“§. ‘0 _, “7’33’3‘in‘v. Jig-.W -‘ ' ‘ . . "-"J'" “Wm: wa- "79‘77'72 _ . twl' ‘lc'. 4—1. - "v2 . V . . .‘v'c :19 “w 25 flood lamps. The lollipop containing the algae suSpension was also illuminated and aerated prior to the 1”C02 fixa- tion.with another flood lamp so that the cells were photo- synthesizing actively at the time the radioactive bicar- bonate came in contact with them. To reduce the heating effect of the floodlamps a five liter diphtheria toxin cul- ture bottle filled with distilled water was placed between the lamps and the apparatus. Routinely 45 ml cells and 50 no NaHluCOB in 10 ml water were used for each time period. An.alternate method of 002 fixation was conducted by placing 25 ml of 1% suSpension of cells in a 400 ml beaker and illuminating from above and from the sides with the incandescent flood lamps. The light intensity at the sur- face was 2200 ft-candles. The suSpension was continually stirred rapidly with a water driven magnetic stirrer; the temperature did not change significantly during the course of the 1L"CO?_ fixation. To assure rapid mixing of the added bicarbonate, the 50 no aliquot was diluted with 0.5 ml distilled water preadjusted to pH 8.0 with dilute NaOH. This solution was then rapidly injected into the suSpension at zero time and the fixation was stopped by pouring in sufficient methanol to give an 80% solution. Subsequent procedures were identical for both methods. The methanol mixture was brought to a boil. Upon cooling the extract was acidified with a few drops of glacial acetic acid and flushed with C02 gas to remove any 26 d lLL'COZ. Aliquots were then counted in Kinard's unreacte scintillation fluid (33) for total fixation. Part of the sample was chromatographed directly to discern the products formed. The rest was used for isolating the amino acids and glycolate. Kinard's scintillation fluid for counting water soluble radioactive samples consisted of the following mix- ture: 10 g PPO (2,5-diphenyloxazole), 0.1 g a-NPO (2-(d- naphthyl)-5-phenyloxazole), 160 g naphthalene, 770 ml xylene, 770 ml p-dioxane and 462 ml absolute ethanol. Separation of.Amino Acid Fraction Free amino acids were separated from the total photosynthetic products by use of ion exchange chromatography (56). Dowex—5OH+ resin which had been recycled at least three times with NaCl and HCl was suSpended in 80% ethanol. The resin was packed in the column as a Slurry which was allowed to settle by gravitation. .An aliquot of the 80% methanol killing solution containing the radioactive materials was placed on the column after removal of sedi- mented debris. Amino acids were bound while all anionic and neutral compounds passed through. The column was washed with 80% ethanol until the effluent was free of radioactiv- ity. These fractions, containing organic acids, sugar phOSphateS and neutral sugars were combined and saved for isolation of glycolate. Elution of the amino acids was accomplished by using 0.4 N NH40H in 80% ethanol. Material 27 was collected until no radioactivity could be detected in several successive 10 ml aliquots. These combined frac- tions contained a mixture of amino acids suitable for further separation by paper chromatography. Chromatography and Radioautography 1LAC-Products of photosynthesis were separated by two dimensional paper chromatography on Whatman No. 1 chromatography paper, 0.16 mm thick with a medium flow rate. The paper was washed with 0.1 M_citric acid and then distilled water prior to use so that products could be eluted off with a minimum of residual material from the paper. Good separation was attained with the solvent system of Benson.gt‘al. (4). Water saturated phenol (88% phenol: water, 4:1) was used in the first direction, and for the second dimension the solvent was prepared fresh by mixing equal parts of nebutanolzwater (1246:84) and propionic acidzwater (620:790). Two dyes provided reference Spots for locating compounds. These were applied as a mixture just below the origin of the chromatogram. The red dye was Crocein Scarlet, MOO, 3B (Acid Red 73) from Matheson, Coleman & Bell and the orange dye was p—(2-hydroxy-1-naphthalazo)- benzenesulfonic acid-Na+ salt from Eastman. The dyes were dissolved in 50% ethanol at a concentration of 0.1 g/100 ml and mixed in equal volumes for Spotting. The glycolate area of the chromatogram was Sprayed soon after drying with 0.001 M_Na2C03 to convert the free 28 acid, which is slightly volatile to the less volatile sodium salt. To locate radioactive compounds the chromatogram was placed in contact with a Sheet of Kodak No-Screen X-ray film for two to three weeks. After development of the film the radioactive compounds were individually counted using a thin window gas flow counter (Nuclear Chicago, Model 161A). Separation of larger amounts of glycolate, glycine and serine was accomplished by initially streaking the organic acid fraction or amino acid fraction off the Dowex-50-H+ column, which had been reduced to a small volume under vacuum, onto the washed chromatography paper, along with a marker near one end of the sample. By developing in one dimension the organic acids were separated with the n-butanol- prOpionic acid-water solvent and amino acids were resolved with phenol-water. The marker glycine and serine were located by ninhydrin Spray, while the commercial glycolate- 14C as a marker was located by its radioactivity. The strip correSponding to the Rf of the marker was then cut out and eluted with distilled water. The sample was again evapor— ated to a small volume and reSpotted on washed paper for the two dimensional separation described above. The final purified compound was located by radioautography. Metabolism of Exogenous Glycolate by Scenedesmus Sterile cultures of Scenedesmus obliquus were grown similarly to the Chlorella described above in 1 L batches 29 of Medium V of Norris 2£.§l- (53), except in this case the growth of the culture was synchronized by maintaining the cells on a 16:8 hour light:dark regime. Cells for study of glycolate metabolism were harvested at the sixth hour of the dark period by centrifugation procedures described earlier and suSpended in 0.01 M phOSphate, pH 6.6. Glyco- late uptake by these cells was linear with time in both light and dark for periods of 30 minutes (Nelsen and Tolbert, unpublished results). For this study 2.5 ml cell suSpension were mixed with 0.5 ml 0.01 M glycolate-i-luc, Na‘+ salt (Specific activity 76 uc/mg for Ca++ salt) from Orlando Res., Inc. After 15 minutes in the dark the sus- pension was transferred into sufficient hot absolute ethanol to give an 80% solution. The 80% ethanol soluble products were then separated by the two dimensional chromatography system described previously. Serine Hydroxymethyltransferase in Chlorella An air-grown culture of Chlorella was harvested and made up to a 30% suSpension in 0.02 M Tris, pH 7.0 contain- ing 0.01‘M B-mercaptoethanol. By passing the suSpension through the French pressure cell twice at 16,000 psi reason- ably good breakage was attained. Aliquots of the extract were assayed for serine hydroxymethyltransferase activity by the procedure of Taylor and Weissbach (75) described in detail in the last section of this thesis. 30 Glycolate Degradation The results of a series of degradations using com- mercial samples of glycolate-i- and 2-140 are shown in Table 4. The method never gave quantitative yield of the carboxyl carbon and showed some cross contamination of C-1 from the d-carbon. These errors were either inherent in the enzymic method, or the distribution of label in the commercial samples was partially randomized. Thus when taken individually the standards indicate that this method does not give unequivocal results. However, when the mixed glycolate-1,2—14C was degraded, the yields from the two carbons were such that the final results were quite close to the expected distribution based on calculation by difference. The glycolate samples obtained from chromatograms of the algal photosynthesis with and without isonicotinyl- hydrazide were degraded, and the percent distribution of label obtained by calculations based on difference are given in Table 5. The distribution of label in glycolate from normal photosynthesis was in good agreement with the results of Hess and Tolbert (see Table 1). The initial labeling pattern was skewed toward C2-labeled glycolate but by 60 sec the glycolate became uniformly labeled. In contrast the glyco- late produced in the presence of isonicotinylhydrazide was 31 Table 4 Degradation of Specifically Labeled Commercial Glycolate Percent Distribution of 1“c Compound Theoretical EXperimental Glycolate-I-luc C-1 100 92.7 (average of 7) C-2 " 703 Glycolate-Z-luC C-l - 9.6 (average of 5) C-2 100 90.4 Mixed Label C-1 42.6 42.3 (average of 3) C-2 5701‘" 5707 Table 5 Percent Distribution of Label in Photosynthetic Glycolate Time (Sec) 2 4 6 10 12 15 60 Photosynthesis in Buffer C-1 - 15 - 31 - 46 51 0-2 - 85 — 69 - 54 49 Photosynthesis With Isonicotinylhydrazide C-1 58 - 50 - 50 0-2 42 - 5o _ 5o 32 somewhat carboxyllabeled initially, but became uniformly labeled much more rapidly. Serine Degradation ' A series of commercial serine-i-luc, serine-3-14C and mixtures of the two were subjected to degradation by the periodate method. The results obtained are Shown in Table 6. The eXperimental values obtained are sufficiently close to the theoretical values to be useful in showing small differences in labeling distribution that occur in the photosynthetically formed serine. The results of the photosynthetic serine degrada- tion are presented in Table 7. Only serine from normal photosynthesis was degraded, since the amount of serine formed in the presence of isonicotinylhydrazide was too small to be detected except in a 30 second sample. The formation of carboxyl labeled serine is the Opposite from the initial labeling pattern in glycolate and glycine. As previously proposed, carboxyl labeled serine is most likely to have arisen from carboxyl labeled 3-P-glycerate. The retention of asymmetric labeling in the carboxyl groups of serine for relatively long periods of time indicatasthat the glycine-serine interconversion is rate limiting. Car- bon flow into these amino acids appears to be by two path- ways: from glycolate to glycine and from 3-P-glycerate to serine. 