Influence of Glucose Light Pyruvate Metabolism · by Starved Cells of Chlorella elipsoida 1 2 Ann Oaks3 Bacteriological Institute of the South German Dairy Research & Experiment Station,

PLANT PHYSIOLOGY

25. MYERS, J. & J. A. JOHNSTON. 1949. Carbon &nitrogen balance of Chlorella during growth.Plant Physiol. 24: 111-119.

26. NEAL, G. E. & H. BEEVERS. 1960. Pyruvate utili-zation in castor-bean endosperm & other tissues.Biochem. J. 74: 409-416.

27. OAKS, A. 1962. Influenice of glucose & light onpyruvate metabolism by starved cells of Chlorellaelipsoida. Plant Physiol. 37: 316-322.

28. SCHLEGF.L, H. G. 1956. Die Verwertung organisherSiiuren durch Chlorella im Licht. Planta 47:;10-5 26.

29. SIMoN, E. W. 1953. Dinitrocresol, cyanide, & thePasteur effect in yeast. J. Exptl. Botany 4:393-402.

30. VICKERY, H. B. & 1. ZELITCH. 1960. The miietabo-lism of organic acids of tobacco leaves. XVII.Effect of culture of excised leaves in solutions ofpotassium pyruvate. J. Biol. Chem. 235:1871-1875.

31. WOLFE, J. 1955. Nichtfluchtige Mono-, Di-, undTricarbonsauren. Modern Methods of PlantAnalysis. K. Paeclh & M. Vr. Tracey, eds.. II.490-538.

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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OAKS-PYRUVATE METABOLISM BY STARVED CHLORELLA

Table IEffect of Pyruvate on Glucose Assimilation

TotalSubstrate . 02 RQ CO, uptake*

(AM) (cpm) (cpm)None 6.7 0.735 ...Glucose- -C14

(0.017 M) 11.31 0.870 13,120 107,470Glucose + pyruvate




(0.1 M) 11.41 1.031 5,072 108,910* Total uptake represents the sum of the activities found

in the CO, and in the cells.Glucose was added 30 minutes after the pyruvate, and

the experiment was stopped 2 hours later.

tion of one reaction of the TCA cycle (18). In thiscase there should be a greater reduction in the dis-appearance of glucose from the medium than in itsrelease as CO2 analogous to the effect of fluoroace-tate or malonate (9), but as shown in table I withoutreally affecting the uptake of glucose an increasingconcentration of pyruvate progressively reduces thecontribution of glucose to CO2. This result is pre-dictable if pyruvate is filling the pools of the TCAcycle, thus reducing the breakdown of glucose. Thetypical effects of the interaction of pyruvate and glu-

Interaction of

cose are illustrated more clearly in table II. In theseexperiments the unlabeled substrate was added 30minutes before the labeled one, and the cells were

killed 2 hours after adding the latter. There aresignificant differences in the proportion of glucoseor pyruvate incorporated into the various cell compo-nents, but the same compounds become radioactive.More pyruvate is found in the CO, and hot water ex-

tract while a greater percentage of the glucose isincorporated into the acid hydrolysate. The dis-tribution within the acid hydrolysate is important too.With pyruvate the activity coincides chiefly withninhydrin-positive spots (proteins) while with glu-cose the main product is glucose derived from long-chain glucose polymers. About 75 % of the glucoseis incorporated into the sugars of the hot water ex-

tract and the acid hydrolysate, which fact agrees wellwith the analytical results of Taylor (28) and theestimations of Myers (16).

