STUD3ES GM PE‘ifiTOSYNTHf-ESES 53A??? 5: Biasynfimsis of Sucrose {mm Géycahfs PART :6: Eicwbonam Ut'ifizafim 32y Washed Aiggaa Theais éo: $11.0 Dogma of H1. D. MiCHiGAN STATE UNEVERSWY Eduarda fiémenez Seen: E962 This is to certify that the thesis entitled Stu-lies on Photcsynth-asis Part I: Biosynthesis of sucrose from 3L33.L L Part II: Utilization of bicarbonate P; w'ggai a1 3;“ <2 presented by Eduardo Jimenez has been accepted towards fulfillment of the requirements for Ph' D'. ._ degree in—Bc’t‘vmy 41. a. W ou4 H soybean or coffee leaves. Supptrate Glycolate ,33014 cgs* 13 95.9 42.1 64.7 29.6 15.0 9.1 1706 8.0 9.4 4.3 14.7 6.9 66.7 50.0 3.1 2.3 25.2 19.0 8.6 6.5 17.1 12.9 12.4 9.3 30.4 54.3 103 2.3 16.7 29.8 5'1 901 1.9 3.4 0.6 1.1 *% Uncorrected for loss due to sublimation. * Values are based on Cl4 content of a paper chromatogram. 36 14 would label carbon atoms 1, 2, 5 and 6, that glycolate-2-C if the hexoses were formed by the reversal of the Embden- Meyerhof Pathway after the labeling of glycerate. That the present results are in accordance with the eXpectations is illustrated by experiments 4 and 6 in Table III. The higher degree of randomization of tracer that was observed when glycolate-1-C14 was fed to wheat (Table III, experiment 5) as compared to soybean (experiment 6) was probably caused by a larger portion of the C14 which was oxidized to 01402 plus an unlabeled 1-carbon fragment during the enzymatic conversion of the 2-carbon moiety to a 3-carbon derivative. The radioactive carbon dioxide thus formed would be fixed photosynthetically to yield uniformly labeled hexoses as in experiment 1 of Table III. Serine-3-C14 Feeding_Experiment The results obtained from the feeding of serine-3- C14 to wheat leaves are shown in Table VI. Even though the substrate was metabolized slowly during the 10 minute expe- riment in the light, an appreciable amount of sucrose was formed (about 5 percent of the total activity). Glutamate, alanine and a phosphate ester, which may be phosphoglycolate, could be detected also on the chromatograms although in trace amounts. Degradation of the hexoses from this sucrose showed that they were labeled predominantly in the 1 and 6 positions (Table III, experiment 3). Carbon atom 5 also contained a significant percentage of radioactivity, while \21 .\] T1333 VI. Products of serine-3-Cl4 metabolism during a period of ten minutes in the light by wheat leaves*. Compound cgs _;L__ Unmetabolized serine 269.5 94.0 sucrose ‘ 16.5 5.0 Glutamate traces Alanine traces Phosphate esters traces * Values are based on the C14 content of one paper chromatogram. 38 atoms 2, 3 and 4 were only slightly active. Since it has been previously demonstrated (60) that glycerate-3-C14 i 14 3 produced from serine-3-C by corn leaves in 15 minutes, the labeling pattern shown in experiment 3 indicates that glycerate was subsequently incorporated into hexoses by reversal of the Embden-Meyerhof Pathway. This route of hexose formation, however, does not account for the signifi- cant randomization of C14 which occurred into carbon 5. Even though the cross-contamination of labeling within the carbon atom pairs 1-6, 1-2 and 2-5 as observed by Busse 23. a1. (12) during the fermentation of fructose by a different strain of Leuconostoc mesenteroides were sustained in these experiments, the significance of the data still would not be invalidated. Any cross-contamination that might have occurred between carbon atoms 1 and 2 or 5 and 6 of the hexoses derived from glycolate-2-C14 would not change the results since these carbon atoms should have about the same specific activity. The same reasoning applies for cross-contamination between carbon atoms 1 and 6 of hexoses formed from serine-3-Cl4. Furthermore, the lack of activity in the 02 + C3 fragment is indicative of the absence of contamination of carbon atoms 2 and l and verifies the fact that bacterial fermentation produced little spreading of labeling in the present degradations. It has been previously shown that a major portion of the photosynthetically fixed C1402 moves through a pathway involving glycolate (7, 50, 60, 69, 80, 84). Glycolate may 39 function in the translocation of carbon from the chlor0p1asts to the cytoplasm (36). Since the enzymatic conversion of glycolate to glyoxylate (18) as well as the subsequent re- actions leading to the formation of sucrose occur in the cyt0plasm, the sucrose labeled from glycolate represents cytoplasmic sucrose in contrast to the reservoir located in the chloroplasts. Due to the fact that glycolate and all of the 3-carbon compounds derived from it during photosyn- thesis are uniformly labeled even for the shortest experi- ments (65), the cyt0plasmic sucrose formed from glycolate produced during C14 02 photosynthesis should also be uniformly labeled. Since the chlor0p1astic sucrose produced under similar conditions is unsymmetrically labeled (Table III, eXperiment 1), it follows that the Glycolate Pathway accele- rates the randomization of labeling in the hexoses within the whole cell, and that the contribution of this pathway to the production of uniformly labeled sucrose would not be obvious except as revealed in these studies. PART II RESULTS The Hashing_Effegt The impairment of the photosynthetic activity of washed algae has been recognized for some time. The origin of such deterioration of the cells has been associated with factors such as damage due to centrifugal force (79), or disturbance of some metabolic system caused by deficiencies of minerals which are induced during preparation of the algae (10, 20, 31, 43, 48).‘ However, in the present report data will be shown which may indicate that washing algae affects also, and perhaps primarily, the utilization of bicarbonate ions. The effect of 3 washings with distilled water upon the photosynthesis in Chlamydomonas reinhardti is illustrated in Table VII. The capacity to utilize bicarbonate ions was reduced by almost 80 percent. The photosynthetic activity of the washed algae could be restored to about a normal rate by the addition of phosphate or phosphoglycolate ions (pH 7) at a final concentration of 3.3 x 10"3 H. Smaller concen- trations of phosphate were less effective, and in fact, 1.7 x 10"3 M was only 70 percent as effective as 3.3 x 10'3 M (Table XIII) and 6 x 10-4 M was much less effective (data not shown). Stimulation of photosynthesis above the rate induced by 3.3 x 10":5 M phosphate was obtained with 40 TABLE VII. Effect of three washings with distilled water on the capacity of Chlggydgmona§.reinhapgti to utilize bicarbonate ions. Total 0l4 Fixed (o/sgplzio min. PSfi)‘ Treatment Unwashed cells** Jashed cells Control 2,700 546 3.3 x 10-3 M phosphate 2,960 3,100 (pH 7) added prior to PS 3.: x 10‘3 x phospho- 2,560 3,050 glycolate (pH 7) added prior to PS * PS stands for photosynthesis. ** Values were corrected for algal dilution, so that they corresponded to the same dilution (1:100) of the washed cells. 