Metabolism of Pyruvate by Starved Cells of Chlorella elipsoida 1>2Ann Oaks 3

Bacteriological Institute of the South German Dairy Research & Experiment Station,Weihenstephan, Post Freising/Germany

The metabolism and function of the organic acidsof the tricarboxylic acid (TCA) cycle in plants andmicroorganisms is not clearly understood. Evidencefrom the effects of inhibitors, the incorporation ofradioactivity from specifically labeled acids, and theisolation of the acid intermediates and the activeenzymes (3, 4, 11, 24, 26, 30) indicate that the cyclecan operate. By feeding acids as a sole source ofcarbon to Chlorella cells depleted of their polysac-charide reserves the functions of the TCA cycle canbe isolated from those of the glycolytic and pentosephosphate pathways. Their efficiency in promotinggrowth or the synthesis of reserve material will be ameasure of the ability of the TCA cycle in supplyingthe carbon skeletons, energy, and reducing power need-ed for the synthetic reactions. In a few instances ithas been demonstrated that the acids of the TCAcycle support neither growth (7) nor the incorpora-tion of NH3 (16, 23) as efficiently as glucose. Con-versely, it has been shown that in a number of micro-organisms the disappearance of the acids from themedium is greatly increased with the change fromresting to growing conditions (18).

In this report the kinetics of pyruvate utilization.the fates of the various carbons of pyruvate, and thefactors limiting its utilization are described in anattempt to indicate the importance of the TCA cyclein synthetic and energy producing reactions.

Materials & MethodsCells of Chlorella clipsoida (culture supplied by

Prof. A. Pirson) were grown in a complete mineralnutrient solution in liter flasks in the light (14).Air supplemented with 5.0 % COO was constantlybubbledl through the flasks. The cells were harvest-ed 3 to 4 (lays later, centrifuged, washed twice withdistille(l w-ater, ancl allowed to shake in the dark for16 to 24 hours. Before the experiment they wererecentrifuged and enough distilled water was addedto give a specific density which was checked photo-metrically.

Aliquots of cells, 1 or 2 ml, together with potas-siumil phosphate buffer (final concentration 0.017 MI,

1 Received Oct. 2, 1961.2 This work was supported by the Alexander von

Humboldt Stiftung, Germany,3 Present Address: Department of Biological Sciences,

Purdue University, Lafayette, Ind.

pH 5.4) were placed in Warburg flasks, and were al-lowed to shake in a water bath at 25 C in the darkfor a minimum of 30 minutes before the substratewas tipped in from the side-arm. A final concentra-tion of 0.017 M glucose or pyruvate was used in thevarious experiments unless otherwise stated. Theradiochemicals were obtained from the RadiochemicalCentre, Amersham, Bucks, England. Oxygen up-take and CO, evolution were measured by standardWarburg techniques. At the end of the experimentthe flasks were placed in an ice-water bath and thecells were quantitatively transferred to centrifugetubes. They were then centrifuged at 0 C, washedtwice with cold distilled water, and were finallv kill-ed by placing the tubes in a boiling water bath for 30minutes. This relatively slow method of killing (ap-proximately 15 min) was necessary because the pyru-vate remaining in the medium interfered with thechromatograms of the hot water extract.

Further fractionation was achieved by centrifug-ing and washing the dead cells several times withdistilled water, hydrolyzing the cells with 0.5 N HCIin a boiling water bath for 30 minutes, and centri-fuging and washing the residue. The first superna-tant, the hot water extract (H.W.E.) contains thewater-soluble sugars, and amino and organic acids;the second, the acid hydrolysate (A.H.), the sugarsderived from the polysaccharides and a ninhydrinpositive fraction. The residue was made up to 1 mlwith distilled water, and the various extracts wereevaporated to 2 ml. Aliquots (50 il) of each frac-tion were placed on aluminium discs, dried over lowheat, and counted. No loss in activity occurred (lur-ing the various extraction processes. The alkali inthe center well of the Warburg flask was quantita-tively removed, converted to BaCO3, and counted.

Duplicate or triplicate samples of the H.W.E.were chromatographed on Whatman No. 1 paper,first in a water saturated phenol solution and then ina butanol: propionic acid: water solution (2). TheA.H. was chromatographed in the butanol: propionicacid: water solution. Further separations of sucroseand asparagine were achieved by developing in apicoline: ammonia: water (78: 2: 20) solution. Thechromatograms were then placed on X-ray plates.After the films were developed, the radioactive spotson the chromatograms were counted and the percent-age activity of each spot was calculated. The spotswere positively identified by co-chromatography withknown compounds.