33 Table 6 Degradation of Specifically Labeled Commercial Serine Serine-i-luc Serine-3-1uC C-1 92.7% .2% c—2 1.3% 2.1% C-3 601% 97.7% Mixtures Theoretical EXperimental C-1 34.2% 30.1% I 0-2 - 2.9% C-3 65.8% 67.0% c-1 20.9% 19.4% II 0.2 — 0.2% C-3 79.1% 78.6% Table 7 Percent Distribution of Label in.Photosynthetic Serine J Time of Photosynthesis 6" 10" 15" C-1 50.9 48.5 63.0 0-2 21.2 23.4 18.3 C-3 27.9 28.1 18.7 34 Glycine Degradation 1) Theory for Planchet Counting The basic equation for determining the Specific activity of the BaCO3 planchet is given by the equation: NS A x k F Nk where A = mac/mg carbon N = sample count, i.e. observed Cpm (gross cpm - background) N = counter constant; equivalent to NS given by a BaCO disk with a Specific activity of 1.00 3 mac/mg carbon at "infinite thickness" _, N Nk - .7- where N = cpm at infinite thickness A': true Specific activity F = "infinite thickness" factor N F=_§ N k _ mg carbon in sample + mg carbon in blank mg carbon in sample 12 correction factor for C in reagents 2) Calibration of Counter and Determination of Nk The self-absorption curve was obtained by plotting cpm vs. thickness of the BaCO3 with the constant Specific activity. The curve is shown in Figure 3; the observable 606 m coo. (L m w ( ooh. ooom OOmN muonosmam moomm How o>aso Soapaaompwlpaom .m 25m; ANEoBEV $65.th mm mm mm vN ON m. N. m A o _ _ A _ _ _ _ _ .l Nd 1. ed 0.0 \\\ ago 1: e .0 IIII .IIIIIIIIIIIPIOIII'II‘I‘x IIIIIIIInIJIIIIIIIIuliluI .I I nY. 36 counts reach a maximum and level off at a thickness of 32 mg/cmz. The 0pm at this value were taken as the 0pm at infinite thickness; N = 2300 cpm. The factor F was then calculated for each point by dividing the observable cpm by N. These values are given on the left ordinate. The combustion of 10.0 mg benzoic-lac acid produced 1.140 milliequivalents of C02 as determined by titration of the Ba(OH)2 trap. This represents a yield of 0.570 mmoles of CO2 out of a possible 0.574 mmoles from the given amount of benZOic acid, which is a yield of 99.2%. The Specific activity of the benzoic acid was given as 5760 dpm/mg which is equivalent to 3.77 mac/mg carbon. Three planchets with a diameter of 1.48 cm of the Ba003 from the combustion were prepared and counted. Then using the equation Ns Nk = -—- FA' where F was determined from the curve in Figure 3 and.A' = 3.77. the counter constant Nk was calculated. Wt. Ba003 Thickness NS F Nk (ms) (ms/0mg) (0pm) 32.4 18.8 528.0 0.950 147.4 30.9 18.0 511.7 0.947 143.3 33.7 19.6 521.4 0.952 145.3 144.0 average 37 Next the correction factor k was calculated from the yield of C02 from citrate buffer and ninhydrin without glycine. In this blank run 0.016 mmole 002 was obtained, which correSpondS to 0.192 mg carbon. The sample size used was 0.100 mmole of glycine which could give 0.100 mmole lucoz in the reaction. This is equivalent to 1.201 mg car- bon. Thus from the equation k = mg carbon in sample + mg carbon in blank mg carbon in sample k = 1.16 3) Degradation of Glycine—luC Standards In addition to the ninhydrin decarboxylation a dimedon derivative was prepared from the formaldehyde produced from the d-carbon during the reaction. This was done by adjust- ing the products of the ninhydrin reaction to pH 8.0 and adding 175 mg dimedon dissolved in 3 ml methanol. The solu- tion was heated to 80-900 for ten minutes. After standing one hour, the mixture was acidified with glacial acetic acid and the derivative precipitated out while standing over night in the cold room. The product was collected by filtration and recrystallized from 50% methanol. The colorless deriva- tive was then collected and dried over P205. This could be counted for radioactivity by means of the toluene rinsing solution described earlier for use with the phenethylamine trapping system. The results with commercial radioactive glycine samples 38 are shown in Table 8. The results for the glycine-i-luc and glycine-Z-luc alone were calculated on the basis of the Specific activities of the BaCO3 and of the dimedon derivative. The method shows virtually no randomization of label between the two carbons. While the dimedon derivative was useful in estab- lishing that no randomization occurred, when compared to the Specific activity of the glycine sample the dimedon derivative was consistently low and the reproducibility was not good. Therefore, in most determinations, the distribution of label in the glycine molecule was based on the difference between the original activity and that obtained as the Specific activity of C-1. The results for the mixed samples in Table 8 were calculated in this way. The eXperimental values are in good agreement with the theoretical distribution. 4) Degradation of Glycine From Photosynthesis by Chlorella The results of the degradation of glycine from the various times of photosynthesis are shown in Table 9. Thus under conditions of normal photosynthesis glycine rapidly became uniformly labeled after 10-15 seconds of photosyn- thesis. These results agree with the data from samples by Hess and Tolbert taken at 12". In contrast, the glycine formed from 14002 in the presence of isonicotinylhydrazide was quite heavily carboxyl labeled and remained so for at least 35 seconds. This carboxyl labeling was also observed 39 Table 8 Degradation of Specifically Labeled Commercial Glycine Compound Theoretical EXperimental Glycine-i-luc C-1 100 100 C-Z 1- - Glycine-2-14C C-l - 003 C-2 100 99.7 Mixed Label I II III I II III C-l 5.0 9.7 29.7 5.4 9.2 31.7 C—2 95.0 90.3 70.3 94.6 90.8 68.3 Table 9 Percent Distribution of Label in Photosynthetic Glycine L -— Time (Sec) 2" 6" 10" 12" 15" 35" Photosynthesis in Buffer C-1 31 42 55 -- 55 49 C-2 69 58 45 -- 45 51 Photosynthesis With Isonicotinylhydrazide C-1 76 60 -- 64 -- 57 0-2 24 4o -- 36 -- 43 40 in the glycolate sample from the shortest photosynthetic time in the presence of isonicotinylhydrazide. Thus the difference in labeling patterns observed under these two conditions seemed real and highly Significant. The dis- tribution patterns for glycine in normal photosynthesis are similar to those for glycolate (Table 5) and strongly support their postulated biosynthesis during photosynthe- sis from a similar source. Glycine and Serine Formation in Scenedesmus The glycine and serine formed from metabolism of 14C by Scenedesmus obliquus in the dark have glycolate-1- also been degraded by the techniques described above. The glycine was totally carboxyllabeled, while the serine also appeared predominantly labeled in this position with 70% of the radioactivity being released by the periodate oxi- dation (Table 10). These labeling patterns are in agree- ment with those eXpected from metabolism of glycolate-i-luc via the glycolate pathway. Serine Hydroxymethyltransferase Activity in Chlorella An investigation of serine hydroxymethyltransferase in Chlorella confirmed that the glycine to serine step is possible. The Specific activity of the enzyme in a crude algal extract is shown in Table 11 along with the activity from a Spinach homogenate for comparison. In crude extracts the algal enzyme appears even more active than the Spinach 41 Table 10 Percent Distribution of Radioactivity fin Glycine and Serine From Glycolate-l-1 C % Distribution Glycine C-1 100 C-2 _ Serine C-1 70 C-2 + C-3 30 Table 11 Serine Hydroxymethyltransferase Activity in Chlorella S.A. of Crude Homogenate (mumoles x mg-1 protein x 10 min‘l) Chlorella 6.1 SpinaCh 1i5 42 enzyme, although the activities are of the same order of magnitude. It would not appear that this enzyme is rate limiting in vitro for the glycolate pathway. Discussion The labeling patterns in both glycolate and glycine from 1“'COZ during photosynthesis in Chlorella pyrenoidosa are similar for identical periods of time, thus confirming their origin from a common C2 precursor. The labeling shows a skewed pattern being more C-2 labeled at short times and attaining a uniform distribution by 12 to 15 seconds. This confirms the labeling distribution for gly- colate reported by Hess and Tolbert (25) and clarifies the possibility of the metabolic sequence from glycolate to glycine in this alga. This observation would support the findings of several workers that glycolate in algae is oxidized to glyoxylate, which in turn is converted to gly- cine. The 1"AC distribution in glycolate and glycine appears similar to that in the top 2 carbons of the sugar phOSphates of the photosynthetic carbon cycle. In brief times of 1“C02 fixation the 14C distribution in hexoses generally is pre- dominately C-3, C-4 labeled and label in C-1 and C-2 is less and nearly equal. In very short times of 14002 fixation the C-1 of the hexose has more 14C than C-2. Since it has been postulated that the C-1 of glucose becomes the d-carbon of 43 glycolate, then the d-carbon should be more radioactive than the carboxyl which arises from C-2 of glycose by this theory. However such extreme differences in Specific activity of hexose carbons 1 and 2 have not been seen as in the glycolate data now being presented. This may be accounted for by the fact that all degradation on C-6 com- pounds has had to be done with accumulated sucrose or polysaccharide and not with the more active phOSphate esters of the hexoses from where the glycolate probably is biosynthesized. The serine at all times investigated remained car- boxyllabeled, which is also similar to the pattern observed by Hess and Tolbert (25). If the glycolate pathway were Operating as shown in the introduction, then the serine should rapidly approach uniform labeling. As this is not the case, the glycine to serine reaction must be proceeding slowly so that the major portion of the serine pool would arise from carboxyl labeled 3-phOSphoglycerate. In an alternate suggestion presented below, it is Speculated that the hydroxymethyltransferase reaction may be operating in a selective way to produce more heavily carbOXyl labeled serine. The labeling pattern reported here for glycine is much different than that observed by Zak (90). Only under conditions of isonicotinylhydrazide inhibition does the labeling pattern shift toward carboxyl labeled glycine 44 (Table 9). In this case the glycolate formed at the Short- est time is also somewhat carboxyl labeled but quickly becomes uniform. Failure to see such heavily carboxyl labeled glycine in normal photosynthesis may reflect physio- logical differences in the strains of Chlorella being investigated which could arise from environmental variances as well as possible genetic differences. Such discrepancies point to the importance of using biologically uniform material in studies to elucidate metabolic pathways. How— ever, since much work was done for this thesis with organ- isms which were assumed to be biologically equivalent, ten- tative conclusions must be drawn from the data on hand. In Scenedesmus the metabolic conversion of glycolate to glycine and serine was demonstrated by feeding glycolate- 1-140 which gave rise to the eXpected glycine-l-luc and serine-1-14C. Thus the glycolate pathway does Operate in this alga for the metabolism of exogenously supplied gly- colic acid. As mentioned earlier, the strain of Chlorella pyrenoidosa used by Merrett and coworkers also converts exo- genous glycolate to glycine and serine (M. J. Merrett, personal communication), while the endogenously formed gly- colate from the photoassimilation of acetate is not metabo- lized to glycine (47). The fact that unicellular