42 1 x 10‘2 M, but higher concentrations were inhibitory (Table‘XIII). Washing 0f the algae, however, is not always detri- mental, for it may serve to remove undesirable metabolites which may accumulate in the suspending medium, as it is illustrated in Table VIII. Even though 1-day old Chlamv- domonas were capable of utilizing bicarbonate ions in the presence of the original media, photosynthesis by Chlorella of the same age and grown in the low-P medium was very slow. The failure of Chlorella to fix bicarbonate when the cells remained in the original medium could not be changed either by lowering the pH of the medium from about 8.2 to 6.9 or by adding phosphate ions to a final concentration of 3.3 X 10'3 M (pH 7) 10 minutes before the photosynthesis eXperi- ment was started. Instead, it was necessary that the cells be washed with distilled water and phosphate ions added back before the photosynthetic capacity could be restored. The response of unwashed Chlorella may be related to the phos- phorus content of the medium during growth, since this same species, when grown in the high-P medium ordinarily used for Chlamydomonas (data not shown) was capable of rapid utiliza- tion of bicarbonate even if kept in the nutrient solution in which the algae had been cultured for one day. An alternative would be that the behavior of unwashed Chlorella may be due to accumulation in the surrounding medium of a substance which is produced by neither of the two Chlamydo- monas species nor formed by Chlorella when grown in the TABLE VIII. 14.3 to utilize bicarbonate ions. Total 014 Fixed* Differential capacity of three species of algae .1 133-23-11- One-day 01d Cultures (o/s/m1/10 min. as) A B Chlamydomonas eugametos 2,284 5.6 6.0 (unwashed) Chlamydomonas reinhardti 2,262 3.3 4.0 (unwashed) Chlopella pyrenoido§g_ 172 8.2 9.0 (unwashed) Ch. pyrenoidosa (unwashed, 256 - 8.5 pH adjusted to 6.9 with HCl) Ch. pyrenoidqgg (unwashed, 344 - 8.5 plus 3.3 x 10‘? H phos- phate (pH 7) added 10 min. before PS) Ch. pyrenoidosa (washed 3 1,712 - 7.8 times plus 3.3 x 10"3 M phosphate (pH 7) added at the beginning of PS) Ch. pyrenoidosa (washed 3 200 - 9.0 times without phosphate) * Values were adjusted for cell dilution on the basis of 1 ml of packed cells per 100 ml of suspension. ** The pH was measured before (A) and after (B) the 10 minute photosynthesis experiment. suspension before PS was about 7. The pH of the washed algal .i"~" fi=_._r7W - ' #3") 44 presence of an excess of phosphate ions. A possible substance could be chlorellin (51, 52, 53, 54) which is known to inhibit photosynthesis, and which seems to be active only when found in the external medium. It is possible that this inhibitory substance may block in some way the mechanism of transport of bicarbonate ions across the cell membrane. .. .. --1~’+ . - materials which stimulated LaHC O_ Fixation by dashed Algae The capacity of washed algae to utilize bicarbonate was restored by a number of compounds. Table IX represents a summary of the substances that were tested for stimulation of bicarbonate uptake by all three species of algae. The substances were grouped into three categories according to their relative efficiency at a concentration of 3.3 x 10"3 M and at pH 7, under the standard experimental conditions. In the first group were put those compounds which restored completely the ability of the cells to fix bicarbonate. Since these compounds were not all phosphate esters, the concept was untenable that photosynthesis in the washed cells was limited by the availability of phosphorus for synthesis of the phosphate esters of the photosynthetic carbon cycle. Compounds grouped under the second category restored the photosynthetic fixation of bicarbonate only partially. In the third category were placed those substances which did not show any appreciable effect. This arbitrary classifi- cation does not imply that all the substances listed under 4? kfl 3T1 m IX. Effect of compounds* on stimulating washed algae to utilize bicarbonate ions. Degree of Stimulation Complete Some_‘ Hone Sodium phosphate Phosphoglycolate Phosphoglycerate Serine lspartate Asparagine Phos erine Ammonium chloride Tris- hydroxymethyl- aminomethane Glycine Glycolate Ribulose 1,5-di phosphate luc se-6- os date ’UQ 0 Ph Calcium chloride Sodium bicarbonate Potassium acetate Hagnesium chloride Potassium chloride Sodium chloride Sodium sulfate Adenosine-tri- phosphate Minor elements as found in Hoagland’s formula (77) Sodium arsenate * The compounds 3 3 X 10-3 H carbonate which the minor eler dents. cell dilution) and at 173.8 ..ere were tested at a final concentration of about pH 7, except for sodium bi- tested at l x 10‘“ Three time washed algae (1:100 used throughout the experiments. ‘ F I l'l (pH 8.1), and 46 a given heading had the same degree of activity. To a limited extent, the order in which the compounds were listed represents theirNdecreasing order of effectiveness. Among the cations which were tested, only anmonium and calcium moderately increased the uptake of bicarbonate. In this connection, Holm-Hansen, 33. El- (31) have suggested that the response of the washed algae to NH: ions may be to relieve a mild nitrogen deficiency. However, this sugges- tion cannot explain the stimulation from non-nitrogen substances. Addition of phosphate and ammonium ions together stimulated radioactive sodium bicarbonate fixation more than that obtained from either one alone (32). This suggests that there is more than one stimulatory effect. The stimulation by ammonium ions has not been further investigated. The rather small effect from calcium ions in the standard procedure might have been explained by the fact that the cation was added after washing. Hence, calcium could not have prevented the leakage of cellular phosphorus com- pounds during the preparation of the algae, as suggested by Schmidt1. In order to test this hypothesis, an experiment was performed in which the effect of both washing and re- suspending with two calcium chloride solutions was compared against distilled water or phosphate added prior to photo- synthesis (Table X). washing and resuspending Chlamydomonas 1See footnote on page 14. TABLE X. Effect of washing ghlamydomonas reinhardti with two calcium chloride solutions*. "0".in “I. -" AA“ _ v—v— v"'-H- ww— l‘l Phosphatew‘ Hashing Addition Qgggggen During_rs Total 014 Fixed__ Final 23 (C/S/mi/io min. 257 Distilled water 0 106 8.5 Distilled water 3.3xlO"3 X 1,400 7.5 2xlO‘2 H CaCl 0 360 7.5 22:10"2 H CaOl 3.3xlO‘9 I 954 6.3 lxlO'l n CaClO o 400 . 