310

OAKS-PYRUVATE METABOLISM BY STARVED CHLORELLA

Results & Discussion> Effect of Pyruvate Concentration. Kandler andErnst (16) found that pyruvate and acids of theTCA cycle are readily utilized by starved Chlorellacells, and that with pyruvate the rate of respirationis roughly proportional to the concentration, althoughit falls to the endogenous level before all the sub-strate is utilized. The results (fig 1) show more

clearly the relationship between the respiratory rateand the disappearance of pyruvate from the medium.Initially the Qpyruvate exceeds the Qo, but with timeboth rates fall off, and with 0.005 M pyruvate theserates are approximately equal between the 2nd and3rd hours (fig la). The pyruvate in the mediumwas determined by the dinitrophenylhydrazine meth-od of Wolf (31) and radioautograms from later ex-

periments showed that pyruvate was the only radio-active compound in the medium. After 3 hours,when the rate of oxygen uptake approaches the endog-enous rate, only 58 % of the 0.005 M, 17 % of the0.017 M, and 9 % of the 0.05M pyruvate are used.Despite the difference in the proportion of addedpyruvate that is used, there is a strict linear relation-ship between the oxygen uptake and the disappear-ance of pyruvate (fig lb). As the pyruvate con-

n.'0-..

.4

cLv

z 4

2x0

LAi.

41a.,-.

IAJ~-

0>4

1oa PYRUVATE (pIM)

Table IEffect of Pyruvate Concentration on CO2 Activity

Conc ofPyruvate Activity in CO2 (cpm) I.Q.*

C, C8 C,/C8 C1 C8

0.1 M 6,740 567 11.85 0.392 9.020.05 M 2,375 462 5.15 0.253 5.660.01 M 506 172 2.94 0.202 2.530.005M 345 156 2.21 0.218 2.44* I.Q. represents the ratio of the activities found in the

Chlorella cells and CO2.Pretreatment as in figure 1.

centration increases both the proportion taken upfrom the medium and the proportion respired drops.

The relationship between the pyruvate incorpo-rated and respired is shown more clearly in table I.In these experiments the cells were incubated for 2hours with several concentrations of specificallylabeled pyruvate-1-C14 and -3-C14. The activities inthe table are corrected for the specific activities of theadded substrates. At each concentration the sum ofthe activities found in the algae and COO is the same

30

261

TIME (HR.)

Fig. 1. The relationship between the disappearance of pyruvate from the medium (A pyruvate) and oxygenuptake. The starved cells were washed, suspended in KH2PO (0.017 Ni; pH 5.2) and allowed to equilibrate in thedark for 30 minutes before the addition of pyruvate.

(b) O.OU

0.05M

i0.017M0.005M

3 Hr.

1- ~~~~~II

311

PLANT PHYSIOLOGY

for both pyruvates showing that the fermentation ofpyruvate is not an important factor at these concen-trations. With a decreasing substrate concentra-tion both the ratio of carbon-1 to carbon-3 (CJ/C3)respired to CO., and the proportion of carbon-3 as-similated into the algal residue are reduced. Themajor cause for this is the more complete oxidationof pyruvate at lower concentrations, which affects theC, little but increases the contribution of C3 to theCO.. fraction.

- Effect of Jodoacetate on Pyruvate Respiration.When iodoacetate is given to Chlorella cells the phos-phoglyceraldehyde pool increases while the phospho-pyruvate and pyruvate pools decrease (17). Aftera 2 hour pre-treatment with iodoacetate (table II), aconcentration of the inhibitor which eliminates glu-cose respiration in Chlorella (10, 15,17) has no ef-fect on the oxygen uptake induced by pyruvate, butboth the C1/C3 ratio in the CO2 and the proportionof carbon-3 assimilated into the algal residue are re-duced. As with glucose (14), the disappearance ofpyruvate from the medium is more sensitive thanrespiration to the inhibitor.