green algae are capable of converting glycine to serine is further substantiated by the investigation of the presence of serine hydroxymethyltrans- ferase in air grown Chlorella pyrenoidosa. Substantial 45 enZymic activity could be detected in a crude cellular extract; on a mg protein basis this activity was greater than the enzymic activity detected in a similar extract from Spinach. Thus the explanation for carboxyl labeled serine in Chlorella during photosynthesis remains obscure. Perhaps all of these observations are best eXplained by invoking compartmentation of metabolic pools such as those suggested by Bassham (68) for amino acids. To fit the given data in this thesis, such a scheme would require two sources or pools of glycolate; one producing predomin- antly carboxyl labeled glycolate from 1-”002 which is some— what Slow initially, and the other very rapidly producing heavily C-2 labeled glycolate. This glycolate-i-luC pool would be rapidly converted to glycine while the conversion of the glycolate-Z-IMC pool would be somewhat Slower. Thus at short times the combined glycolate in the methanol extract appears more C-2 labeled than the combined glycine. Both of these pools may be turning over very rapidly rela- tive to the accumulation of 3,4-labeled hexoses. This trend is seen until the combined pools of both glycolate and glycine give the appearance of uniform labeling. In the case of isonicotinylhydrazide inhibition, both glycolate biosynthetic routes are operating rapidly, hence the uniform labeling at 6 seconds; however, little glycolate-Z—luc is converted to glycine so that the C-1 labeling pattern in glycine remains constant over a longer period of time. It 46 would be attractive to propose that one site in Chlorella is equivalent to a peroxisome-like particle or Site to account for these differences. Then the glycolate-i—luc from 1”C02 would be rapidly tranSported from the chloro- plast to the compartmentalized site for oxidation and sub- sequent transamination to glycine-i-luC. The glycolate-2- 1LAC would be excreted across the chlorOplast membrane (as phosphoglycolate?) into the cytoplasm where some might also be converted to glycine by cytOplasmic enZymes, although more slowly, and the rest would be excreted. Isonicotinylhydrazide, an inhibitor of aminotransferase enzymes, would inhibit this cytOplasmic route, but because of the impermeability of the peroxisome-like particle to such compounds, the glycolate-i-luc to glycine conversion would continue to operate. In view of this proposal it would be interesting to investigate the effect of Triton on the inhibition of algal photosynthesis by isonicotinyl- hydrazide, Since this detergent apparently makes the perox- isome permeable to large molecules such as NAD (R. K. Yamazaki, personal communication). To carry the model further, it is possible that the glycine-i-lu C is preferentially converted to serine via the serine hydroxymethyltransferase system, thus helping to account for the heavy C-i labeling of this molecule. The cytoplasmic glycine-Z-luc would be used for such processes as protein synthesis or purine Synthesis or might return to 47 the chloroplast for incorporation into porphyrins. From these labeling studies and enzyme investigations I conclude that the glycolate pathway does Operate in the unicellular green alga Chlorella pyrenoidosa. These results do not account for the totally carboxyllabeled glycine reported by Zak (90) in photosynthesis, nor the apparent lack of glycine formation from glycolate in the photoassimi- lation of acetate observed by Merrett (47) in Chlorella pyrenoidosa. It might be significant to note an abstract by Fott (19) in which the taxonomy of the genus Chlorella is reviewed. In this abstract the author states, "Chlorella pyrenoidosa is a mixture of Species; its physiological investigations lead, therefore, to contradictory results." Thus it is quite conceivable that future investigations may show further differences in metabolism of glycolate by algae and provide additional support for more than one route of glycolate biosynthesis. SERINE FORMATION IN PLANTS WITHOUT PHOTORESPIRATION Currently higher plants are considered as belonging to one of two groups, those with and without photoreSpira- tion. More recent evidence has shown that all plants may exhibit a photoreSpiration as manifested by oxygen uptake in the light (18, 28), but that the two groups of plants are differentiated by the presence or absence of CO2 evolu- tion in the light. The plants which do not evolve C02 in the light appear to have an alternate pathway of C02 fixa- tion involving B-carboxylation of phOSphoenol pyruvate (22), This has sometimes been referred to as the Cu-dicarboxylic acid pathway. Since the glycine to serine reaction has been posed as a possible mechanism for C02 evolution in photoreSpiration, the pathway of serine formation in plants without photoreSpiration was investigated. Literature Review Serine From C02 Fixation in Higher Plants In higher plants the observed labeling pattern of 140 in intermediates of the glycolate pathway has been more consistent with the proposed reactions than in the algal studies mentioned earlier. Vernon and Aronoff (84) initially reported the occurrence of uniformly labeled glycine from 1”C02 in soybean leaves during photosynthesis in a time 48 49 period of 15 seconds. At 50 seconds the molecule remained uniformly labeled. These results were compatible with the observations that glycolic acid was also uniformly labeled at short times in barley (65) and tobacco (24). In con- trast the serine in the eXperiments of Vernon and.Aronoff (84) was predominantly carboxyl labeled at both 15 and 50 seconds (49% and 46% of the label respectively). This observation is not consistent with Operation of the glyco- late pathway utilizing the uniformly labeled glycine. Later eXperiments of Tolbert and coworkers (24, 60) showed essentially uniformly labeled serine from 1L"C02 in higher plants at a photosynthetic time of 20 seconds or in the case of tobacco, 11 seconds. This data is shown in Table 12. The only one of these plants lacking photoreS- piration is corn; its labeling pattern appeared somewhat carboxyl labeled but not significantly so. It is signifi- cant to note that the soybean did not exhibit the predomin- antly carboxyl labeling observed by Vernon and Aranoff. Prior EXperiments With Isolated ChlorOplasts Using isolated Spinach chloroplasts Chang and Tolbert (13) found carboxyl labeled serine but uniformly labeled glycine after a ten minute period of photosynthesis. They proposed that two routes of serine biosynthesis existed, one starting from 3-phOSphoglycerate and the other starting from glycolate. The latter route would be predominant in the whole leaf, while formation of carboxyl labeled serine occurred in the chloroplasts. 50 Table 12 Labeling Pattern of Serine From Higher Plants Percent Distribution Plant C-1 C-2 C-3 20 Sec Photosynthesisx Soybean 28 38 34 Coleus 25 29 46 Peppermint 37 39 24 Barley 26 41 33 Corn 43 28 29 11 Sec PhotosynthesisO Tobacco 4O 39 20 6 Sec Photosynthesis+ Pea Bean 30 34 36 X Ref. 60 ° Ref. 24 + Chapter 3, this thesis 51 Metabolism of Glycolate in Leaf Tissue In addition to investigations of labeling patterns from lL'LCOZ, much work has been done with metabolism of radioactive glycolate and other related substrates, which lead to the concept of the glycolate pathway as Shown in Figure 1. Tolbert and Cohan (79) originally showed the direct conversion of glycolate to glycine in both green and etiolated wheat leaves and homogenates of these using glycolate-Z—luc. Glycine-Z-luc and serine-2,3-1uc were formed during a one hour period. Work by Wang and Burris (86) confirmed the conversion of glycolate to glycine and serine in wheat leaves. Tolbert and coworkers (30) looked at further products of glycolate metabolism in soybean and wheat. The hexose from sucrose was degraded after a ten minute incubation period. In soybean glycolate-2-170 pro- duced hexose-1,2,5,6-1uc and glycolate-i-luc gave rise to predominantly hexose-3,4-14C. However glycolate-i-luc in wheat produced essentially uniformly labeled hexose. Whittingham has also worked extensively on the evi- dence for the glycolate pathway by use of labeled substrates. A recent paper (44) showed that in pea leaves glycine-Z-luc produced hexose-1,2,5,6-14C whereas both glycolate-l-luc and glycine-1-140 were metabolized to uniformly labeled hexose. These results seem incompatible. Glycolate-i-luc gave rise to glycine-l-luC (80:20) and to serine-l-luC (79:13:8) indicating that randomization of 14C in hexose 52 must have occurred at a step later than serine biosynthesis. 1“'0 was lost as 002 from both glycolate and glycine-- Some 25% and 28% reSpectively. Other work Showed that the metabol- ism of exogenous substrates may be Spatially separated from the metabolism of glycolate and glycine produced photosynthe- tically. If this is indeed the case, then all feeding eXper- iments with exogenous labeled substrates would have to be reconsidered in terms of the permeability of the particular compound into the various organelles of the cell. Wang and Burris (86) studied the glycine to serine reaction in wheat leaves. In the dark glycine-2-140 was converted to serine and organic acids, primarily malate and glycolate, with a small amount of radioactivity appear- ing in sugars. This could be increased by adding cold gly- colate; in this case 14% of the added radioactivity appeared in sugars. In the light the major organic acids produced were glycerate and malate. When serine-i-luc was fed to the leaves, only 0.2% was converted to glycine; thus in ELLE this reaction seemed quite irreversible both in light and in the dark. In contrast to the studies above, the data of Marco (45) was interpreted to indicate another route of glycolate metabolism in grape and geranium leaves. In the light gly- colate was converted to tartaric acid, glyceric acid and serine, but no glycine could be detected. These results could be explained by invoking a glyoxylate condensation to 53 form a C4 compound which could either be reduced to tartaric acid or be decarboxylated to tartronic semialdehyde. This intermediate, in equilibrium with hydroxypyruvate (37), might be transaminated to serine or reduced to glycerate. large amounts of radioactivity from glycolate-i-luc were lost as CO2 in accordance with the scheme. Materials and Methods Photosynthetic 14002 Fixation All plants used in these eXperiments were grown in the greenhouse and were fertilized with Hoaglands nutrients. The plants used were sugar cane (Saccharum Sp., var. CL41223); corn.