7.o xlO‘l m CaCl2 3.3xlO'9 m 1,170 6.7 0» "~ The algae were washed 3 times according to the standard procedure. J; ¢ \‘o C. ‘ The pH of the potassium p-0sphate solution was 7. 48 reinhardti in either 2 x 10'2 or 1 x 10"1 H Ca012 could not prevent the loss of photosynthetic activity. Phosphate in the external medium was required in order to restore activity. That pH could not have been a limiting factor in the experi- ment is illustrated by the pH values recorded at the end of the 10 minute photosynthesis period. The values fell within the physiological range (pH 4 to 9) determined by Emerson and Green (22) for Chlorella, and by Calvin 23. El. (16) for Scenedesmus. A slight stimulation of 014 fixation was observed by the addition of excess carrier bicarbonate during an experi- ment with completely washed Chlorella pyrenoidosa (Table XI). The total amount of bicarbonate added per treatment was increased stepwise but the specific activity of 014 was held 12 and C14 were used, a correction constant. Since both C for isotOpic dilution was made according to the formula given below, in order to calculate total fixation rates. 12 14) + pmgles of C Correction factor = LEEOIES 0f 0 14 C umoles of Since increasing bicarbonate concentrations had some stimulatory effect upon photosynthesis, the influence of the q presence of this anion during washing of the algae was investigated (Table XII). Chlorella pyrenoidosa cells were both washed three times and resuspended with 1 x 10"3 H (Suspension A) or 1 x 10"1 M HaH003 (Suspension B). The pH of these solutions was 7.9 and 8.1, respectively. Also, 49 TABLE LI. Effect of increasing amounts of bicarbonate ions on photosynthesis of washed Chlorella pyrenoidogg, Treatment Total 014 Fixed* Final_pH tc/S/m17io min. To) Control (6.7 x 10-4 H 168 7.4 Ha10140~) 9 3.3 x 10‘5 3 hand 3 + 3.3 230 6.5 x 10-5 H HaHCl 03 3.3 x 10"4 n HaHCO; + 3.3 268 7.1 x 10-4 n rancl403 3.3 x 10-3 n ra3003 + 3.3 600 9.0 x 10‘3 n wancl403 * Values were corrected for isotOpic dilution according to the formula given in the text. The correction factor was 2. TABLH XII. Photosynthesis by algae washed with bicarbonate. _‘.._- Treatment Total Cl4 Fixed* Final p? (b/s/ml/lO min. To) sus pension A + 6.7 x 10'4 189 7.4 H anclaow 3 SuSpension A + 3.3 x 10'3 1,390 8.1 H phosphate (pH 7 + 6.7 X 10-5 M HaHC£403 SuSpension B + 6.7 x 10"1‘L 3,620 8.6 n IJchl403 Suspension B + 3 3 x 10 3 4,220 8.7 M phOSphate (pH 7) + 6.7 x 10-4 n HaH31403 Suspension A: Chlorella pJ'L rinpidosa both washed (3 times) and resuspended with l x 10 H HaHCOB. ‘: Same as suspension A except that l x 10"1 H * Values were corrected for isotopic dilution. The correction factors used were 2.5 and 151, respectively for suspensions A and B. 51 the effect of the addition of the standard amount of phosphate ions was tested during photosynthesis with the bicarbonate treated cells. Preparing the algae with 1 x 10-3 H bicar- bonate solution did not prevent the loss of photosynthetic ability, but activity could still be restored by 3.3 x 10.3 M phosphate (pH 7). Preparation with 1 x 10'1 H bicarbonate, on the other hand, was effective for controlling the dehierious action of washing. This amount of bicarbonate was much larger than the amount of phosphate necessary for restoring photosynthetic activity. The stimulation of 0‘4 fixation by 0.1 H bicarbonate (alone or in combination with phosphate) occurred at the high pH values, 8.6 to 8.7. Speculation why this occurred will be discussed in more detail later. The combination of phosphate and bicarbonate waS' further explored. Accordingly, increasing amounts of phosphate (pH 7) were added to Chlorella cells which had been both washed (3 times) and resuspended with either dis- tilled water or 1 x 10‘3 M 17ch03 (Table XIII). The presence of 1 x 10"3 H bicarbonate in the medium resulted in a small 14 increase of C fixation which cannot be ascribed to pH. In the presence of phosphate and bicarbonate, the rate of C14 fixation increased until the phosphate concentration 3 reached a level of 6.6 x 10- H and the pH had dropped to about 7.5. When phosphate was added to cells resuspended in water, the highest rate of fixation was attained with a concentration of 1 x 10"2 H. In the absence of bicarbonate, .w- ‘7 - TABLE XIII. Effect of increasing phosphate concentrations on the photosynthetic activity of algae washed either with distilled water or a bicarbonate solution.* Phosphate** Addition pigshinngreatment During PS” Total 014 Fixed F rg;_jfii (c/s/ml/lO min. PS) Distilled water 0 47 8.4 Sodium bicarbonate O 210 8.4 Distilled water 1.7xlo-3 x 817 7.4 Sodium bicarbonate 1.7x10-3 n 1,010 7.8 Distilled water 3.3x10‘é H 1,210 7.6 Sodium bicarbonate 3.3x10-3 H 1,890 7.6 Distilled water 6.6x10-3 x 1,730 7.3 Sodium bicarbonate 6.6xlO-3 n 3,280 7.5 Distilled water lxlO‘2 n 2,000 7.3 Sodium bicarbonate lxlO-2 H 2,630 7.4 Distilled water 1.3xlo-2 H 1,310 7.2 sodium bicarbonate 1.3::10-2 n 3,180 7.3 t Sodium bicarbonate .012 at 1 x 10-3 M was used both for washing and resuspending the alga Chlorella pyrenoidosa, according to the standard procedure. ** The pH of the potassium phosphate solution was 7- :13 a slight decline in effectiveness of the phosphate ions occurred at the highest concentration, which may reflect some unbalanced condition of the medium. The Effect of Inhibitors Inhibitors were used to provide data which might help to explain the phenomenon of stimulation or recovery of radioactive bicarbonate utilization by thoroughly washed algae. Uranyl acetate and sodium arsenate were investigated. According to Few gt. 3;. (23), uranyl ions cause a major reduction in the charge density of purely phosphate-type colloids such as protoplast membrane lipid. With respect to carboxyl-type colloids, on the other hand, uranyl ions are not as effective. Frenkel (24) reported that photosyn- thesis in Chlorella was not inhibited by uranyl concentrations as high as 1 x 10"2 M. This is remarkable because of the generally high sensitivity of photosynthesis to heavy metals (58). Although Frenkel indicated that endogenous respira- tion in Chlorella was only slightly affected by uranyl chloride, respiration attributed to added glucose was 80 percent inhibited by a 1 x 10"3 H concentration of this salt. The latter observation suggests that transport of glucose across the cell membrane was more drastically inhibited by the uranyl ions in the external medium than subsequent metabolism