- Utilization of Specific Carbons of Pyruvate. Theresults in table III show the typical distribution ofeach of the carbons of pyruvate in starved Chlorellacells after a 2 hour period, a time when the rapiduptake is just completed (fig la). The total as-similation is the activity found in the CO2 and algalfractions. After correction for specific activity, it isessentially the same for each of the pyruvates. Fromthe contribution of each carbon to CO, and from theratio of the activities found in the cells and CO,(incorporation quotient; I.Q.), it is clear that C? ispreferentially incorporated, C2 is intermediate, andthat C1 appears chiefly as CO,. With C3-labeledpyruvate a measurable activity is found in malic,citric, fumaric, succinic, and a-ketoglutaric acidswhich suggests that the full TCA cycle is operating.Since activity from carbon-i is found in citric andglutamic acids, a carboxylation reaction followed by

a randomization of the activity in the carboxyl car-bons of malate (5) must account for some of the en-try of pyruvate into the TCA cycle.

Carbons two and three of pyruvate contribute ap-proximately twice as much activity to alanine ascarbon one. That derived from C1 probably rep-resents a direct incorporation (20) and the extrafrom the other two carbons a less direct synthesis,possibly including assimilation into the tricarboxylicacids and then a decarboxylation of malate or oxalo-acetic to yield the 3-carbon precursor of alanine.

The incorporation of pyruvate into sucrose couldinvolve either a direct reversal of glycolysis (8,9)or the energetically more feasible route over malateto phosphopyruvate (the malate bypass) (8, 19, 26).The almost linear increase in the contribution of car-bons 1, 2, and 3 to sucrose in(licates that a majorpart of the sucrose is synthesized in these cells bythe malate-by-pass. Glutamate whiclh is synthesizedduring pyruvate feeding probably acts as a reservoirfor the TCA cycle and as a precursor for the proteinswhich comprise most of the activity in the acid hy-drolysate and residue fractions. The contribution ofC1 to the glutamic and tricarboxylic acid fraction isalmost the same as that to the sugars, while C., andCo contribute approximately twice as much activityto the acid fraction. This shows that the oxidativedecarboxylation of pyruvate is an important reactionand suggests in agreement with McConnel, et al.(20) that Co and C3 are important as precursors ofprotein synthesis.

- Time-Course of Pyruvate Assimilation. Thekinetics of pyruvate incorporation and release as CO,are summarized in table IV. The proportion ofpyruvate-3-C14 going to CO2 increases with timewhile the incorporation quotient falls off drastically.Thus pyruvate is supplying a minimumi of carbon tothe CO, during the peak of the pyruvate-inducedrespiration (fig la). When the activities found inthe crude fractions are plotted as a percentage of thetotal activity against time the slope of the activity in

Table II

Effect of lodoacetate in Pyruvate Metabolism in Dark

Total* C. ciiIAA O., uptake CO. I.Q.**

Pyruvate (M) (M) (AM) RQ (cpm) C1 C3 CI/C30.1 M0.01 M0.1 M0.01 M

...

..

1o-310-3

14.011.712.710.4

0.7830.7390.7930.720

24,6368,17014,6244,830

19.1224,25611.7604428

3,4851,3904,6402,310

6.893.063.161.91

6.104.862.161.09

0.017 M None 8.9 0.749 220,168 144.996 22,486 6.45 8.8910-6 12.6 ... 201,288 ... 20,328 ... 8.9010-5 12.8 ... 197,664 ... 19,024 ... 9.39

3 X10-5 12.6 ... 178,256 ... 18,036 . . . 8.910-4 12.4 0.706 122,299 ... 17,899 ... 5.8

The cells were allowed to shake in the dark with the inhibitor and 0.017 M KH,PO4 (pH 5.2) for 3 hours beforethe substrate was added. The experiment was stopped 2Y2 hours after adding substrate.* Total uptake represents the activity in cpm found in the CO, and in the algae.

** I.Q. represents the ratio of the activities found in the Chlorella cells and in the CO, for pyruvate-3-CI4.

312

OAKS-PYRUVATE METABOLISM BY STARVED CHLORELLA

Table IIIUtilization of Specifically Labeled Pyruvate

by Starved Chlorella Cells

Pyruvate- Pyruvate- Pyruvate-J_C14 2-C; 4 3_C14

Totalassimilationi(cpm) 155,384 155,389 165,678

% CO, 63.3 24.1 8.6% H.W.E.* 25.1 47.7 53.0% A.H.* 7.5 17.1 23.1% Residue 4.1 11.1 15.1I.Q.* 0.581 3.15 11.06