Glucose increases the uptake of pyruvate from themedium without altering the proportion respired toCO.. There is a drastic reduction in the proportionof the pyruvate remaining in the hot water extract,which is accounted for by the increase in the nin-hydrin-positive fraction (the proteins) of the acidhydrolysate. Glucose also reduces the proportion ofpyruvate incorporated into sucrose and increases thatfound in the TCA cycle acids and glutamate. How-ever, there is little change in the absolute amount ofpyruvate incorporated into the acids. In the con-verse experiment pyruvate reduces the contribution

Table II

Glucose & Pyruvate in Starved Chlorella Cells

Pyruvate-3-Q4

Control + glucose C12

Glucose-U-C04

Control + pyruvate C'2

Total uptake (cpm)Algae/CO.,% CO,% H.W.E.% A.H.-% Residue

% Activity within hot uwater extractSugar-PSucroseAlanineGlutamicAspartic

CitricMalicSerine-glycineGlycolic

Sucrose (cpm)T.C. Acids, glutamic & aspartic (cpm)

Sucrose/TCA*

37,59511.098.3

42.640.68.5

7.7436.527.27

30.691.91

2.292.931.862.88

5,2365,222

1.00

59,84511.917.8

18.465.28.6

5.5113.356.85

53.501.05

5.544.943.120.75

1,3116,851

0.19

125,85913.876.7311.9971.289.00

49.810.414.6

10.419.1

8,0905,849

1.38

121,03118.495.13

12.6774.247.99

71.98.319.06

1.973.87

13,2104,080

3.24

The starved cells were washed, suspended in KEH2PO4 (M/60; pH 5.2) in Warburg flasks (Total vol. = 3 ml),and were allowed to equilibrate in the dark for 30 minutes before adding the substrate (0.017 i,r).* TCA represents the acids of the TCA cycle and glutamic and aspartic acids.

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PLANT PHYSIOLOGY

of glucose to CO. and increases the proportion foundin the hot water extract and acid hydrolysate. Thereis a marked increase in the proportion of glucose go-

ing to sucrose in the hot water extract, and a cor-responding decrease in that found in the acids. Add-ed pyruvate is behaving as a true intermediate ofglucose metabolism. In addition, glucose is con-

tributing to the better incorporation of pyruvate intothe protein fraction of the acid hydrolysate.- Effect of Succinate on Glucose, Pyruvate, & Ace-

tate Metabolism. The typical effects of succinate onthe nmetabolism of glucose, pyruvate, or acetate areillustrated in table III. Acetate is incorporated intothe same compounds as glucose and pyruvate, butagain the distribution of activity shows certain spe-

cial features. A greater proportion of the acetate isfound in CO, and the tricarboxylic acids and signif-icantly less in the sugars. As with pyruvate, theactivity in the acid hydrolysate coincides with theninhydrin-positive spots.

Succinate reduces the contribution of glucose toCO. but other changes are less clearly defined thanin the case of pyruvate-glucose feeding. Addingsuccinate drastically reduces the uptake of pyruvate,a result expected if both substrates are directly de-pendent on the rate of turnover of the TCA cycle fortheir incorporation. In addition, succinate lowersthe proportion of pyruvate oxidized to CO., and in-creases that found in the acids. It may be significant,although the activity is low, that with succinate a

greater percentage of the activity from pyruvate isfound in malic, citric, and a-keto-glutaric acids,those acids associated with the initial assimilation ofpvruvate and the svnthesis of glutamic acid. Simi-

larly, with the addition of succinate an increased pro-portion of the activity from glucose is found in citricand a-keto-glutaric acids. Succinate appears to besparing pyruvate and glucose for the synthesis ofamino acids.

Any effect that succinate may have on the rate ofdisappearance of acetate from the medium would notbe detected in such long experiments, since nearlyall the available acetate is utilized, but the changesinduced in the distribution of the radioactivity areimportant. The proportion of the activity found inthe CO, in the tricarboxylic acids, and in glutatnicand aspartic acids is reduced while that in sucrose isdoubled and that in the acid hydrolysate is increasedslightly. By supplying a dicarboxylic acid the in-corporation of acetate into the synthetic fractions isincreased and less is respired to CO, thus a mecha-nism for the net synthesis of dicarboxylic acids fromacetate is rate limiting in these Chlorella cells.