inside the cell. Arsenate, on the other hand, is known to interfere in the metabolism of phosphorus, and specifically with the 54 formation of high energy phosphate compounds (58). Presuma- bly, arsenate might also act as a competitive inhibitor of phosphate during uptake of bicarbonate. Uranyl Inhibition The effect of uranyl ions on the utilization of bicarbonate by 3-time washed algae was studied first by adding increasing amounts of uranyl acetate1 to Chlamydomonas reinhardti, simultaneously with the standard concentration of either phosphate, phosphoglycolate or serine (Table XIV). Uranyl, at a concentration of 6.6 x 10"4 H, inhibited the rate of HaHCj403 fixation over a 10 minute period of illumi- nation in the presence of 3.3 x 10'3 M phosphate, phospho- glycolate or serine, respectively by 32, 27 and 48 percent. Increasing the uranyl concentration to 1.7 x 10"3 H caused no appreciable change in the degree of inhibition in the presence of phosphoglycolate. However, this same uranyl concentration reduced the photosynthetic activity 75 percent in the presence of phosphate, and almost 95 percent in the presence of serine. At 3.3 x 10"3 H, uranyl proved to be equally toxic in the presence of any of the three substances, although phosphoglycolate still showed some activity. The extreme sensitivity of the serine stimulation to uranyl ions cannot be explained by a comparison of the differential f the solution of this salt could be brought only .1 with KOH, for beyond this point it began to form a ipita U1 U1 TABLE XIV. Effect of uranyl acetate on the stimulatory action from addition of phosphate, phosphoglycolate and serine* to washed Chlamydomonas reinhardti. ===————4——=areg======================;“a=:l: .rl Treatment Total 014 Fixed (c/s/ml) Final pH 5_minutes 10 minutes Control 155 475 7.4 3.3xlO"3 H phosphate 1,064 2,240 7.0 6.6x10'4 M uranyl + phosphate 806 1,520 6.9 1.7xlo-3 n uranyl + phosphate 292 570 5.8 3.3xlC"3 H uranyl + phosphate 100 0 4.7 3.3x10‘.3 H phosphoglycolate 810 1,740 7.0 6.6x10"4 M uranyl + phospho- 852 1,274 6.0 glycolate l.7xlO"3 M uranyl + phospho- 688 1,236 5.9 glycolate 3.3x10"3 H uranyl + phospho- 180 200 5.2 glycolate 3.3::10"3 H serine -- 1,480 7.0 6.6x10'4 H uranyl + serine -- 770 -- 1.7x10'3 M uranyl + serine -- 100 4.7 3.3x10'3 H uranyl + serine -- O -- * The pH of the phosphate, phosphoglycolate and serine solutions was adjusted to 7. The algae were washed 3 times and resuspended with distilled water. 56 ability of uranyl to reverse the charge density of the anionic groups, as discussed by Few gt, 3;. (23). The concentration of uranyl ions which inhibited the utilization of bicarbonate in the presence of all three stimulatory compounds was 3.3 x 10"3 M, or three times less than the concentration which Frenkel reported as harmless to photosynthesis in Chlorella. If it is assumed that Frenkel was correct and that Chlamydpmonas and Chlorella respond in a similar manner towards uranyl ions, it follows that the activity of the stimulatory compounds was not concerned with the photosynthetic fixation of 01402 but with some other related process. In a second experiment (Table XV), uranyl acetate was used at a constant concentration of 1.7 x 10'3 H, in conjunction with 3.3 x 10'3 H alanine or ammonium chloride, as well as phosphate, phosphoglycolate or serine. The alanine stimulation was inhibited by uranyl ions exactly the same as serine, while the ammonium stimulation was not affected. In fact, it seemed as if the ammonium and the uranyl ions had acted in a synergistic fashion. The higher resistance of photosynthesis to uranyl inhibition in the presence of phosphoglycolate may be explained by the assump- tion that phosphoglycolate, which has two anionic groups, could bind more uranyl than either phosphate, serine or alanine. Inhibition by binding of uranyl ions to some anionic group on the cell surface would occur only when an excess of free uranyl ions were available. Presumably, the 57 TaBLE XV. Differential response of enhanced photosynthesis in presence of a constant concentration of uranyl acetate.* —__. I *- l4 m . ,_ , n m. c th refitting; 11:38:13.7 W Control 324 7.0 3.3x10'3 H phosphate + uranyl 100 5.2 3.3x10'3 H p-glycolate + uranyl 1,360 5.7 3.3x10~3 H serine + uranyl 100 4.7 3.3x10'3 H alanine 1,680 7.6 3.3x10"3 H alanine + uranyl 176 5.0 3.3x10'3 H ammonium chloride 1,000 6.7 3.3::10‘3 H ammonium chloride + 1,540 6.9 uranyl * Ura yl acetate was added at a final concentration of 1.7 x 10' H. Chlamydomonas geinhardti, prepared as usual, was utilized in these experiments. 58 site affected by the uranyl ion is active in the mechanism of bicarbonate transport across the cell membrane or some other process related to photosynthesis. The stimulation of photosynthesis by ammonium ions is probably a different phenomenon than the stimulation by phosphate, as already suggested (page 41). In the first place, one is a cation and the other an anion. Secondly, phosphate stimulation was inhibited by uranyl ions, while ammonium stimulation was not sensitive to this cation. In fact, a higher rate of stimulation of photosynthesis was obtained in the presence of both uranyl and ammonium ions. Arsenate Inhibition The influence of arsenate on the utilization of radio- active bicarbonate by algae was studied first with Chlamyd - monas eugametos. The cells were centrifuged once and the supernatant was discarded. The algal pellet was then resuspended in distilled water. After adaptation in the photosynthetic apparatus sodium arsenate was added to the algal samples (B-ml aliquots) at a final concentration of 6.6 x 10-4, 1.7 X 10.3 M or 3.3 x 10'3 M. Immediately afterwards, 20 ul of 0.1 M HaH01403 was added and the algae were allowed to photosynthesize for 5 or 10 minutes (Table XVI). The addition of arsenate up to 3.3 X 10'3 M concen- tration had no inhibiting effect on the photosynthetic activity of partially washed algae. In another experiment (Table XVII), arsenate was .0 lABLL AVI. Effect of sodium arsenate* on the photosynthetic activity of partially washed algae. v—-.---'IU ..-l‘ v Treatment __Total 014 Fixed Final 03 (67S/mi710 min. Pd) Control 1,808 6.9 r / -‘£4- .. 4. " O 6.6 x 10 h arsenate :,l+0 0.2 7 ‘v 1.7 X 10‘) g arsenate 1,776 -__ 3.3 X 10') h arsenate 1,562 --- t The p? was 8.2. Arsenate was added to Chlamydomonas eugam~t cells which were centrifuged once and then ‘e os nded with distilled water. 60 TABLE XVII. Effect of sodium arsenate of the photosynthetic activity of completely washed algae*. Treatment Total 0 Fixed__ ina pH (c/s/ml710 min. Po) Control 215 6.9 6.6 X 10"!