Sugar-P 2.49 5.38 2.40Sucrose 6.20 10.25 15.50Alanine 3.82 3.52 2.58Glutamic-glutamiiie 2.10 17.15 23.0

Aspartic- Compoundsasparagiiie ... 0.76 0.97 in hot

waterGlycine- extract asserine 0.85 2.03 0.23 % of totalMalic 1.42 1.75 2.54 activityCitric 1.95 0.46 0.87Fumaric ... 0.40 0.04Succinic ... 0.92 0.95

a-keto-glutaric ... ... 0.58Unident.Polysacc. ... 0.26 0.16Unknown17 0.88 0.75 0.20Unknown19 1.05 1.36 0.94

Sucrose 9,633 15,946 25,640TC Acids, TotalGlutaniic activity& aspartic 8,502 33,992 47,229 (cpm)The starved cells were washed, suspended in KH2PO4

(0.017 M; pH 5.2), and allowed to equilibrate in the darkfor 30 minutes before adding pyruvate (0.017 M). Thecells were killed 2 hours after adding pyruvate.* H.W.E. is the hot water extract; A.H. the acid hy-

drolysate and I.Q. the ratio of the activities found inthe algae and CO2.

the hot water extract (H.W.E.) is negative, show-ing that pyruvate is first incorporated into this frac-tion and then is either transformed to the productsof the acid hydrolysate (A.H.) and residue or res-pired to CO2. A further plot of the percentage ac-tivity within the hot water extract indicates thatmalate and alanine are the first products of pyruvateassimilation. The proportion of the activity in glu-tamic acid, which is a major component of this frac-tion, reaches a peak by 60 minutes. The incorpo-ration into sucrose, the acid hydrolysate, and residueis most rapid during the first 60 minutes, but con-tinues during the experiment.

Of these products the amino acids, glutamic, as-partic, and alanine (27) and the sugars (28) are ac-tively synthesized during pyruvate feeding. By

so

70

60

.304

I->204

-J 40

0

11.30

0

20I0

460

(r 50

w

0

2

0

U.

0

-2t

30 60 120 240 300TIME (MIN.)

Sucrose

I@ /

_______________X___________ TC. AcidsL_AanineI// ~~~~~~Molic AcidI

30 60 20TIME (MIN.)

---~~~~4030

Fig. 2. The distribution of pyruvate-3-C14 within themajor cell fractions. The experimental conditions wereas described in table III.

Fig. 3. The distribution of pyruvate-3-C14 within thehc,t water extract.

Table IVKinetics of Pyruvate-3-C14 Incorporation

Time (min) 30 60 120 300

Total uptake(cpm) 76,900 103,700 166,900 256,100

I.Q* 39.4 17.5 10.0 5.1% CO2 2.54 5.41 9.09 12.40% H.W.E.* 74.61 66.04 57.95 48.00%0 A.H.* 16.09 20.50 24.06 28.45% Residue 6.76 8.05 8.90 11.15

Major components of hot weater extract as% of total activity

Sucrose 7.85 16.87 20.60 228.05Glutamic 36.78 33.51 27.21 15.09Aspartic 0.95 1.29 1.15 0.97Alanine 9.35 3.98 3.41 1.83Malic 6.96 0.86 0.30 ...T.C. Acids* 10.32 7.45 4.23 2.03

Pretreatment as in table III.* The T.C. acids include malic, citric, fumaric, succinic,

and a-ketoglutaric acids; I.Q. is the ratio of activitiesfound in the algae and CO,; H.W.E. the hot waterextract; A.H. the acid hydrolysate.

313

Hot watHrExtract

AcidHydyso1e

CO2_/ ==!RRidu.---1a--

/.II

240 300

PLANT PHYSIOLOGY

analogy to Mothes' work with yeast (23) and fromthe effect of pyruvate on the assimilation of NH3 inChlorella (16), it may be assumed that only a smallpart of the activity incorporated into the proteins ofthe acid hydrolysate and residue represents a netsynthesis. Also from the similarity between the in-corporation patterns of pyruvate-3-C14 and CO, (22),it may be assumed that there is little change in thesize of the organic acid pools. Thus the amino acids,principally glutamic, and sucrose may be consideredas competing for the pyruvate initially assimilated.In the early stages of pyruvate feeding (30 or 60min) glutamate is favored in this competition.