- Effect of Fluoroacetate on Metabolism of Glucose& Pyruvate. If the major part of glucose and pvru-vate respiration involves the TCA cycle, specific in-hibitors of the cycle should inhibit the respiration in-duced by each to the same degree. Fluoroacetatewas used in preference to malonate because of itsspecificity (15, 19) and because malonate is also arespiratory substrate (10). With equivalently label-ed pyruvate-3-C14 and glucose-6-C14 a direct com-parison of the effect of the inhibitor on oxygen up-take and the evolution of C140., can be made. The re-sults in table IV show that 10-4 M fluoroacetate in-hibits glucose- or pyruvate-induced oxygen uptakesimilarly. The inhibition of the release of C14O2from pyruvate-3-C14 and the oxygen uptake is the

Table IIIInfluence of Succinate on Utilization of Glucose-6-C14, Pyruvate-3-C14, & Acetate-2-C'4

Glucose-6-C14 Pyruvate-3-C14 Acetate-2-C'4Control 4- Succ. Control + Succ. Control -.- Succ.

Total uptake (cpm) 169,020 169,516 18,756 7,666 185,807 179,729Algae/CO., 17.58 22.8 7.8 10.8 4.14 5.02% CO., 5.7 4.2 10.9 8.5 19.4 16.6% H.W.E. 19.3 20.6 54.5 57.3 40.7 41.0% A.H. 72.7 72.2 27.3 27.3 22.9 25.3% Residue 2.6 3.0 7.3 7.5 7.0 7.1

Major comitponients of hot zepater extract as % of total actizitySucrose 12.9 12.8 20.7 21.5 6.38 13.5Glutamic 2.41 1.94 18.8 19.5 19.7 16.5Aspartic 0.29 ... 3.46 4.72 3.05 2.30Alanine 0.27 0.46 4.73 5.95 2.8 2.70Malic 0.10 0.02 0.85 1.52 1.02 1.22

Citric 0.04 0.19 0.75 1.76 1.75 1.07Fumaric 0.04 0.07 0.55 ... 0.60 0.46Succinic ... ... 0.75 ... 0.69 0.98ay-Ketoglutaric 0.02 0.11 0.83 2.40 2.07 0.72TC Acids 0.20 0.38 3.74 5.68 6.14 4.44

The pretreatment was described in table II. Succinate (0.017 M) was added 30 minutes before the radioactivesubstrate and the cells were killed 2 hours after the latter was added. Substrate concentrations were glucose andpyruvate 0.017M, and acetate 0.002 M.

318




Table IVEffect of Fluoroacetate on Pyruvate &

Glucose Metabolism

Pyruvate-3-C14 Glucose-6-C14

Control FA* Control FA10-4 M 10-4 M

Endogenous 02CUM) 3.12 4.16 3.12 4.16

Substrate 02(AM ) 7.29 6.71 12.11 8.80

% Control** 100 61 100 52CO2 (cpm) 406 240 3,605 1,879% Control 100 59 100 30

* FA is fluoroacetate.** Substrate respiration is corrected for the endogenous.

The starved cells were treated with FA for 2 hoursbefore adding the substrate (0.017 M), and the experimentwas stopped 2 hours later.

same, but with glucose-6-C'4 there is a much greaterreduction of the release of C140. When uniformlylabeled glucose and glucose specifically labeled in the1 and 6 positions are used (table V) this curious re-sult is explained. The C140 derived from C6 isreduced while that from either C1 or Cy is not af-fected by 10-4 or 10-3 M fluoroacetate. The higherCj/C6, ratio for the CO2 released indicates that withfluoroacetate a greater proportion of the glucose isoxidized by the pentosephosphate pathway (3).Such results indicate the greater flexibility of glu-cose-induced respiration, a factor which may be im-portant in its complete utilization under differentconditions.