(§3§ gays, W64A x Oh43) and pigweed (Amaranthus hybridus). Leaf sections of corn and sugar cane 6-8 cm in length were cut from fully eXpanded leaves of young plants and were placed in either 0.01 M c-hydroxy-Z-pyridinemethanesulfonate (OHPMS), pH 5.5 or in distilled water for one hour. The sec- tions were completely submerged in a tall cylinder of solution to enhance uptake of the inhibitor and were aerated continu- ously to avoid anaerobiosis. This method was found effective for uptake of metabolites by such tissue (R. H. Hageman, personal communication). During this time the leaves were shaded. Whole leaves of pigweed were excised and the petioles were placed in either water or 0.1 M OHPMS for uptake of the inhibitor. Photosynthetic experiments were carried out in full 54 sunlight out-of-doors in a plexiglas chamber having a lid fitted with a stopcock for evacuating the chamber and a separatory funnel for generating 14C02. For each leaf 50 uc of 1”C02 were prepared by pipetting that amount of NaHlL‘co3 and 1.0 ml 1 y; lactic acid in opposite ends of the separatory funnel while laying on its side. The fun- nel was closed off and the two solutions were mixed, thus releasing the 14002 gas. The lid was then placed on the chamber with the leaf inside, and the System was partially evacuated for a few seconds by a small portable vacuum pump. By Opening the stopcock of the separatory funnel and removing the stOpper simultaneously, the 1”€02 rushed into the chamber and photosynthetic fixation occurred under atmOSpheric conditions. The tissue was killed in about 1 second by dropping off the lid and rapidly pouring in methanol at the designated time. The corn and sugar cane leaf sections were blotted dry before being placed in the chamber for the 11+C02 fixa- tion eXperiments. The extent of stomata opening after the treatment was not determined, but the total fixation (Table 13) was great. After photosynthetic periods of 15 and 30 seconds, the leaf and the added methanol for killing were transferred to a beaker and boiled a few minutes until the leaf was free of green color. The methanol was then decanted and the leaf was extracted by boiling in water. The alcoholic and 55 aqueous extracts were combined, the volume measured, and the radioactivity determined. The dry weight of the leaves after extraction was measured. Chromatography and Radioautography of Photo- synthetic Products Portions of the extract were chromatographed by the procedures described previously and compounds were located by radioautography. Amino acids were separated by the Dowex-50-H+ion exchange technique for large-scale isolation of serine. Serine Degradation Serine was degraded by the method described in sec- tion 1. Results Total 1”(:02 Fixation and Radioactive Products The amount of 14002 fixation and the distribution of radioactivity into photosynthetic products are presented in Table 13 for all three plants. The heavy labeling in the C-4 compounds, malate and aSpartate, is significant. OHPMS caused an inhibition of total photosynthesis in pigweed and corn but showed little effect in sugar cane; this may reflect low uptake of the inhibitor by the sugar cane leaf. In both pigweed and corn the sulfonate caused a decrease in incorpor- ation of label into glycerate with an increase in alanine and sugar phOSphates. No significant accumulation of glycolate Products of Photosynthetic 1400 With B-Carboxyla ion PIGWEED PHOTOSYNTHESIS Table 13 56 Fixation in.Plants Treatment Control a-Hydroxypyridine- methanesulfonate Time (Sec) 15 30 15 30 Fixation (cpm/mg x 10-3) 14.6 15.9 2.4 11.5 Compound 2 of Total 1"C x of Total 11‘C 34P-glycerate tr tr tr tr sugar phOSphates 18.8 16.5 34.4 37.7 sucrose 2.9 7.0 - tr glycolate tr - tr 1.4 glycine 5.9 9.0 serine 5.9 6.5 {9.4 {7.2 unknown.B 1.7 5.5 glycerate 11.3 13.5 3.1 1.4 alanine 13.8 17.5 29.7 21.7 aSpartate 7.9 7.0 7.8 8.7 malate 27.2 11.5 14.1 21,7 fumarate tr tr - - succinate - - - - glutamate - - - - threonine 2.5 2.5 - - y-aminobutyrate - - — - misc. - 3.5 1.6 - salve. sly ser, sucrose 14.? 22.5 9.4 8.6 Table 13 (Continued) 57 SUGAR CANE PHOTOSYNTHESIS Treatment Control a-Hydroxypyridine- methanesulfonate Time (Sec) 15 30 15 30 Fixation (cpm/mg x 10'3) 25 20 24 54 Compound % of Total 11"C A of Total 140 3-P-glycerate sugar phOSphates sucrose glycolate glycine serine unknown.B glycerate alanine aSpartate malate fumarate succinate glutamate threonine v-aminobutyrate misc. N PH Nd HHHOHowH-floopx‘iol-‘mfl I I I I I I I I I d perm wmpmnmmowpv N flH nonlppmpmmmmpowm: I I I I uomomoommq o N o \1 H I I H N A Ol—‘HI I OWMCDNI HHHI 4:0 Uri-{:0 H mNHHOHNmmeHpmpmN hue Unptmoca \orrp- Ono kn d' WUUI \AJMI-‘OO‘sknCDOOGJ-C‘WU glyc, 81y: ser, sucrose 13.5 11.9 p I V w I N Table 13 (Continued) CORN'PHOTOSYNTHESIS Treatment Control a-Hydroxypyridine- methanesulfonate Time (Sec) 15 30 15 30 Fixation (cpm/mg x 10'3) 95 202 74 98 Compound 2 of Total 14C % of Total 1“C SAP-glycerate 4.9 1.4 0.4 1.6 sugar phOSphates 8.2 4.8 9.7 10.4 sucrose 2.2 11.7 10.5 3.8 glycolate 1.1 0.1 0.8 1.1 glycine 1.1 1.8 0.8 1.4 serine tr 1.3 0.4 2.2 unknown.B tr 1.1 1.2 - glycerate 16.5 27.7 3.2 8.4 alanine 14.8 21.0 30.6 11.2 aSpartate 1201 9014' 1009 1301‘" malate 36.8 17.9 27.0 43.0 fumarate 0.5 0.3 - tr succinate - tr - tr glutamate - 0.2 tr tr threonine 0.5 0.7 tr 1.9 y-aminobutyrate 1.1 0.7 tr 0.8 misc. - tr 4.4 0.8 SIPC. 81y. ser, sucrose 4.4 14.9 12.5 8.5 59 occurred in any of the plants, in contrast to the studies of OHPMS inhibition of photoreSpiring plants where large amounts of glycolate accumulated. The low amount of radioactivity in 3-phOSphoglycer- ate and the high percentage in glycerate stand in sharp contrast to earlier reports of sugar cane photosynthesis (22, 38) where 16-36% of the 14C fixed was found in 3-phos— phoglycerate while little was detected as free glycerate. It is possible that the killing procedure used in the present work did not rapidly inactivate all phOSphatase activity; and, therefore, label was shifted from 3-phos- phoglycerate to glycerate during killing. Thus the differ- ences observed in the amount of glycerate which accumulated in the presence of OHPMS cannot be unambiguously interpreted. Serine Labeling in.Plants with B-Carboxylation The intramolecular distribution of radioactivity in serine from luCOZ during photosynthesis in pigweed, sugar cane and corn is given in Table 14. This C-3 labeling is most pronounced in corn and in the short time fixation by pigweed. Although Rabson‘gt El: (60) did not report a similar pattern in their photosynthetically formed serine from corn, the phenomenon seems real and reproducible at two time periods in the present work. The reliability of the serine degradation procedure was excellent (see methods) and was checked repeatedly during the course of this work. Since these are plants without photoreSpiration, the results 60 Table 14 Labeling Pattern of Serine From B-Carboxylation.PlantS Percent Distribution Plant C-1 C-2 C-3 15 Sec Photosynthesis Pigweed 22 19 59 Sugar Cane 34 36 30 Corn 17 14 69 30 Sec Photosynthesis Pigweed 32 30 38 Sugar Cane 25 28 47 Corn 17 16 66 61 may suggest something unique in serine biosynthesis in this group of plants. Discussion The 002 fixation studies indicate a rapid flow of carbon into products of the glycolate pathway. The amount is not particularly large, and the effect of OHPMS is dif- ferent than the effect of this inhibitor observed in photo- reSpiring plants, namely the accumulation of glycolate with a decrease in glycine and serine. Either the inhibitor did not reach the site of glycolate oxidation, or a typical glycolate pathway is not operative in these plants. The labeling patterns of serine from these plants do not lend themselves well to clear interpretation. The heavy label in the B-carbon has no precedent except for the case of Coleus reported by Rabson.§§H§l. (60). Whether or not Coleus has photoreSpiration is not known; the plant is unusual in that it accumulates sedoheptulose in large amounts equal to that found in Crassulacean.plants (81). Whether this is correlated with the occurrence of B-carboxy- lation of phOSphoenolpyruvate, which also occurrs in Crassulacean.plants, likewise is not known. In the case of pigweed and sugar cane it is quite likely that the typical glycolate pathway is Operative, together with the route producing serine-3-1uC, leading to a less asymmetric label- ing pattern for serine. In corn. however, the C-3 pathway 62 is predominant. Concrete evidence for the Operation of the glycolate pathway in these plants has been accumulating in this laboratory from work of other investigators on peroxi- somal activity and the enzymes for glycolate metabolism. The most probable route for incorporation of label into the C-3 position of serine is via a tetrahydrofolate mediated transfer of a C-1 group onto glycine. To account for the heavy C-3 labeling, this THF-C moiety would have 1 to be derived primarily from the newly fixed lucoz. At the present time there is no biochemical justification for hypothesizing any kind of tetrahydrofolate mediated CO2 fixation. However, when considering the elusive nature of the "transcarboxylation reaction" posed by Hatch and Slack (22) to account for transfer of a carboxyl group from oxalacetate to a C2 or C5 moiety, it is attractive to con- sider whether this C1 transfer at the level of formate may in some way be linked to the tetrahydrofolate mediated sys- tem. There is evidence for the incorporation of formate into the C-3 of serine (81). Assuming that this C1 pool were small and rapidly saturated with 140, this could account for the heavy C-3 labeling of serine where the glycine moiety would be more slowly labeled via the flow of carbon through sugar phOSphates and glycolate. However, in view of the fact that there are no anal- ogous reactions known for the flow of carbon from C02 directly to a tetrahydrofolate-C1 unit, an alternate eXplanation 63 invoking compartmentation is probably just as plausible, eSpecially when considering the previously mentioned irreg- ulatities in the distribution of label in serine from a variety of plant sources. This may reflect a lack of the smooth uninterrupted flow of carbon from glycolate to serine and glycerate. Rather, at certain stages smaller pools such as the glycine serving as the substrate for photoreSpiration may be rapidly saturated with label while the bulk of the glycine serving as the precursor of serine may be in a larger, more slowly labeled pool. Thus the initial serine would appear more C-3 labeled and would rapidly approach a uniform distribution. GLXCOIATE BIOSYNTHESIS IN MANGANESE-DEFICIENT BEANS Introduction and Literature Review Manganese deficiency in plants is manifested by chloro- sis of the leaves (6) and abnormal chlorOplast develOpment (57, 76). A deficiency of this element has been shown to cause an impairment of oxygen evolution during photosynthesis in both algae (14, 32) and higher plants (70, 71). Also man- ganese deficient algae have been reported to exhibit an inhi- bition of glycolate biosynthesis (25, 74). More recently manganese deficiency has also been shown