+ M arsenate 340 8.2 1.7 X 10'3 H arsenate 350 8.2 3.3 x 10"3 m arsenate (pH 71) 140 7.2 * The alga Chlamydomonas reinhardti was used in this eXperiment. The preparation of the cells was as usual. Except in the last treatment, the pH of the arsenate solution was 8.2. 61 added as above to B-time washed Chlamydomona§=Epiphardti. Arsenate had no inhibiting or stimulating effect on completely washed cells. The results from these experiments suggest that arsenate ions were not taken up during the duration of the experimental conditions. If arsenate had been absorbed to any appreciable extent, it should have inhibited photo- synthesis in Chlamydomonas, since at least two steps in the photosynthetic carbon cycle are known to be susceptible to arsenate poisoning. One is the reduction of 3-phospho- glycerate to B-phosphoglyceraldehyde, which is catalyzed by triosephosphate dehydrogenase and requires both ATP and TPHH. The other step is the phosphorylation of ribulose- 5-phosphate to ribulose-1, 5-diphosphate, which is catalyzed by phosphOpentokinase (70). Arsenate inhibition was also studied with 3-time washed Chlamydomonas reinhardti, in the presence of phosphate or phosphoglycolate (Table XVIII). Incubating the algae for 10 minutes in 3.3 x 10-3 M arsenate prevented phosphate or phosphoglycolate from stimulating the rate of photosyn- thesis when added later. However, when the algae were first incubated for 10 minutes in a phosphate or phosphoglycolate solution, the subsequent inhibition by arsenate did not Occur. The simultaneous addition of phosphate or phospho- glycolate and arsenate just prior to the addition of ITaHCMO3 was nearly as effective as incubating the a gas with phosphate and then adding arsenate. These results may be indicative of a type of competitive inhibition between the 62 TABLT XVIII. Effect of incubation* with arsenate or some stimulator; substance on the capacity of washed Ch amydomonas reinhardti to utilize bicarbonate ions. Treatment Total 014 Fixed '“ ' (c/s/ml/lO min. rs) Control 262 3.3 x 10"3 H arsenate (pH 8.2) 190 3.3 x 10-3 n phOSphate (pH 7) 2,360 10 min. incubation with arsenate 470 followed by phosphate 10 min. incubation with phosphate 1,575 followed by arsenate Simultaneous addition of arsenate 1,395 and phOSphate 3.3 x 10“3 n phosphoglycolate (pH 7) 1,347 10 min. incubation with arsenate 124 followed by p-glycolate 10 min. incubation with p-glycolate 1,357 followed by arsenate Simultaneous addition of arsenate 950 and p-glycolate * Incubation consisted of adding the substance under study during the 10 minute period of light and temperature adapta- tion prior to the photosynthesis experiment. In all cases, arsenate, phosphate or phOSphoglycolate were added in the proportion of 10 umoles per 3 ml of algal suspension, or 3.3 x 10‘9 n. 63 two anions for the same site, although more detailed research needs to be done before this idea can be developed. ‘ Ipflgpnce of_ph on the Utilization of Bicaroonate long It was found in previous experiments that the stimu- lation of bicarbonate utilization by completely washed algae occurred at pH values near neutrality. Hence, it appeared logical to examine the influence that the pH of the medium may have on the effectiveness of the so-called stimulatory compounds. Several factors determined the final pH of the algal suspension after a 10 minute photosynthetic experiment. One factor was a slow and small increase in pH during photo- synthesis as if dissolved 002 as carbonic acid were being removed from the medium. Another factor was the 20 ul of 0.1 H Ea301403 solution which was added to the algal culture. A 0.1 M lZaHCO3 solution has a pH of about 8.2. Generally the 014-bicarbonate mixture was prepared in a small excess 14 of free base in order to insure against loss of C 02. The solution was therefore a mixture of Hal-1003 and NaO 3 and as a result even a small amount of the bicarbonate solution when added to the algal suspension raised the pH to above 8. To counteract the alkalinity of the bicarbonate potassium hydro- genphosphate and trishydroxymethylaminomethane (tris) buffers were chosen. The pH of-0.1 M buffer solutions was adjusted with HCl to predetermined values and 10 umoles of each were added to 3 ml of completely washed Chlorella pyrenoidosa (Table XIX). Both phosphate and tris buffers were effective 64 TABLE XIX. Influence of pH of the medium on the stimulation of photosynthesis by two buffer solutions*. Treatment Total 014 Fixed Final 03 (c/s/ml/lO min. Pd) Control 203 8.3 Potassium phosphate, pH 7.0 1,953 7.4 7.5 938 7.9 8.0 375 8.3 8.8 300 8.5 Tris, pH 7.7 816 7.8 8.0 524 7.9 8.5 210 8.6 900 96 901 * The buffers were adjusted to the desired pH with 0.1 N H01. Ten umoles of each buffer were added to B-ml aliquots of Chlorella pyrenoidosa cells which were prepared as usual. 65 stimulators of CO2 fixation when the final pH was maintained around 7 to 7.8. Above pH 8 there was no stimulation of photosynthesis by these buffers. The lack of stimulation at higher pH values cannot be explained solely on the basis .0 Or an adverse pH effect on normal photosynthesis by unwashed or partially washed algae since these pH values fell within the accepted physiological range of 4 to 9 (16, 22). When the effectiveness of the phosphate buffer was investigated at pH values less than 7 (Table XX), it was found that the rate of photosynthesis was equally stimulated between the range 4 to 6. In this lower pH range the predominant ionic form of the buffer was H2r0; (11), which was necessary to neutralize the alkalinity of the bicarbonate. The above results indicate that a pH effect could partially explain the stimulatory action from the buffer. To investigate this possibility without buffer, it was nece- ssary to add experimental amounts of 0.01 N H01 to a washed algal suspension (Chlorelgi pyrenoidosa), so that when the HaHCMO3 was added, the final pH was in the range of 4 to 7 (Table XX). The results of this experiment indicated that there was a stimulation of C14 fixation when the final pH was 7 or less, even though no phosphate buffer was added. It appears, therefore, that the Lng; ions acted as a weak acid which in the presence of H0140; ions would release C1402 according to the equation ,_ "' 'r 14 - a - 2 ,, 14 £21304 + RC 03 (“—- 111304 4" £120 + C 02 66 TABLE XX. Effect of pH on the utilization of bicarbonate ions by washed algae. PH 14 Adjustment* Total 0 Fixed Final pH (c/s/ml/lO min. P5) Control 570 7.5 Potassium phosphate, pH 4 2,565 6.0 5 2,540 6.2 6 2,620 6.6 7 2,120 7.6 -2 ‘ _ p q l x 10 N n01, pa 3 2,020 9.7 3.8 1,330 7.0 4.8 540 8.2 * A 0.1 H solution of KHQPO4 was adjusted to the desired pH values with KOH. Then each buffer was added to completely washed Chlorella pyrcnoidosa cell, prepared as customary, at a final concentration of 3.3 x 10‘) H. when H01 was used, the pH of about lO-ml aliquots of the same algal suspension was adjusted before the period of adaptation. 