- Effect of Carbon Source on Concentration of KeyAmino Acids. Starved cells treated with glucose,pyruvate, or acetate for 4 hours in the dark werekilled in the usual way (22); the hot water extractwas concentrated to 2 ml, and quantitatively trans-ferred to Whatman No. 4 paper for chromatography.The chromatograms were developed first in isopro-panol, water, and acetic acid (75: 15: 10) and thenin picoline, ammonia, and water (78: 2:20). Afterdrying, the papers were dipped in a ninhydrin solu-tion (75 mg CdCM,, 6 ml H2O, 0.3 ml acetic acid,100 ml acetone, & 1 mg ninhydrin), and were allow-ed to dry over night in the dark in an H,2SO3 atmos-

Table VEffect of Fluoroacetate on C1/C6 Ratio of Glucose

Control 10-4 M 10-8 MFA* FA

Endogenous (Mm) 3.29 3.91 3.94Glucose O2 (OM) 14.12 12.71 12.45C1 in CO2 (cpm) 4,954 4,457 4,681Cu in CO2 (cpm) 6,160 6,024 6,024C. in CO2 (cpm) 2,474 1,780 1,873C1/C6 2.49 3.38 3.20* FA is fluoroacetate. The conditions are described in

table IV.

phere at room temperature ( 11 ). The spots wereeluted with methanol and the color density measuredat 510 myA. The results summarized in table VIshow that with glucose there is no change in theconcentration of alanine, and glutamic and asparticacids, but with either pyruvate or acetate there is atwo- or threefold increase. A similar increase isfound with the two concentrations of pyruvate used.Thus the inability of the TCA acids to replace glu-cose as a carbon source is not due to any failure inthe synthesis of the primary amino acids. On thecontrary, from the results it is suggested that thesuperiority of glucose as a carbon source may lie inthe further metabolism of these amino acids.

- Effect of Light on Metabolism of Pyruvate &Acetate. The results from a typical experiment com-paring the utilization of pyruvate or acetate in the

Table VIEffect of Pyruvate, Acetate, & Glucose on

Amino Acids in Hot Water Extract

Treatment Aspartic Glutamic Alanineacid acid

Control 8 66 16+ Pyruvate (0.017M) 22 180 33+ Pyruvate (0.083M) 21 184 33+ Acetate (0.002M) 22 92 20+ Glucose (0.017M) 6 53 12

The concentration of amino acids is in ,utg/100 tml ofhot water extract. 0.5 ml of packed cells were suspendedin 9 ml of 0.017 M phosphate buffer (pH 5.2).

light and dark are summarized in table VII. Beforethe light experiments the flasks were flushed with aircontaining 0.5 % CO. in order to reduce the re-aXsimilation of C140, released from the substrate.The CO, concentration had no effect on the distribu-tion of the activity in the dark. A light intensitywhich just saturated the incorporation of NaHC'402was used.

Light increases the uptake of both substrates:this appears to be related principally to the higherincorporation into the alcohol extractable fats. Thisconfirms earlier results for both pyruvate (17) andacetate (25). There is also a measurable increasewith both substrates in the proportion of the activityin the ninhydrin-positive fraction of the acid hydroly-sate. The most significant changes induced by lightwithin the hot water extract are the marked reduc-tion in the proportion of the pyruvate incorporatedinto sucrose and the new formation of radioactivevaline and leucine. There is little change in the ab-solute activity incorporated into the acids and im-mediate derivatives of the TCA cycle, although thereis a slight reduction in the proportion of the total ac-tivity found in aspartate, malate, and alanine, whichare early products of photosynthesis. As with freshcells (17), light does not inhibit the synthesis ofcitrate and glutamate from pyruvate. Except for

319



PLANT PHYSIOLOGY

Table VIIEffect of Light on Assimilation of

Pyruvate & Acetate

Pyruvate-3-C14 Acetate-2-C14

Dark Light Dark Light

Total uptake 187,560 289.080 185,807 239,208(cpm)

% Co2 10.9 19.4% H.W.E.* 54.5 34.2 40.7 29.4% A.H.* 20.1 26.1 20.9 26.7% A.E.* 7.2 26.2 12.0 32.7% Residue 7.3 13.5 7.0 11.2

% Activity of miiajor componienit 7ithin hot water extractSucrose 35.6 23.2 14.3 12.8Glutamic 32.2 45.5 44.5 61.0Aspartic 6.3 2.4 6.9 5.6Alanine 8.2 8.3 6.4 4.6Malic 1.5 2.1 2.3 3.1