to result in altera— tions in amino acid metabolism in a higher plant (20). The effect of manganese deficiency on glycolate biosynthesis in higher plants has not been reported; therefore, an investiga- tion was undertaken with manganese-deficient beans (Phaseolus vulgaris) to determine the effect of this condition on glyco- late biosynthesis and formation of other products of the gly- colate pathway. Materials and Methods Manganese Deficiency Pea beans (Phaseolus vulgaris. var. Sanilac) were grown in May in the greenhouse. The plants were grown in 7 inch pots, each containing 700 g oven dry soil (Houghton muck) and 64 65 1300 ml water at field capacity. Manganese deficiency was obtained in two ways: by withholding manganese from the nutrient and by lime treatment in addition to withholding manganese. A fertilizer application of 300 ppm N, 250 ppm P and 400 ppm K was mixed with the soil in each pot one day prior to planting. At planting a micronutrient mixture, with or without manganese,was added in a band one inch below the seeds. Plants grown in this media for three weeks with- out lime treatment produced mild manganese deficiency. Severe manganese deficiency, as evidenced by chlorosis, was produced by adding 40 g pure CaC03 to pots prior to plant- ing. Control plants which Showed normal greening were pro- duced by addition of 40 ppm manganese as MnSOh. I am indebted to Dr. B. G. Ellis of the Soils Department at Michigan State University for growth of these plants. IsotOpic Procedure For 14002 fixation experiments young single leaflets of about the same size and degree of chlorosis were selected from a number of plants. The amount of chlorophyll in these leaves was not quantitatively measured but similarly defi- cient leaves contained about 5-10% as much chlorOphyll as normal leaves. The leaves were removed from the plants between 9:30 and 10:30 A.M., quickly weighed on a pan balance, and immediately eXposed to 14002 or placed in inhibitor solutions. All work was done in the greenhouse area and stomata should have remained Open during most of 66 the eXperiments except for the 600 second eXposure to 1“C02. The leaflets varied in fresh weight between 0.4 and 0.6 g. For eXperiments with inhibitors the petioles of leaves were placed in 0.1 M a-hydroxy-Z-pyridinemethanesulfonate (OHPMS), adjusted to pH 5.5 with KOH, or in water for one hour before eXposure to 11+C02. This treatment probably caused the stomata to close (94). The isotOpic procedures as described by Hess and Tolbert (24) and given in greater detail in the second section of this thesis involved eXposure of a leaflet inside a plastic chamber to about 0.2% 14002 containing 200 no 14C (52 uc/mg BaCOB). EXperiments were run in sunlight which varied between 5000 and 10,000 ft-candles. Fixations were stOpped by rapidly flooding the chamber with methanol followed by extracting the leaflet with hot methanol and then boiling water. Aliquots of the extract were counted for total fixation and analyzed by the previously described two-dimensional paper chromatography system for percent dis— tribution of 1LAC among the products. Serine Degradation The periodate method of Chang and Tolbert (13) described earlier in this thesis was used. Results Total 1“Cog Fixation and Radioactive Products The total 14C02 fixation and distribution of radio- activity among the photosynthetically formed compounds in 67 the two types of manganese-deficient plants are shown in Tables 15 and 16. In all cases there is a marked decrease in the amount of radioactivity in glycolate and a corres- ponding increase in the percentage of sugar phOSphateS formed. When the individual metabolites of the glycolate pathway are compared under the two conditions of manganese deficiency, however, the results are not similarly consis— tent. In the case of the severe manganese deficiency (lime treatment) the amount of glycine formed is less while serine formation increased. Sucrose formation also increased at short times butat longer times appeared unaffected by the deficiency (Table 15). In those plants with the deficiency induced by withholding manganese there was a general decrease in radioactivity in glycine, serine and sucrose (Table 16). This difference in the effect of manganese deficiency on the formation of serine was confirmed by comparing the ratio of glycine to serine in the 6 second photosynthesis eXperi- ment from chromatograms of the amino acid fraction. These ratios are shown in Table 17. Obviously, in the severely manganese-deficient plants there is a marked increase in serine accumulation. Serine Degradation There are two possible routes of serine biosynthesis in plants, one with glycolate as the precursor (78) which gives rise to uniformly labeled serine from luCOZ, and the other starting with 3-phOSphoglyceric acid (13) which results 68 m.m m.m w.m 5.0 «.« m6 Nam o.m mango Nam «.N 9m N.« m.o« m.« 0.3 0...... 055: Tn NA 9.2 2m 2. oi so NJ 32830 m6 m.m o.w« «....~« mfiw «.3 06m m.«m mougauonm humSm 4.3 To: Tau Tom 95 4.9 99 9: 323m m.m« 3.: m.«« N.m :.w« a.« o.~.« 3.: canton w.w« sow oém 5.3 «.3 06m 0.x. mi...“ , 05088 m6 06 0A 9m on o.m on w.m opadoohac m a u a a a a a boa on SA e3 x 4.4m 62 w Nod 63 w 84. a: w 8.6 e3 w 93.. boa w 2.6.6 63 w 84“ Rog means 9: ace :2... 8:. :8: £1. :2... :2... at: :2... peaches oo« ow cu w «003 083. 385.5 83 nobuoq seem gso«o«.«onlouo:¢w§x mp 203183 m« 0.5.9 an 8538 8:38.83 N003 no 30.60am wag 0.: .«o Scampsntvua usogom 69 NJ «.N. I w.o 0.: I 0 ~50 2230 .3 m6 0.: o5 Nun m; «A o.~ 235$ n.m 9N m.m m4. m.m Nam N4. «.3 39305.8 m.~m «.m 98 3.2 «.3 93 9? N5 movafiuofi 2mg... u.m« «6n O.N« N.«N o.: «.m« 0.0 u.m« ongoam {N m.~ 0A w.~ on «.m 0A m.« ofihom m.nn ER 9? 93 m Sm Qmm QR m .3 0593b 3.0 n; o. m6 EH ma 3 o; 3383... m m N u . u a n. mg n 3A 03 x mfi a: x Ra wS x 8.: mg x 94 m3 x 8.~ a: x 3672 x mo.~ 8.3 33.2 oi 3% as: at... 2:... :2... at... 5:. £1 — c2... «napuoua om« ow cm «003 3.; 7353...: 9:322 «3 F 3538 59.33.35 3»qu :dom #:3033335”?! hp «835 003 .«o 3260.5 wean—4 03 .«o 2033....“an accouom 3 3.3. 70 Table 17 Relative Amounts of Glycine and Serine Formed During Photosynthesis By Bean Leaves at 6" Treatment Glycine/Serine + Mn 21.5 + Mn (Lime) 10.2 Mn (Lime) 0.56 71 in an.accumulation of label in the carboxyl group of serine. If the glycolate biosynthesis is being inhibited by condi- tions of manganese deficiency, it is conceivable that the glycine and serine observed under these conditions are com- ing from 3-ph03phoglycerate and the change in the glycine to serine ratio reflects an effect of manganese on the serine hydroxymethyltransferase. To discern which of these routes may be operating in the manganese-deficient beans, the serine from the six second period of photosynthesis was subjected to chemical degradation. These results are shown in Table 18. It is apparent that there is no particularly heavy carboxyllabeling which would support the view that serine was formed from 3-ph03phoglycerate; rather, the labeling distribution appears fairly uniform suggesting that the glycolate pathway is Operating even under condi- tions of severe manganese deficiency, although at a reduced rate. When the percentages of the major metabolites of the glycolate pathway appearing on the chromatograms are added (Table 19), this lowered rate of carbon flow through the pathway is evident eSpecially at the shorter times. The data of Hess and Tolbert (25) and of Tanner, 23.2;, (7%) with Chlorella are included in the table for comparison. In their eXperiments the total flow of carbon through the glycolate pathway was not reduced even though the relative amount remaining in glycolate was significantly less in the manganese-deficient algae. The effect of the deficiency in this alga may have been on the subsequent metabolism of 72 Table 18 Distribution of 140 in 6" Serine From.Beans L L - - % Distribution 140 Treatment + Mn - Mn + Mn (Lime) - Mn (Lime) C—1 28 26 31 38 0-2 36 34 33 39 C-3 36 39 36 23 73 i. use Ti. mm .3933 a.um o.m: N.:m 3.3m omouosm .ocapom .oCHohao .opaflookao :2 I a: + :2 I :2 + nae ow ads 0« mm .mom Afiofloa us. 305 0.8 Now vim mad ocflom .oCdomHu .opuHoohHo «HHoAOHzo fi socoaoflonéz :.mm «.om 0.5m m.ms «.0: m.:m m.m¢ o.dn omonosm .ocanom UHonnpaz .ocdohao .opuaoohaw c: m.om o.mm o.~w o.ms o.mm «.mu w.um n.0w . omouowm .ocanom popuoha nonwodsoo ca Honda mo omapcoopom Hence ocdohao opuHoohHw mean as I :z + a: I :z_+ a: I a: + as I gz.+ unospuoue om” ow om w Aoomv mafia fluofifimfiofi mafia vapors tag...“ 338ch 93 no 3938302 3 canes 74 glycolate rather than on its biosynthesis. Effect of Hydroxypyridinemethane Sulfonate That glycolate biosynthesis might be inhibited in manganese-deficient beans was examined further by using leaves which had imbibed OHPMS for one hour prior to eXpO- sure to 1b’COZ. This sulfonate is an inhibitor of glyco- late oxidase (91), but it also exhibits inhibitory proper- ties on other enzymes such as aminotransferases (80). In tobacco leaves a large accumulation of glycolate during photosynthesis has been observed in the presence of OHPMS (24, 92, 93). This effect has been interpreted to indicate the inhibition of the glycolate pathway in 1112 by this compound. Under these conditions again differences were observed in the two types of manganese-deficient plants. In the less severely deficient leaves, there is a marked inhibition of glycolate accumulation in the presence of OHPMS (Table 20), consistent with the earlier mentioned general decrease in glycolate pathway products. The lime-treated plants, however, accumulate as much glycolate under deficient conditions as the manganese-grown controls and exhibit a correSponding decrease in glycine and serine. Thus in the case of the less severe deficiency it appears that glycolate biosynthesis was inhibited, whereas in the lime treated plants it was not. 75 cannon» egos nobcoH on» was» a .Nooam op cyanogNo ouomon opunchHamocunposocdvduhmbwonchnIt and: use: H how 0 as .3 a; 3 can: 5. 8 3:32.98 8335 «8.: tag o.m d.«a N.w m.m o o o o .oufiz u.NN a.w H.BN w.wH w.nm :.m n.mm :.m osdqua< m.: m.N o m.N o.m o.N o m.m oucuoohflo N.mm m.:fi N.N: d.wa m.m: w.w~ n.00 0.0N monogamonmnnuwsm o.NH m.:~ 0.0 :.w« n.m m.w o m.m ouchosm o.n Nam o o o o.m o NA ofinom m .o as: o c 0 man 0 T3 ofiohu m. E mam Ta 93 v.3 an: :3 can opuaoofio u a u a m m u m ooo.oH#.a ooo.omN.« ooo.mom ooo.ooa.« ooo.m#a coo.HHm oom.Nm ooo.m¢m Mica wB\COHpQNdh Emu n: I a: + n: I GZI+ :EII nz.