67 Thus, at pH values below or near neutrality, the stimulatory activity of phosphate, phOSphoglycolate and the amino acids can be best explained in terms of availability of 01402. The data suggest that at the low 002 concentra- tions normally encountered in photosynthesis, the 002 is the active species which enters the cell. If bicarbonate ion enters the cell it does so at a slower rate. This concept, however, does not account for the arsenate inhibition during bicarbonate utilization (Table XVIII). The stimulation due to excess carrier bicarbonate (Table XII) would have to be accounted for by mass diffusion or active transport of bicarbonate ions. Additional data are given in Table XXI concerning photosvnthesis rates in the presence of higher concentrations of carbonate buffers which were added just prior to the 10 minute test period. The rate of 014 fixation was highest at about pH 8.5, when both phosphate and carrier bicarbonate were added at a final concentration of 3.3 x 10'"3 M. These results seem to be in disagreement with the idea that only 002 which can exist below pH 8 was effective in photosynthesis. It is necessary to assume that bicarbonate ions can also diffuse or be transported into the cell. The decline of the rate of 014 fixation which was observed at pH values above 8.5 might be due to the adverse effect of the higher pH values. feeding Radioactive Compounds which Stimulate Photosynthesis In order to determine whether phosphate or serine TABLE XX . Influence of high pH values on the stimulatory effect of phosphate. Treat;ent* Total 014 Fixed Final p: Zc7é7m1/io min. P3) 6.7 x 10-4 n waxol4o3 and 140 8.6 Nacl403 3.3 x 10‘3 d phOSphate (pH 7) 1,760 7.9 3.3 x 10‘4 M Hawco3 + phosphate 1,815 8.2 1.7 x 10-3 n Na3003 + phOSphate 2,770 8.4 3.3 x 10-3 n wa3303 and "321003 + 3,470 8.5 phOSphate 3.3 x 10-3 M Hal—loo3 and raco3 + 1,740 8.7 phosphate 3.3 x 10-3 M NaHC 3 and Ka003 + 995 9.0 ‘ phosphate 3.5 x 10'3 n Ka3005 and Ha003 + 432 9.6 phosphate 3.3 x 10-3 w Na003 + phOSphate 100 10.3 * The same amount of 6.7 x 10"4 M NaHClZ‘LO3 was present in all treatments. The values were corrected for isotopic dilution as needed. The same amount of 3.3 x 10'3 K phOSphate buffer was present in all treatments except the first one. 69 were absorbed by the washed algae during the 10 minute photosynthesis experiments, P32-labeled phosphate (1 mc/ml) and serine-3-014 (100 uc/ml) were added to washed Chlorella pyrenoidosa cells. The experiments were run in the presence of the corresponding carriers, so that the final concentra- tions were 3.3 x 10'3 M. The P32 experiments were done with algae grown either in the standard low-P medium, or in the high-P medium used for Chlamvdomonas. Both media were used to investigate whether the phosphorus content of the original culture solution could affect to any appreciable extent the uptake of phosphorus by washed algae (Table XXII). Even though the photosynthetic activity of the washed algae was markedly stimulated by adding either phosphate or serine P32 14 to the medium, only a very small amount of or serine-0 was found with the cell fraction (extract plus residue) after the short experimental period. In the case of P32, similar results were obtained whether the algae were grown in a low-P or a high-P medium, thus suggesting that in both kinds of Chlorella cells, the internal reservoirs of phos- phorus did not influence the amount of phosphorus uptake during the experiment. The amount of P32 found in the supernatant and the washing fractions together, represented about 97 Percent of the total added radioactivity. Less than 1 percent was detected in the cell extract, and the remaining 2 percent was associated with the algal residue and the millipore filter. This fractionation procedure was described in the Methods section. To what extent the amount 7O ABLE XXII. Absorption of labeled phOSphate or serine by washed Chlorella pyrenoidosa during stimulation of bicarbonate uptake. __ Treatment Total 014 Fixed (c/s/ml/IO min. P5) Controls Distilled water 188 Low-P cells + 3.3 x 10-3 x 2,486 phosphate (pH 7) High-P cells + phosphate 2,444 Low-P cells + 3.3 x 10"3 M 897 serine (pH 7) Labeled Compounds Activity/ml pg/sZlelO min. of whole Super- wasn- Ex- Re- sample nat. ing__tract sidue Low-P cells + 19 uc P32 975 98 9 24 1,027 + 3.3x10'3 H carrier phOSphate ’3 High-P cells + 10 no P3“ 967 91 8 37 1,045 + carrier phosphate Low-P cells + 10 £0 of 345 36 7 28 -- serine- -01 + 3.3XIO“ M carrier serine The treatments receiving P32 or serine—3-0l4 also received 10 umoles of 012 sodium bicarbonate in order to stimulate photo- synthesis. 71 of radioactivity left on the filter represents actual meta- bolism of the added P32-phOSphate was not determined. But even if all this residual radioactivity was attributed to the cells, the total amount of P32 that was taken up was very small compared to the strong stimulation of photosyn- thesis caused by phOSphate. In considering this finding, one must remember that rather large concentrations of phos- phate ions were used to restore photosynthesis to the washed algae, and that just catalytic amounts were not sufficient. Jhen serine-3-C14 was used, the pattern of distri- bution of radioactivity among the various cell fractions was essentially the same as for the P32-labeled phosphate experiments. There was, however, a slightly higher per- centage of 014 associated with the cell extract and the residue left on the filter. The very low absorption of phosphate by washed Chlorella may reflect the fact that the algae were mostly mature cells, since the youngest ones were probably lost during the process of washing. Another limiting factor for phosphorus uptake may have been the absence of any nitrogenous compound in the medium. Corroborating data for these two assumptions has been reported by Schmidt (66), who studied the uptake of phosphate by synchronized cultures of Chlorella pyrenoidosa. He observed that the time and amount of phosphate absorbed by the cells were related to the nitrogen source and age of the culture. Thus, ammonium ions were more effective than nitrate ions, and the rate of 72 absorption decreased during the last stages of cellular growth (nuclear and cell division). Therefore, it is not surprising that washed Chlorella cells did not absorb appreciable amounts of phOSphate during the 10 minute experiments. The important fact is that a