Succinic 1.3 ... 1.6 3.6Citric 1.3 3.4 4.0 1.6a-ketoglutaric 1.4 1.3 2.9 2.3T.C. Acids 5.5 6.9 10.7 10.6

Serine & glycine 1.54 4.8 3.5 1.05Valine-leucine ... 4.3 0.48Sucrose (cpm) 36,350 22.,900 10,810 9,020TCA* (cpm) 44,900 54,100 54,520 54,400* TCA represents acids of the TCA cycle, and glutamic

and aspartic acids: H.WV.E., the components solublein hot water; A.H., those soluble after 1 hour of treat-ment with hot 0.5 N HCI; and A.E., those extractedwith 80 % ethanol.

the less striking reduction of the inicorporation of ac-tivity into sucrose, light affects the metabolism ofacetate in a similar fashion. Light promotes thebetter utilization of pvruvate an(d acetate which isassociated with an increased incoriporation into thefats and proteins.

DiscussionOne important fact (lemonstrate(l by these ex-

periments is the variability in the utilization of pvru-vate under different conditions. With pyruvatealone, sucrose is a major productt: when glucose isadded, a greater proportion of the pyruvate carbonis found in the ninhydrin-positive fraction of theacid hydrolysate, and with light there is an increaseboth in the ninhydrin-positive fraction of the acidhydrolsate and in the fats of the alcohol extract. Ineach case the absolute contribution of pyruvate to theacids of the TCA cycle is the same. The increaseduptake of pyruvate-C14 induced by glucose or light isaccounted for by the higher incorporation of pyru-vate carbon, respectively, into proteins or fats. Theeffect of pyruvate in reducing the contribution ofglucose to CO2 and the acids of the TCA cycle wouldbe expected in a reversible system if pyruvate were anintermediate in the degradation of glucose. This issupported by the similarity in the percentage inhibi-tion of pyruvate- and glucose-induced respiration by

fluoroacetate. Thus, the major pathway for the oxi-dation of the three substrates used here appears to bethe full TCA cycle.

When equivalently labeled substrates are compar-ed. about 5 % of glucose-6-C'4. 10 % of pyruvate-3-C14, and 20 % of acetate-2-C'4 are found in the CO_.This suggests either a decreasing efficiency or lesschance for dilution with the endogenous pools as amore terminal substrate is used. The chance for thedilution of pyruvate and acetate carbon should beabout equal, since each is incorporated initiallv intothe acids of the TCA cycle. Both are unable tosupport the synthesis of proteins. With acetate theformation of dicarboxylic acids to replace the carbon(Irained to the synthetic reactions is an additionallinlitation. It is possible that under such conditionsa greater part of the potential energy available fromthe oxidation of the substrate will be lost. In starvedcells the proportion of pvruvate oxidized to CO, isthe same in the presence and absence of glucose. butthe incorporation into the proteins is greatly en-hanced by glucose. The contribution of acetate toCO.. is reduced slightly by the addition of a dicar-boxvlic acid while considerably more acetate carbonis found in sucrose. Tn both cases where a limitationis partially overcome the efficiency of the suibstratein coupling the synthetic to the energy prodtucingreactions is increased.

Twvo possibilities exist for the discrepancy betweenthe abilities of glucose and pyruvate in promotingthe assimilation of NH, or under proper conditionsgrow-th. Either the TCA cycle is more important asa supplier of carbon skeletons than of energy (16,27), or another pathway active in the degradation ofglucose supplies a factor that is limiting when therespiration is confined to the TCA cycle.