+ :2 I nz_+ pcufipdoua 0.85.29 33 3 1 03 om A88 8: ; unbuoqnecoaoauonIononuwsdx an mp05p0hm cavonpshuoponm so pcoapuoue opuspmHamonunpoaoadvnummhxohvhm mo vacuum 0N oanue 76 Discussion Although manganese deficiency has been correlated with an inhibition of glycolate biosynthesis in Chlorella, the results of this investigation do not conclusively support this hypothesis in pea beans. Also a reexamina- tion of the data with Chlorella suggests there may be an alternate eXplanation of the results obtained by these other workers. The data presented above indicate a moderate reduc- tion in the amount of 1"c flowing through the glycolate pathway under manganese deficient conditions. The amount of this reduction ranged from as much as 50% less in one case to as little as 8% less in another. Also with the OHPMS inhibited plants, the results show in one set of experiments that glycolate biosynthesis occurred signifi- cantly less in the manganese-deficient plant. However, the total fixation by this leaf in a 20 second period of photosynthesis was only 6.3 x 10“L opm. Perhaps the stomata were closed and prevented adequate 1”C02 from reaching the cells. Because of the low amount of radioactivity fixed, the radioautogram showed only a few faint spots. Thus the absence of glycolate is not well verified. More definitively, the occurrence of essentially uniformly labeled serine in both controls and manganese- deficient plants indicates that carbon flow through the glycolate pathway was still occurring, but that the relative 77 amounts of 1”C in the various intermediates varied. Thus the manganese deficiency may have a more general effect on the activity of several enzymes. This might be somewhat analogous to the work of Gilfillan (20) where manganese deficiency in macadamia caused the accumulation of certain amino acids. Also as mentioned earlier, in the eXperiments of Hess and Tolbert and of Tanner, gt_al. with Chlorella, manganese deficiency resulted in an increased accumulation of amino acids, namely glycine and serine. The conclusion from these eXperiments is that the glycolate pathway does operate in manganese-deficient plants, but the amount of carbon flow through this route is somewhat reduced. Since sugar phoSphates accumulate to a greater extent in the deficient plants and such compounds are believed to be the precursor of phoSphoglycolate which is hydrolyzed to glycolate (78), it is suggestive that one of the metabolic effects of manganese deficiency is related to the sequence of reactions leading to the formation of glyco- late. INTRACELLULAR LOCATION OF SEBINE HYDROXYMETHYLTRANSFERASE Subcellular particles containing glycolate oxidase from green leaves have been identified by Tolbert §t_§l. (82) as peroxisomes. Initial observations indicated that the leaf peroxisomes contained in addition MADE-glyoxylate reductase and catalase. In subsequent work Kisaki and Tolbert (34) also reported a glyoxylatezglutamate amino- transferase in these particles. Thus the flow of carbon from glycolate to glycine could take place within a single compartment of the cell. The question of whether additional reactions of the glycolate pathway were also located within the peroxisome was posed, and because of the significance of this pathway in photosynthetic carbon metabolism,an investigation of the next reaction of the pathway was begun. This reaction is the conversion of two glycine molecules to a molecule of serine and carbon dioxide, catalyzed by serine hydroxymethyltransferase (EC 2.1.2.1). Literature Review Serine Hydroxymethyltransferase in Plants The enzymic conversion of serine to glycine in plants was first observed by Wilkinson and Davies (88) using turnip hypocotyls and homogenates of cauliflower buds. The enZyme required tetrahydrofolate and pyridoxal phOSphate. Subse— 78 79 quently the enzyme has been reported to occur in corn seed- lings (23), wheat leaves, carrot roots, pea leaves, castor bean endoSperm (15) and tobacco roots (58). A more detailed report on the enzyme from cauliflower buds has appeared recently (#6). These latter authors Speculated on the pos- sibility of isoenzymes since their preparation showed two pH Optima and there were indications of two enzyme bands off a Sephadex column. However, Hauschild (23) has been the only worker to examine the intracellular location of the enzyme. Using a sucrose isolation medium, he found about 10% of the activity in a 15,000 x g pellet containing mito- chondria from etiolated corn seedlings. Upon washing this pellet, all activity was solubilized. Glycine-Serine Interconversion in Animals The properties of this enzyme and the intracellular location in animal tissue have been studied more thoroughly. Nakano‘gglgl. (49) have reported both a mitochondrial and a soluble form of the hydroxymethyltransferase in rat liver. The two isoenzymes could be separated by electrophoresis and DEAE-cellulose chromatography. Of all the folate-linked enzymes studied in rat liver, only serine hydroxymethyltrans- ferase showed a higher Specific activity in the mitochondrial fraction than in the cytoplasmic extract (87), indicating a concentration of the enzyme in mitochondria. Sanadi and Bennett (63) found that the entire comple- ment of enzymes and cofactors necessary for the synthesis of 8O serine from glycine was present in a mitochondrial fraction from chicken liver. Other workers (45) also showed that a particulate fraction from pigeon, duck, or chicken liver could produce stoichiomentric amounts of C02 and serine from glycine in the presence of PAIP and NAD. Tetrahydrofolate was stimulatory only in homogenates from birds raised on a folate deficient diet. The enzyme activity was shown to be in the mitochondria by using differential centrifugation of a sucrose homogenate. Recently Sato 32 21° (64) have examined the glycine to serine system in rat liver mitochondria and have shown that this system requires tetrahydrofolate, PALP and NAD. The reaction was stimulated by dithiothreitol. The stoichiometry of COZ evolved and serine formed was very close to 1:1. It then follows that it would not be unreasonable to eXpect some conversion of glycine to serine to take place in plant mitochondria. However, in view of the fact that the conversion of glycolate to glycine has been shown to occur in that organelle of the Spinach leaf termed peroxi- some, this particle could also appear to be a probable site for the serine hydroxymethyltransferase, particularly since it has often been present as contamination in previous mitochondrial preparations prepared.by differential centri- fugation. 81 Serine Formation in Isolated ChlorOplasts As mentioned earlier, Chang and Tolbert (13) reported the formation of serine in isolated Spinach chlorOplasts. It is quite likely that the preparation also contained mito- chondria and peroxisomes. New improved methods of chloro- plast isolation have been reported (29) and C02 fixation studies have been carried out. It is interesting to note that in chlorOplasts prepared by the Bassham and Jensen method no glycine and serine were formed from 14002 (2). Bassham did Show that glycolate-Z-luc is converted to gly- oxylate and glycine by these chloroplast preparations (12), however, in 17 minutes the total conversion was only 0.07% (0.05 umoles) for glycine. Early work of Tolbert and Kearney (31) with a Simplier salt or sucrose medium for chlorOplast isolation had Shown a 0.9% conversion of glyco- late to glycine in their chlorOplast preparation and a 1.6% conversion in the suSpension medium. These authors dismissed this as an inconsequential amount probably arising from cyto- plasmic contamination. In light of recent work from this laboratory (82) these reactions could be reinterpreted to be a manifestation of peroxisomal contamination in the chloroplast preparation. Gibbs gt 2;. (17) also observed no glycine or serine produced from llJ’COZ in isolated Spinach chloroplasts at times as long as 32 minutes, although both 3-phoSphoglycerate and alanine were labeled in one of the experiments Shown. 82 Shephard gtflgl. (66) used Acetabularia chloroplasts isolated by a very careful procedure to examine photosyn- thetic C02 fixation by whole plastids. Contrary to the data of other workers chloroplasts and whole cells showed essentially the same products, and glycine and serine were apparent in small amounts on the chromatograms. Insoluble material was subjected to acid hydrolysis, and 40% of the strong HCl hydrolysate from a 70 minute photosynthesis eXperiment was recovered as glycine and serine. In terms of total fixation, this amount represented about 1.3% of the total 140 incorporated. The authors stated without showing data that in these chloroplasts oxygen evolution was unaffected by addition of exogenous glycolate, so that it might be argued that no cytoplasmic enzymes were contam- inating the preparation which could convert excreted gly- colate to glyoxylate and subsequently to glycine. However, the lack of an effect on oxygen evolution does not rule out the possibility of a glycolate oxidoreductase such as that seen by Nelson and Tolbert (51) in Chlamydomonas which might be utilizing some endogenous electron acceptor other than oxygen. Recently published electron micrographs (5) of the Acetabularia chloroplast preparation showed some mitochondrial contamination. Thus it is not clearly established whether glycine and serine could be synthesized in chlorOplasts. 83 Materials and Methods Fractionation of Subcellular Particles by Isopycnic Sucrose Density Gradient Centrifugation The fractionation of subcellular particles from Spinach leaves has been described previously (82). The same procedure was used in this investigation and is only briefly described. Spinach leaves (var. Dixie Market) from plants grown in a growth chamber on an 8:16 hour light:dark cycle were harvested and stored in the dark several days to minimize the amount of starch granules in the chloroplasts. After the washed leaves were rinsed with distilled water and blotted dry on paper towels, they were deribbed and weighed. The leaves were ground 10 seconds at full Speed in a Waring blendor with 1.5 volumes 0.8 M sucrose in.0.02 M glycylglycine, pH 7.5. The brei was squeezed through nine layers of cheesecloth, adjusted to pH 7.5 and centrifuged as follows: Filtrate l100 X 89 30 min | ‘ fi 100 g Pellet Supernate (whole chloroplasts) 6000 x g, 20 min V 6000 g Pellet Supernate (broken chloroplasts) 39,000 X g, 20 min r “1 39000 g Pellet Supernate (mitochondria) 84 The pellets were resuSpended in small volumes of the buf- fered 0.8 M sucrose. A sucrose density gradient of the given composition was prepared: Sucrose Volume (ml) 2.5 M 4.0 2.3 M 8.0 1.8 M 10.0 1.5 M 15.0 1.3 M 15.0 With a pipet 1.5 ml of the resuSpended 6000 g pellet con- taining broken chlorOplastS, peroxisomes and mitochondria was carefully layered on top of the gradient. The centrifu- gation was then run in a Beckman Spinco Model L-2 Ultracen— trifuge at 25,000 rpm for 3 hours using the SW-25.2 rotor. Nine fractions were collected by draining the tube from the bottom upon completion of the centrifugation. Preparation of Linear Sucrose Density Gradient The linear sucrose density gradient was prepared on an apparatus fabricated at this department from a design reported by Britten and Roberts (7). This mixing apparatus consisted