significant absorption of phosphate or serine apparently was not an obligatory step in the process of bicarbonate utilization by the washed algae. Products of Photosynthesig The alcoholic extracts of washed Chlamydomonas eurangpgs and Ch. reinlardti were obtained from eXperiments in which the algae were treated with phOSphate or phospho- 4. as W ‘ . ‘ ~~r --q14 rm glycolate, bObJ at pa 7 and tnen exposed to paxv 0;. ins insoluble cell residues were removed by centrifugation at full speed for about 5 minutes in a clinical centrifuge. The supernatant fractions were then evaporated and subjected to the standard procedures of chromatography and autoradio- 14-labeled products of 10 minute PhOtO' /. graphy (o). The 0 synthesis were identified according to their relative Rf values from inspection of the chromatograms and autoradio- grams. In addition, glutamate, aSpartate and serine were also identified by cochromatography. Poor separation of glycine and serine made impossible the separate determination of radioactivity in these two compounds (Table XXIII). In general, both species of ggggggggggyggg produced about the same compounds regardless of treatment,. Perhaps / TAB"? XXIII. Percentage distribution of 01* among products of photosynthesis as affected by different stimulatory compounds*. Ch. eugametos 93;,T91933rdti Phhs- P-gly- Phos- T:gly- Compound“; Eater phate colate Eater phate 221g§§_ PhOSphate esters 25.3 21.7 18.5 17.3 29.4 16.0 Glycolate 17.3 16.8 2.2 2.2 0.5 1.4 Glutamate 3.9 3.2 14.9 4.7 2.5 21.2 Glycerate 3.1 1.8 10.9 20.9 14.5 7.5 Aspartate area 6.8 3.9 15.4 7.2 4.7 13.0 Scrine + glycine 1.8 4.5 5.4 0 4.0 0 Lipids 11.6 17.2 2.9 13.0 18.0 8.5 Others 23.4 12.9 17.6 15.9 10.2 20.4 Origin 6.8 18.0 12.2 18.8 16.2 12.0 * Phosphate and phoSphoglycolate were added to 3-washed algae at a final concentration of 3.3 x 10‘) M. The duration of the photosynthesis experiments was 10 minutes. 74 when the algae were stimulated with phosphoglycolate, they had a tendency to produce different amounts of glycolate, glutamate, glycerate and aspartate, but the significance of this observation is doubtfull on account of the small number . . . . 14 OI repetitions. Incorporation of 0 into phosphate esters was not stimulated by addition of orthophOSphate or phospho- glycolate. If the water washed algae had had a phosphorus deficiency, one would expect to find a much less percentage of the 014 in the phosphate esters and an increase in the percent of radioactivity incorporated into this fraction upon addition of phosphate ions. These changes did not occur, suggesting that the washing procedure had not caused any phosphorus deficiency which may have reduced the reservoirs of the sugar phOSphates encountered in the path of carbon in photosynthesis. DISCUSSION Huch of the photosynthesis research in recent years has been done with washed algae cultures to which was added a tracer amount of Ea301403. The cultures were generally washed one or more times by centrifugation in order to remove most of the nutrient salts which otherwise would ‘overload subsequent paper chromatograms. In addition, un- washed cultures often gave poor photosynthetic rates which may be explained by such toxic substances as Chlorellin ( subsequent photosynthetic tests some investigators have at 1, 54), which thaalgae excrete into the medium. During U1 times added back to the washed algae a small amount of phosphate buffer, generally around pH 7. The effect of the washing treatment on photosynthesis has not been thoroughly investigated. I have found that algae washed three times with water lost 80 to 90 percent of their photosynthetic ability as measured by the fixation of HaHC140 at pH 8.2. 3 - At first this phenomenon was not attributed to a pH effect, because of numerous reports that photosynthesis by algae was unaltered between pH 4 to 9. However, examination of those compounds which were most effective in restoring photosynthesis to the washed algae indicated that they all were potential buffers below pH 8. Phosphate, phosphoglycolate, serine and tris buffer were used the most often to restore photo- synthesis for the washed algae. The addition of such compounds at pH 7 to the algal cultures did indeed lower the 75 76 . . . . n , 14 final pH of the suspension after addition or the nadC 03 to values between 7 and 8. In this pH range part of the bicarbonate would be converted to CO2. Consequently, the data can be explained best by the working hypothesis that 014 140 O2 enters the algae much faster than HO 3 ions. From the action of the buffers a constant amount of C14 02 would be released and disolved in the water. As the algae absorbed the 002 more would be released from the bicarbonate without a significant change in pH because of the action of the buffer. The results emphasize the need for buffer and pH control during tracer photosynthesis experiments with algae. A pH range between 5 and 7 but not over 7.5 is recommended. Too low a pH would cause the loss of much of the C1402 as gas to the atmosphere, or would necessitate working in closed and aerated apparatus. Of course, one could gas the algae cultures with C14 02, but this procedure would involve a much more elaborate apparatus. The above hypothesis was substantiated by several additional types of experiments. (a) Buffers were not necessary for restoring activity to the washed algal cultures providing the final pH was lowered to about 7 by addition of HCl. (b) Radioactive labeled buffers, phosphate and serine were not abosorbed by the cells in significant amounts during the course of the experiments. Thus, there was not evidence that these buffers entered the cell or participated in the photosynthetic process. (c) Even though the rate of photosynthesis was reduced 90 percent at pH 8.2 and above, 77 the C14 labeled products of photosynthesis did not seem to be significantly altered. This suggests that availability of 01402 was the limiting factor rather than alteration of the fixation products. In the course of this work several deviations arose from the postulate. (a) At high bicarbonate concentrations, around 10-2 and 10'3 H, normal rates of photosynthesis occurred even at pH 8.2 or above. This may be explained by mass diffusion of bicarbonate into the cells which occurs too slow at 10"4 and 10'5 M bicarbonate concentrations which are encountered with the tracer research method. (b) Uranyl (U02++) at concentrations equal to that of the buffers completely negated the stimulatory effect from the buffers even though the pH remained far below 8. Uranyl ions have been reported to be without effect upon photo- synthesis by partially washed algae. (o) Arsenate