The results of Merrett and Svrett (15) suggestthat the TCA cycle contributes both energy and car-hon under conditions which promote growth. Theyfoun(d that in nitrogen-starved cells glucose had littleeffect on the contribution of acetate to CO., but thatacetate reduced the evolution of C140.2 from glucose.Acetate appears to be sparing glucose-carbon fromthe reactions of the TCA cycle in a manner similarto pvruvate. In growing cells, hoiwever, where boththe drainage of carbon and the need for energy areincreased, there is a greater mixing of the carbonsfrom glucose and acetate. The TCA cycle is ap-parently supplying both of these needs. Generallvwhen respiration is stimulated [e.g. by dinitrophenolpoisoning (2), by inducing growth (4), or by wash-ing storage tissue (24)] that respiration is more sen-sitive to malonate indicating that the full TCA cycleis active under conditions of high metabolic activity.

The general shift in the distribution of the activityof pyruvate in the light is basically the same as thatinvoked by the simultaneous feeding of glucose andradioactive pyruvate: the contribution of pyruvateto sucrose is reduced while the synthetic reactionsderived more directly from the TCA cycle are stimu-lated. Yet the effect of either glucose or light on

320




the metabolism of pyruvate is not an interaction atthe substrate level alone, for in both cases new syn-thetic reactions are induced. The clue to the inabilityof pyruvate to support these reactions is seen in thelight-stimulated incorporation of activity into valine,leucine, and the fats, reactions which require re-duced TPN (26, 29). It seems reasonable that thereduced TPN needed for these new reactions in thelight is derived from the photosynthetic pools. Theoxidation of glucose by the pentose-phosphate path-way can also supply reduced TPN. Moreover, thissupply is probably more subtly controlled, as seen bythe increased activity of the pentosephosphate path-way on the addition of NH3 (7) or NO3 (5). If thereduced DPN formed by the TCA cycle were notreadily converted to reduced TPN, a shortage of re-duced TPN could prevent the synthesis of fats orof the secondary amino acids and hence of protein ina system limited to pyruvate respiration. The extracarbon energy and reduced DPN formed by the oxi-dation of pyruvate could be diverted to sugar syn-thesis.

In starved cells glucose induces an increased res-piration that cannot be further stimulated by dinitro-phenol (9), NO3 (10), or pyruvate, but its assimila-tion varies in each case. With dinitrophenol the po-tential energy is lost to the synthetic reactions (1,9,20); with pyruvate a greater part is spared for thesynthesis of polysaccharides. In a complementarytype of experiment with inhibitors (9), or withsubstrates which cannot support the synthetic re-actions, a greater part of the substrate is respiredrelative to the work performed. Lynen et al. (14)have shown that with dinitrophenol the mechanismcontrolling the degree to which a substrate is respir-ed is the level of ADP, which regulates the rate ofthe triose dehydrogenase reaction and hence the rateof glycolysis. The experiments described here withpyruvate and acetate indicate that a supply of dicar-boxylic acid which limits the drainage to the syn-thetic reactions or of a specific pyridine nucleotidemay also regulate the efficiency with which a sub-strate is utilized.

Summary

Glucose increases the incorporation of pyruvateinto the ninhydrin-positive fraction of the acid hy-drolysate (proteins), and light the incorporation intoboth proteins and fats. The interaction of succinatewith pyruvate or glucose, and of pyruvate with glu-cose, and the inhibition of glucose- or pyruvate-in-duced respiration by fluoroacetate indicate that thetricarboxylic acid cycle is the major pathway forglucose and pyruvate oxidation. The incorporationof pyruvate into the proteins or fats appears to belimited by the supply of reduced TPN. The metabo-lism of acetate is further curtailed by the rate of syn-thesis of dicarboxylic acids. The efficiency withwhich the energy produced by the oxidation of asubstrate is coupled to synthetic reactions (CI402/

algae C14) appears to be dependent on a balancedsupply of carbon skeletons, energy and specific pyri-dine nucleotides.

Acknowledgments

I should like to thank Prof. 0. Kandler for the use oflaboratory space and for stimulating discussions through-out this research; Prof. H. Beevers for criticism of themanuscript, and the Deutschen Forschungsgemeindschaftfor its financial assistance.

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Influence of Glucose Light Pyruvate Metabolism · by Starved Cells of Chlorella elipsoida 1 2 Ann Oaks3 Bacteriological Institute of the South German Dairy Research & Experiment Station,