of two chambers mounted on a flat piece of plexi- glas and connected at the bottom with a Short section of tubing. Chamber I in which the mixing took place by means of a magnetic stirring bar, had an outlet with a narrow piece of tubing leading to the nitocellulose centrifuge tube. The solutions at the extremes of the concentration range desired were placed in the two chambers, using 85 Chamber I for the more dense solution. The connecting tube between the chambers was Opened as the magnetic stirrer was started and the solution was allowed to flow out by gravity into the centrifuge tube. A total of 26 ml was used in the final gradient. One freshly poured gradientywhich contained a layer of 2.0 ml 2.5 M sucrose at the bottom, was immedi- ately collected from the bottom in approximately 1.5 ml fractions; a plot of refractive index vs. fraction number showed reasonable linearity. A similar gradient from 1.5 M to 1.8 M_sucrose was used to rerun an aliquot of Fraction V (mitochondrial fraction) from the stepwise gradient after dilution to 1.5‘M with 0'8.! sucrose. This sample was run for 18 hr at 25,000 rpm in the same preparative centrifuge using the SW-25.1 rotor. After centrifugation 1.5 ml frac- tions were collected from the bottom of the tube. Mitochondrial.Preparation in Mannitol A method for isolation of plant mitochondria in a buffered mannitol medium has been worked out by G. G. Laties (personal communication to N. E. Tolbert). This method was adapted for use with spinach leaves to investi- gate the serine hydroxymethyltransferase reaction in such a mitochondrial preparation. The isolation medium consisted of 0.37 M mannitol, 0.25 M sucrose, 0.025 M TES, pH 7.8 (N-tris-(hydroxymethyl)-methyl-2-aminO-ethanesulfonic acid), 0.1% bovine serum albumin, 4‘mM B-mercaptoethanol and 5.2% ethylenediaminetetraacetic acid. The wash medium was the 86 same except that the B-mercaptoethanol was omitted. The washed and deribbed leaves were ground in two volumes of isolation medium for 10 sec at full Speed in a Waring blender at 4°. Debris was renmved by squeezing the homog- enate through eight layers of cheesecloth. Then.the mito- chondria were concentrated by collecting the 1.000-10,000 x g pellet which was resuSpended in a small volume Of wash medium. ,A purified mitochondria fraction was obtained by collecting the particles which sedimented between 250 to 6,000 x g. Although rich in mitochondria, this pellet also contained much chlorophyll. It is unlikely that any of this could be attributed to whole chloroplasts. Enzyme.Assays 1) Serine to Glycine Conversion An.assay utilizing the formation of radioactive for- maldehyde from serine-3-1uC has been reported (75). The method was adapted with minor modifications. Solutions were pipetted into a conical centrifuge tube to give the following mixture: phOSphate buffer, pH 7.5, 7.5 umoles; PAIP, 0.25 umole; tetrahydrofolate, 0.05 pmole; enzyme; and water to a volume of 0.50 ml. The solutions of cofac- tors were adjusted to pH 7.5 before use. The tetrahydro- folate was stabilized by preparing the solution in 0.03% B-mercaptoethanol. After incubating the mixture at 25° for 5 min in a water bath, the reaction was initiated by adding 0.8 micromOles serine-3-14C with about 200,000 cpm radio- 87 activity. The reaction was stOpped after ten minutes by the addition of 0.5 ml 1.0 M_Sodium acetate, pH 4.5. Then 0.2 ml 0.1 M_carrier formaldehyde and 0.3 ml 0.4 M dimedon in 50% ethanol were added and mixed. The tubes were placed in a boiling water bath for five minutes and then cooled; and the dimedon derivative was extracted by shaking the reaction mixture for 1 min with 5.0 ml toluene. The tubes were centrifuged for 2 minutes in a clinical centrifuge to separate the emulsion. The toluene solution as the upper layer was then carefully removed with a diSposable pipet. Because of the great amount of unreacted radioactive serine remaining in the aqueous solution, care was taken to prevent accidental contamination of the toluene sample by droplets of the aqueous solution clinging to the tube walls. Three ml of the toluene solution were pipetted into a scintilla- tion vial with 10 ml scintillation fluid made Of 5.0 g PPO and 100 mg POPOP in.one liter of toluene. Because of the chlorophyll extracted into the toluene, it was necessary to determine the amount of quenching for each sample by means of the external standardization. From the amount of radio- activity found in formaldehyde, the molar quantity could be calculated. A unit was defined as that amount of enzyme catalyzing a reaction Of 1 nmole/min/mg protein. 2) Glycine to Serine Conversion Since the flow of carbon in the glycolate pathway requires the conversion Of glycine to serine, it seemed 88 advisable to check the enzyme reaction in this direction also. The assay was based on the disappearance of formal- dehyde by incorporation into serine via methylene-tetra- hydrofolate. Preparation and determination of formaldehyde solution (69) A weighed amount of D-glucose, capable of reducing no more than 0.5 mmole periodate, was dissolved in 2.0 ml water. The oxidation was started by adding 2 ml 0.3 M periodic acid and 2 ml 1 M NaHCO3. After one hour at room temperature the solution was acidified with 15 ml 0.5 M H2804, and 5 ml 1‘M sodium arsenite were added. The solu- tion was agitated about five minutes while the iodine color disappeared. The standard solution was then diluted to a formaldehyde concentration of about 1 umole/ml. A reagent blank was prepared similarly omitting the D—glucose. Mea- sured aliquots in a total volume of one ml were mixed with 2.0 ml Nash reagent, heated to 600 for 10 minutes, cooled, and diluted to 10 m1. Nash reagent is 2.0 m1 2,4—pentane- dione, 150 g ammonium acetate and 3.0 ml glacial acetic acid made up to a volume of one liter with water. The Optical density was read at 410 nm on a Beckman DU Spectro- photometer using a path length of one cm. The reagent blank showed no Significant absorption at this wavelength. The determination Showed good linearity up to 1.2 umoles formal- dehyde. 89 Enzymic incorporation of formaldehyde into serine The assay as described by Nakano gg'al. (49) was modified to the following procedure of pipetting into a conical tube: 12.5 umoles glycine; 0.05 umoles PALP; 75 umoles potassium phOSphate buffer, pH 7.5; enzyme; water to a volume of 0.4 ml. The mixture was flushed with nitro- gen gas for three minutes; then the enzyme reaction was initiated with the addition of 0.10 ml of a solution con- taining 0.40 umoles tetrahydrofolate and 1.25 umoles for- maldehyde prepared under nitrogen. The reaction proceeded for 30 min at 25°, and was stopped by the addition of 0.10 ml 15% trichloroacetic acid. The precipitate was packed by centrifugation in a clinical centrifuge. For the formal- dehyde determination a 0.30 ml aliquot of the clear solution was pipetted into a test tube, and 2.0 ml Nash reagent were added. The samples were heated for ten minutes at 60°, cooled and diluted to 10 ml. The absorption was read at 410 nm. 3) Cytochrome c Oxidase The method is the same as that used by TOlbert §£_§l. (82). Using micro-cuvettes with 1.0 cm path length, 0.5-2.0 ul enzymes were pipetted into one corner Of the cuvette, and 5 ul of 4% digitonin were deposited on the cuvette wall. The two droplets were then mixed and allowed to stand for exactly 60 seconds, at which time the material was diluted with 0.20 ml 0.1 M phOSphate, pH 7.0. A 50 ul aliquot Of a 90 solution containing 50 mg cytochrome c in 10 ml water with a few micrograms of dithionite was pipetted unto the diluted enzyme solution. The solutions were rapidly mixed by Shak- ing and the time course Of cytochrome c oxidation was fol- lowed automatically, using a Gilford recording SpectrOphotom- eter, by the decrease in absorption at 550 nm. 4) Glycine Decarboxylation The procedure for examining glycine decarboxylation was adapted from methods reported for avian liver (62, 63) and bacteria (36). The reaction was carried out in a Warburg flask which contained the following components: Tris buffer, pH 7.5, 20 umoles; B-mercaptoethanol, 60 umoles; tetrahydro- folic acid, adjusted to pH 7.0 in 0.1% B-mercaptoethanol, 3.0 umoles; NAD, 0.3 umoles; PAIP, pH 7.5. 0.25 umoles; and enzyme preparation. The reaction was initiated by tipping in 1.6 umoles glycine-i-luC with 44,000 cpm radioactivity. lbC02 was trapped in 0.2 ml 1.M NaOH in the center well with a filter paper wick, and the reaction was stOpped by adding 0.2 ml 8-0.§ HZSOu from the other Side arm. The enzyme reac- tion proceded for 30 or 60 minutes at 25°, and after killing the flasks were shaken another 60 minutes to assure complete trapping of the 1L"C02. The wicks were then transferred to scintillation vials containing Kinard's scintillation fluid (33); a 0.2 ml volume of water used to rinse the center well was also added. The amount of decarboxylation was calculated from the radioactivity released. 91 Protein.Determination Protein was determined by the Lowry procedure (41). Although both sucrose and the green color in the chloro- phyll-containing fractions interfered with the test, it was felt that the determination was sufficiently accurate to give values which would be useful in comparing enzyme activities on a mg protein basis. Materials — Reagent A: 2% Na2C03 in 0.1.M NaOH; Reagent B1: 1.0% Cusou-S H20; Reagent 132: 2.0% sodium tartrate; Reagent C: 50 ml A - 0.5 ml B1 + 0.5 ml B2, prepared fresh daily; Reagent D: 1.0 M phenolmreagent (commercial preparation); protein standard: 100 ug/ml commercial bovine serum albumin. Procedure - A 1.0 ml volume of the protein containing solution was mixed with 5.0 m1 Reagent C; after standing 10 minutes or more at room temperature, 0.5 ml Reagent D was rapidly added with immediate mixing. The color was allowed to develOp for 30 min at room temperature. Absorption was read at 660 nm. Results Distrubtion of Serine Hydroxymethyltransferase Among Subcellular Particles The relative amounts of enzyme activity found in the fractions obtained by differential centrifugation and in the discontinuous sucrose density gradient are Shown in Table 21. The assay procedure for the conversion of serine to glycine .osaIOOmsmOHmaaom op OdaIMIosaaom mo noamaoazoo one up Oommmmw ** .mw .poa mom ommampmo UGO ommpaxo opmaoomam .HHzSQOHOHSO mo mcodpsnaapmau Hmoaamp Mom * 92 H.0m oawm m.NN mom pCOpOflthfim fine omam a5 mam poflom w ooomm 0.3 8% {mm 33 poflom m coon m6 or: 0.3 no: aoflom m ooa Asa8\moaoaonmnv Anda\moaoaondsv 535233 a mafia: Heeoa soapsflfina a mafia: Hoeoa aofloeam **®W$.H0.H mQGHP IHmspOamaOhOmm osaaom ommoaxo o machzoopmo Sofipmwzmahpzoo Hmapconomwam 2H Soapsnahpmam mazwzm *msoapomam opmazoaphmm_za moammsm no soapsnaapman HN OHDGB 93 .DJHIOUmSOOHmaaom op USHIMIOnHaom mo SOHmHO>SOO Os» an commons ** .mm .moa mom mmmampmo paw omwpaxo oudaoomaw .Hamsaoaoaso mo mzoapsnahpman