ions if added to the algae 10 minutes before addition of the phos- phate or phosphoglycolate buffers also blocked photosynthesis. This inhibition by arsenate did not occur if the buffers were added first. As mentioned in the result section, the arsenate was probably not absorbed by the cells. There is no ready explanation for these latter two observations. The three algae used in this work were selected because of their ability to synthesize and secrete large amounts of glycolate. Tolbert and 2111 (76) showed that Chlorella pyrenoidosa converted 3 to 12 percent of the total 014 fixed during 10 minutes of photosynthesis into glycolate 78 and excreted it into the supernatant fluid. The two Chlamydomonas cultures synthesized 50 percent of the 014 fixed during 1 to 10 minutes of photosynthesis into glycolate and excreted most of it‘. One hypothesis about the glycolate excretion was that it involved an active glycolate- bicarbonate shift or transport mechanism across the cell membrane (76). A major difference between the photosynthesis experiments on glycolate excretion mentioned in the preceed- ing paragraph and all the experiments in Part II of this dissertation was in the preparation of the algae for experimentation. The glycolate excretion studies were done with either unwashed or once washed algae from which treatments no reduction in photosynthesis rate was imposed. All of my experiments were done with algae which had been washed three times and which had a severely inhibited photosynthetic rate at pH 8.2 when bicarbonate was added. The washed algae, however, would photosynthesize normally at pH values (7 or below) where 002 could exist in the medium. These considerations generate speculation that the unwashed or once washed cells could produce and excrete their own acid or buffer for releasing 002 from bicarbonate. This acid would be the excreted glycolic acid. This release of glycolic acid would occur near the cell surface and be titrated by the HaHCOB to give sodium elycolate and 002. L) 1P. C. Kearney, d. imenez and I. B. Tolbert, unpublished. 79 inis speculation is supported by the fact that the glycolate excretion phenomenon occurred only above pH 5.0 to 5.5 and in the presence of low 002 and bicarbonate concentrations (70). The present hypothesis about glycolate excretion differs from that proposed by Tolbert and Zill, in that it does not involve a glycolate-bicarbonate anionic shift, but ~ather the release of acid by the cells and the generation A of 302 in the medium which diffuses into the cells. SUHIARY AKD CONCLUQIOHS a Glycolate Pathway has been prOposed from the meta- bolic conversion of glycolate to sugars. This scheme first involves the synthesis of serine from two glycolate molecules through the intermediates glyoxylate and glycine. The serine was further converted to glycerate, which in turn was in- corporated into hexoses by reversal of the Embden-Meyerhof Pathway. This route was substantiated by feeding leaves for short periods of time CIA-labeled glycolate and serine-3-Cl4, 1 o ‘ 14 and then isolating and degrading tne C -labeled sucrose which was formed. Glycolate-Q-ClLr produced hexoses labeled in car- bon atoms 1, 2, 5 and 6. Serine-B-Cl4 was converted into 3, 4-labeled hexoses. These data corroborate other investiga- tions which purport to show that glycolate is a major photo- synthetic product which enters the cytoplasm from the cloroplast. The enzymes of the Glycolate Pathway are cytoplasmic. Since the g ycolate produced by 01402 photosynthesis is uniformly labeled, its subsequent metabolism would produce uniformly la- beled hexoses in the cytoplasr. This is in contrast to the predominately 3, 4-labeled hexoses of the photosynthetic carbon cycle inside the chlor0p1asts. The rate of randomization of label in the hexoses of the whole cell is therefore accelerated by the contribution of the Glycolate Pathway. PART II The effect of washing al3ae with water before subse- quent photosynthesis experiments :ith LaCl405m as investigated. An explanation was sought for the reason why better photo- synthetic rates were obtained when the washed algae were re- suSpended in phosphate buffer. Upon washing algae three times with distilled water, the photosynthetic rate of fi: cation of #403 at pH 8. 2 or above was inhioited 80 to 90 percent. f“ F): However, the rate of photosynthesis could be restored to normal when the pH was lowered to 7 or below by buffers such as phos- phate, phosphoglycolate, or amino acids. This phenomenon was best explained by the assuxxLation that @1402 could enter the 1an the bicarbonate ions. Thus he cells many times faster t f :‘3 (L) rs selved the purpose of holding the pH in a range where , "“14 12+ o . ~ tne flaflv 03 was converted to C 02. The buffers when laoeled with tracers were shown not to enter the cells. The products of OJ"r fixation by the washed algae were not altered by the presence or absence of the buffers. At bicarbonate concentra- 014 tracer re- tions lOO fold higher than generally us ed with search, a normal rate of photosynthesis occurred even at pH values of 8.2 or hi her. Presumably bicarbonate ions could enter the algae at a slow rate which was compensated for by higher conce ntrat'ons. The restoration of active photosynthetic rate sby phosphate or serine buffers was prevented by uranyl ions or arsenate even though the pH was lowered well below 7. :2 If the algae which were washed three times produced less glycolate du~in3 photosynthesis than algae washed only once, these results could then be interpreted to indicate that normal production and excretion of glycolic acid by algae may be a mechanism for converting the bicarbonate of the medium to 002, which in turn can be absorbed by the cells. U1 0 O\ O \1 CD 0 \I) O 10. 11. 12. Al Kholy, A. L., Elysiol. Plantarun, g, 137. Aronoff, 3., Arch. Biochem. Biophys., 32, 257. Aronoff, 8., and Gaile‘, F. 3., Plant thsiol., 491. 1955. Barber, D. A., and Russell, R. 5., J. Exp. (35), 252. 1961. Pot., H \C) U1 H U1 0 lg Bassham, J. A., and Calvin, M., The Path of Carbon in Photosynthesis. Prentice-Hall, Inc. Englewood Cliffs, H. J. 1957. Benson, A. A., Basshan, J. 1., Calvin, H., p. 16. Goodale, T. Haas, V. 1., and btepka, 5., J. .§22-..Z§. 1710. 1950. and Calvin, 3., J. Exp. Bot., 1, 63. J. Am. Chem. Soc.,llfl, 4477. Am. Chem. 1950. Raw guchi, 5., Hayes, P., and Calvin, H., 1952. Berstein, I. 1., Lentz, 2L, Ham. Schambye: Pu T "1 and Jood, R. u., J. Biol. Chem., 215, 157. 1955. Bjorn Lindahl, P. 3., Nature, 191 (4783), 51. 1961. Brey, Jr., J. 8., Principles of Physical Chemistry. Appleton-Century-Crofts, Inc., E. Y. 195 p. 186. 311388, 3:. , I