- Effect of Glucose & Light on Pyruvate Metabo-lism. If the TCA cycle supplies most of the energyfor living cells, and if it represents the terminalstages of glucose catabolism, pyruvate should be asefficient as glucose as a carbon source in starvedChlorella cells. Although pyruvate appears to bemetabolized initially via the TCA cycle, it inducesonly half the respiration (table V) and onlv a frac-tion of the growth (7) that glucose does; it does notsupport the assimilation of NH3 (16); and unlikeglucose it induces the accumulation of glutamate, as-

partate, and alanine (27). The disappearance ofglucose from the medium is complete at all con-centrations (14) while with pyruvate it becomes pro-portionally less with increasing concentrations. sug-gesting either that higher concentrations of pyruvateare inhibitory, or that some factor is limiting its as-

similation. If pyruvate is inhibiting cellular metabo-lism it might be expected to reduce either the normalor light-stimulated uptake of glucose but, as shownin table V, this is not the case. On the other hand,pyruvate does reduce the proportion of glucose res-

pired to CO., a result expected if it were a true in-termediate of glucose catabolism. Conversely, glu-cose stimulates the uptake of pyruvate. These resultswith glucose and pyruvate show the same trends ifthe unlabeled substrate is added before, at the same

time, or after the labeled substrate. Light too, at in-tensities which saturate the fixation of CO, enhancesthe incorporation of pyruvate, but is without effectwhen glucose is also present.

ConclusionsPyruvate is readily metabolized by starved

Chlorella cells. In common with tissues from higherplants ( 1, 6, 12, 20, 26, 30) carbon 1 is recovered chief-ly as CO2, while carbon 3 is preferentially incorpo-rated, and the pattern for carbon 2 utilization lies be-tween these two extremes. Two mechanisms are sug-gested for the initial incorporation of pyruvate: a

carboxylation reaction which results in the early ac-cumulation of activity in malic acid, and a decarboxyl-ation reaction which accounts for the much greaterproportion of carbons 2 and 3 in glutamic acid. Withtime the activity found initially in malic acid spreadsto other acids of the TCA cycle. Thus the evidencesuggests that the full TCA cycle is active.

Unlike glucose (14) pyruvate is never complete-ly utilized and it does not support the assimilation ofNH3 (16, 23) or growth (7). Thus pyruvate iseither inhibitory or it fails to supply in sufficientamounts some factor, which is indispensible to thesynthetic reactions. The first alternative seems un-

likely since pyruvate does not reduce the uptake ofglucose, but acts rather as an intermediate of glucosecatabolism.

Since both glucose and pyruvate utilization appearto include the TCA cycle some factor produced priorto the TCA cycle in the catabolism of glucose may benecessary for NH3 assimilation or growth. Of thesethe rate of carboxylation of pyruvate as opposed tophosphopyruvate seems an unlikely candidate sinceboth light and glucose stimulate the utilization ofpyruvate, while the addition of a dicarboxylic aciddoes not (27). Although light increases the incorpo-ration into fats (21, 27) and glucose that into pro-

teins (27) their effect is not additive. Thus withthe extra drainage from the TCA cycle this factorappears to be rate limiting, but in the dark withpyruvate as the sole source of carbon, it should besufficient. The interpretation favored here is thatan inadequate production of energy or reducing pow-er, or perhaps an improper balance of these two fac-tors results in the poor utilization of pyruvate whenit is the sole source of carbon. Both are formedsince pyruvate promotes a net synthesis of sugars

Table VIniteraction of Pvrusvate & Glucose in Light & Dark

Dark Light

Substrate O., RQ Total Total(tiM) CO, uptake uptake(cpm) (cpm)* I.Q.* (cpm)

None 3.44 0.910Pyruvate-3-C-' 4 (0.02 M) 6.56 1.075 784 6,690 5.92 9,420

+ glucose (0.02 M) 11.92 0.985 624 13,610 20.85 12,840Glucose-,u-C14 (0.02 M) 9.91 0.973 12,730 89,780 5.26 111,040

"~ + pyruvate (0.02 M) 11.34 1.024 7,920 88,040 9.36 123,900The unlabeled substrate was added 30 minutes before the radioactive one and the experiment continued for 2 howu

after adding the second substrate. The pretreatment was as described in table III.* Total uptake is the activity in the cells pluis CO2; the I.Q. is the ratio of these activities.