Hwoaamp Mom * N.m umm :.m m.ma xH :.w mom m.m o.HH HHH> m.m see m.oa m.am HH> o.ma mmoH m.oa e.om HS w.Hd odmm H.:d m.aw > H.m 0mm m.w m.HH >H :.m awn 0.0 o.a HHH m.m Ham 0 I HH a H Aaaa\moaoaosmnv Azaa\moaoaoswsv soapsaaepnan a means annoy soaeapaaenao a heads Hoeoa eoaeoeae omwvawo O maOHSOopmo ommaowmswap Iamnpmamxoavmn weapon pumacmhw omoaosm msoamapsoomaa no soapsnaapwam Ill 'W xeoesaesoov Hm oases 94 was employed. The major portion of the enzyme was contained in the 6000 g pellet and sedimented with the mitochondrial band on the sucrose gradient. The table also shows cyto- chrome c oxidase activity, which was employed as a marker for mitochondria. From the table it is apparent that the serine hydroxy— methyltransferase activity closely parallels the activity for cytochrome c oxidase. There is almost no activity in the peroxisomal fraction (III) which was marked by glycolate oxidase and catalase (data not shown), and the activity in the chlorOplast fractions (VII and VIII) is of the same magnitude as the cytochrome c oxidase, which probably indi- cates mitochondrial contamination in these fractions. While fraction V is the major mitochondrial band, it also contains some chloroplast material which is thought to be whole chloroplasts with an outer membrane intact. In order to attain better separation of these particles, a portion Of this fraction was diluted to 1.3 M and recentrifuged on a linear gradient extending from 1.5 to 1.8 M with a layer of 2.5 M sucrose at the bottom. The material was collected from the bottom of the tube in 1.5 ml fractions. Assay results are Shown in.Figure 4. It is apparent that serine hydroxymethyltransferase and cytochrome c oxidase moved down the gradient together. The refactive index of fractions from a similar sucrose gradient are plotted to Show the linearity of the gradient. A great amount of the activity 95 Figure 4. Enzyme Distribution on a Linear Sucrose Gradient Enzymes were assayed as described in Methods section; serine hydroxymethyltransferase was mea- sured in the direction of serine to glycine. For ease of comparison the units of activity were reduced to a similar Scale. To obtain actual units the values for serine hydroxymethyltransferase should be multiplied by 0.30; those for cytochrome c oxidase by 48.8. Relative Units Activity 4.0- 3.0 - 2.0- 96 L452 |2.| I5.6 L425 L423 |.42| |.4|9 l.4l7 \\\\\\\\V\\\\\ \\\\\\k\\\\\.\'.\\\ |.4|5 |.4|3 |.4|I I23456789l0|ll2l3l4l5i76|7l8 Bottom Fraction NO. TOD C] Serine hydroxymethyltransferase Cytochrome c oxidase — Refractive index Estimate Refrative Index 97 was solubilized suggesting that this enzyme is associated with the outer membranes. Serine Hydroxymethyltransferase Activity in Mannitol Prepared Mitochondria The Specific activities of cytochrome c oxidase and of serine hydroxymethyltransferase, as assayed by the serine to formaldehyde reaction, are shown in Table 22. There is a 4.5-5.0 fold increase in both enzyme activities in the 6,000 x g pellet; however, the enrichment of enzyme in the various fractions is not exactly parallel. Even so the low enrich- ment in the 1,000 x g pellet excludes the whole chlorOplast as a possible Site for these enzyme activities, and the high activity on the fractions from the resuSpended 10,000 x g pellet strongly favors the mitochondrion or the peroxisome as the intracellular Site for the glycine to serine conver- sion. Serine Hydroxymethyltransferase in Peroxisomes A peroxisome pellet from a large-scale sucrose den- sity gradient prepared by A. Oeser of this laboratory was examined for hydroxymethyltransferase activity with the radioactive serine assay. Only 1.3% of the activity in the 6000 g pellet sedimented with the peroxisomes. This experi- ment, as the one shown in Figure 4, excludes peroxisomes as the site for this transferase. 98 Table 22 EnZyme Activity in Mitochondria Prepared in Mannitol Specific Activity (nanomoles/min/mg protein) Serine Hydroxymethyl- Fraction transferase Cytochrome c Oxidase Homogenate 0.38 11.9 1,000 g Pellet 0.67 10.4 10,000 g Pellet 250 g 0.76 94.7 6,000 g 1.87 53.2 wash 0.85 31.0 Supernatant 0.48 _* * Not determined 99 Serine Hydroxymethyltransferase in Chloroplasts A chlorOplast preparation from destarched Spinach leaves was prepared in sorbitol according to a method of S. Vandor (unpublished). The leaves were disrupted in the Waring blendor using buffered sorbitol as the isolation medium. The chlorOplast pellet obtained by centrifuging at 1950 x g for 50 sec was washed twice. Assay results showed 7.4% and 6.7% of the activity reSpectively for serine hydroxymethyltransferase and cytochrome c oxidase in the first chlorOplast pellet based on the activity in the super- natant fraction. After the second wash the chlorOplasts exhibited no serine hydroxymethyltransferase activity and 1.6% of the cytochrome c oxidase activity. If the activity were associated with chlorOplastS, it is certainly easily removed. Reversibility of the Enzyme The alternate assay whereby the disappearance of for- maldehyde in the presence of glycine with pyridoxal phos- phate and tetrahydrofolate under anaerobic conditions is measured was used with a homogenate Of Spinach leaves. Figure 5 shows the enzyme dependent formaldehyde utilization. The net rate calculated from the linear portion Of the curve is 80.0 nmoles formaldehyde/ml enzyme/min. Since protein was not determined in this preparation, an exact comparison cannot be made; however, the rate in the reverse direction using serine-3-14C with approximately the same amount of .40 C) I .30 o I 3 '5 .20 E 1 .I0 Figure 5. 100 O ’/,/’/::(3hnfine O - Glycine A (Endogenous) l l l l .05 .I 0 .l 5 .20 ml Enzyme Enzymic Incorporation of Formaldehyde into Serine 101 plant material was calculated to be 21.0 nmoles/ml/min formaldehyde-14C produced. Thus the reaction seems freely reversible lg vitro. Glycine Decarboxylation A Spinach leaf homogenate in 0.8 M sucrose with 0.02 M Tris, pH 7.5 was separated into three fractions: 1,000 x g pellet, 1,000 to 10,000 x g pellet and supernatant. Glycine decarboxylation was observed only in the particulate fractions: Fraction Opm 1“C09 Released 1,000 x g 9,500 10,000 x g 13,300 supernatant 0 The second fraction presumably contained the majority of the 'mitochondria; 62% of the glycine decarboxylating activity was found in this fraction. After 24 hours at 4° this frac- tion retained only 12% of the initial activity; thus the enzyme system is quite unstable under these conditions. A fresh Spinach mitochondria preparation taken from a typical isopycnic sucrose density gradient centrifugation (Fraction 5) was also examined for glycine decarboxylation. The enzyme was likewise found in this fraction. Similar decarboxylation was detected using the same cofactors and rat liver mitochondria (isolated by Dr. L. Bieber). The rate in this case was 14.4 nmoles/hr/mg protein. Thus it is concluded that serine hydroxymethyltransferase occurs in the mitochondria; the glycine decarboxylating enzyme appears in the mitochondria also. 102 Discussion By a combination of several isolation methods it has been shown that serine hydroxymethyltransferase activity closely parallels that of cytochrome c oxidase during sepa- ration Of subcellular particles, indicating that this trans- ferase is a mitochondrial enzyme. The transferase does not exhibit activity in either purified peroxisomes or washed chlorOplastS. During homogenization of the tissue a consid- erable portion of the enzyme is released into the soluble (supernatant) fractions; it has not been established whether this is simply a result of "leakage" from disrupted mito- chondria or whether a separate cytoplasmic enzyme is also present in the leaf. Independent studies by Dr. T. Kisaki (35) have confirmed the reported conversion of glycine to serine in the mitochondria. It has already been determined that plant peroxisomes contain the aminotransferase for glycine formation from gly- oxylate (34). The next reaction of the glycolate pathway-- the conversion of glycine to serine--has now been located in the mitochondria. This result was not entirely uneXpected since the enzyme from several animal sources has been found in mitochondria of the livers (62, 63, 64), and it is known to occur in non-green plant tissue such as cauliflower buds (46), corn roots (23), and carrot storage tissue (15), suggesting that at least one form of the enzyme is not chlorOplastic. 103 The failure to observe glycine and serine formation from 140 02 during photosynthesis by isolated chloroplasts (2, 17) except in one case (66) is consistent with the above observations. Recent attempts to establish compartmentation of metabolites in plant cells have utilized the technique of labeling compounds with 14002 by photosynthesis, rapid kill- ing with liquid nitrogen and isolating subcellular particles by nonaqueous techniques. This procedure has the advantage that only a small percentage of the water soluble components are leached out Of chloroplasts during the isolation (54). By such a method Stocking (73) showed that after a 20 second period of photosynthesis, all of the sucrose was located in the chlorOplast, but considerable glycine and serine appeared in both chloroplast and nonchloroplast material. Furthermore, in the study of the light-dark transitions of label through these compounds, it was found that serine in both the chloro- plast and nonchloroplast fractions followed the same trends whereas phOSphorylated compounds did not. These authors interpreted this to mean that the chloroplast membrane was freely permeable to serine (54). By this technique of pulse labeling in the whole leaf, it is not possible to establish the site of serine synthesis; one can only say that serine appears in the chloroplast as well as in other parts of the cell. Thus serine may be synthesized in the chloroplast or it may be moving freely into the chloroplast from another Site for further metabolism to sucrose. This latter possi— 104 bility would be consistent with the results of this report showing the mitochondrion to be the Site of serine synthesis. Another implication of these findings is that the mitochondrion is the site of C02 evolution during photoreS- piration. Glycolate has been proposed as the substrate for photoreSpiration (96), and indeed considerable radioactivity is released from glycolate-i-luC in the light (44, 96). Recently glycine has been suggested as the more immediate source of the C02 evolved (35). The cleavage of glycine to C02 and methylene tetrahydrofolic acid would be consistent with the evolution of C02 from glycine-i-luc and could also account for the release of C02 from glycolate in plant tis- sue. The oxygen uptake in photoreSpiration has been attrib- uted to peroxisomal Oxidation of glycolate to glyoxylate via the enzyme glycolate oxidase (82). 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