314

OAKS-PYRUVATE METABOLISM BY STARVED CHLORELLA

(28). This synthesis itself, however, may be a

feature of a disturbed metabolism since the incorpo-ration of pyruvate into sucrose is much greater instarved than in fresh cells (Oaks & Kandler: unpub-lished results).

Early experiments have shown that CO2 in thelight is the best carbon source for growth (25), thatin the dark glucose is a suitable substrate (24), andthat the acids of the TCA cycle support only a

meager growth (7). Each of these systems pro-

vides energy, reducing power and carbon skeletons indifferent proportions. The results with pyruvate sug-

gest that with a system limited largely to the TCAcycle the supply of energy or reducing power may bethe rate limiting factor. Under such conditions notonly are the respiration and utilization of the sub-strate inferior, but distortions in the metabolic pat-tern may also be found.

Summary

Pyruvate is rapidly metabolized by starved Chlo-rella cells. The initial incorporation products are

the acids of the tricarboxylic acid cycle, alanine, andglutamic and aspartic acids. The contributions ofcarbons 1, 2, and 3 to CO, and the algal residue in-dicate the operation of the full tricarboxylic acidcycle. After 2 hours sucrose is a major product.Its synthesis appears to be by the malate-bypass tophosphopyruvate. The rate of disappearance ofpyruvate from the medium parallels the rate of thesynthetic reactions, and ceases apparently because ofthe inability of pyruvate to support a good proteinsynthesis. This deficiency is partially remedied bylight or glucose.

Acknowledgments

I should like to thank Prof. 0. Kandler for the useof laboratory facilities and for helpful discussion duringthe course of this work; Prof. H. Beevers for criticismof the manuscript, and the Deutschen Forschungsgemeind-schaft for its financial assistance.

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Influence of Glucose & Light on Pyruvate Metabolismby Starved Cells of Chlorella elipsoida 1 2

Ann Oaks 3

Bacteriological Institute of the South German Dairy Research & Experiment Station,Weihenstephan, Post Freising, Germany

In Chlorella cells the oxidation of glucose pro-ceeds by the glycolytic and pentosephosphate path-ways to phosphoglyceraldehyde an(l pyruvate (12);the resulting pyruvate appears to be oxidized by thetricarboxylic acid (TCA) cycle (22). Since therespiration in plant cells and microorganisms is gear-ed primarily to the synthetic requirements, a delicatebalance must be established between those reactionswhich require carbon skeletons and those which pro-duce the energy and reducing power necessary todrive the synthesis of new materials. With glucoseas a substrate there is a high rate of respiration untilall the substrate is gone (8) showing that such abalance is achieved. On the other hand, the increas-ed respiration induced by pyruvate falls before thesubstrate is completely utilized (22). If the rateof uptake of pyruvate from the medium is limited byits ability to support synthetic reactions, then the ad-dition of either glucose or light partially overcomesthe deficiency (22). Indeed the inability of pyru-vate to support the assimilation of NH3 (10) orgrowth (6) indicates that the acids of the TCA cyclealone cannot promote the synthesis of proteins. Inorder to define the factor linmiting, the utilization of

1 Received Oct. 2, 1961.° This work was supported by the Alexander von

Humboldt Stiftung, Germany.3 Present Address: Department of Biological Sciences,

Purdue University, Lafayette, Ind.

pyruvate more clearly, experiments are describedwhich show the influence of the substrate on the poolsizes of the direct amino acid derivatives of the TCAcycle, the interaction of glucose and pvruvate. andthe effect of light on acid metabolisimi.

Methods

Cells of Chlorella elipsoida Gerneck (culture sup-plied by Prof. A. Pirson) were grown as previouslydescribed (22) starved overnight in distilled water,centrifuged, washed, and placed in WVarburg flaskswith 0.017 M phosphate buffer (pH 5.6). At the endof the experiment (2-2y2 hr) the cells were extract-ed first with hot water (H.W.E.), then with hot0.5 N HCl (A.H.), and then in the light experimentswith hot 80 % ethanol (A.E.). The H.W.E. con-tains the amino and carboxylic acids and sugars, theA.H. sugars derived from the polysaccharides, anda ninhydrin positive fraction (proteins) and the A.E.a material which runs with the front in the butanol-propionic acid-water solution (fats). The killingprocedure, estimation of radioactivity, and identifica-tion of the components within each fraction have al-ready been described (22).

Results- Interaction of Glucose & Pyruvate. The poor

utilization of pyruvate may be caused by the inhibi-

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