THE ACTION OF STREPTOMYCIN

VI. A NEW METABOLIC INTERMEDIATEW. W. UMBREIT

Merck Institute for Therapeutic Research, Rahway, New Jersey

Received for publication February 2, 1953

In attempting to explain the results obtainedwhen certain strains of Escherichia coli weretreated with streptomycin, a reaction betweenpyruvate and oxalacetate was proposed as thesensitive reaction. The action of streptomycin onthis reaction was regarded as being related toits action in inhibiting the organism since onlyantibiotically active forms of streptomycin wereinhibitory and complete inhibition was obtainedat concentrations comparable to those requiredto inhibit growth (Umbreit, 1949; Oginsky et al.,1949). The nature of the "oxalacetate-pyruvate"reaction has remained obscure since furtherstudies (Umbreit et al., 1951) have shown that itwas not identical with any of the known reactionsof these compounds. Since the conversion ofpyruvate to active acetyl and the condensationof this with oxalacetate to form citrate havebeen studied intensively, and since this reactionseries would seem to account for the oxalacetate-pyruvate effect, it was quite unlikely that a newreaction would be found. However, we wereunable to demonstrate any effect of streptomycinon the known reactions of pyruvate or oxalacetate(Umbreit et al., 1951; Umbreit, 1952a,b) even atconcentrations of streptomycin far above thoserequired to inhibit growth. To the best of ourknowledge no one else has been able to demon-strate inhibition of the known metabolic reactionsof these compounds. One paper (Barkulis, 1951)reports streptomycin inhibition of pyruvate me-tabolism under anaerobic conditions, but thedata available do not specify the reaction in-volved. Since the effect is maximal in the presenceof bicarbonate, and since it may be shown thatthe reactions pyruvate to acetate plus formateand formate to hydrogen plus carbon dioxideare not sensitive to streptomycin per se, we havesupposed that these studies pointed to a differentway to measure the same reaction and were areflection of streptomycin inhibition of the oxal-acetate-pyruvate reaction.

If streptomycin did not inhibit the known reac-tions, what was the nature of the unknown reac-tion? However, the experimental problem wasnot easily approachable because of two verylarge difficulties. First, it proved most difficultto obtain any kind of preparation which wouldcontain a streptomycin sensitive reaction meas-urable by the rather indirect methods it wasnecessary to employ (either the oxidation systemof Oginsky et al., 1949, or the anaerobic system ofBarkulis, 1951). There were indications that thiseventually would be possible since a few activepreparations were obtained (Umbreit, 1949), butas an experimental tool this type of approachwas, and still is, impossible. Preparations couldbe obtained which were capable of carrying outthe known reactions of pyruvate and oxalacetate,but these reactions were not inhibited by strepto-mycin. It appeared that the streptomycin sensi-tive reaction was dependent largely upon theintact cell which would suggest either that thecell was conducting an additional reaction(s) ofsome quantitative significance or that the actionof streptomycin had something to do with the"intactness" of the cell. Second, there was thelack of any way to measure the sensitive reactionexcept its inhibition by streptomycin. Underthese circumstances the only way one could iden-tify the sensitive reaction was to stop it withstreptomycin so that a search for end productswas fruitless. It seemed probable that one wasdealing with an active intermediate, probablypresent in small amounts, for which one had nomethod of measurement except to prevent eitherits formation or metabolism with streptomycin.Always, however, when streptomycin showed

inhibition, the presence of both pyruvate and afour-carbon dicarboxy acid was required; thelatter either added or the conditions requiredwere such as to permit its formation. Attentionwas turned therefore to the possibility that aseven-carbon intermediate played an important

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ACTION OF STREPTOMYCIN

role in metabolism; a possibility that had neverquite been eliminated but for which there wasno positive evidence. Studies of the oxalocitra-malic acid of Martius (1943) or of the dihydro-oxalocitramalic acid as possible intermediatesproved negative. Similarly the metabolic proper-ties of several other seven-carbon compounds(including shikimic acid and its relatives)' couldnot be related to streptomycin inhibition. Ofcourse, other seven-carbon intermediates of meta-bolic significance might exist. We therefore turnedour attention to the seven-carbon phosphorylatedcompound, isolated by Rapoport and Wagner(1951) from dog liver, whose structure was in-dicated as 2-phospho4-hydroxy4-carboxy-adipicacid (illustrated in figure 1) which we shall callthe new compound in the remainder of this paper.Because of the content of phosphorus in the newcompound it was possible to trace its formation.Three problems were especially pertinent. First,how may one isolate and determine this com-pound? Second, is it an active metabolic inter-mediate? Third, is it related to the action ofstreptomycin? It is the purpose of this paper toprovide the experimental answers to these prob-lems.

MATERIALS AND METHODS

Chemical methods. Procedures for the isolationand characterization of the compound are out-lined in the first section. One point is of consider-able importance when dealing with compoundsof this structure and requires some modificationof technique. The usual method of determiningradioactivity of inorganic phosphate in the pres-ence of organic phosphate is to precipitate theformer with magnesium. It is assumed generallythat very little organic phosphorus is carrieddown in the precipitate especially during a re-precipitation. However, because the new com-pound is highly acidic and readily adsorbable, webecame suspicious of this procedure. It wasfound by experiment that while 15 per cent ofthe phosphorus found in the first magnesiumprecipitate was organic rather than inorganicwhen prepared from the original trichloraceticacid extract of liver (11 per cent from esterphosphorus fraction), some 78 per cent of the

1We are greatly indebted to Doctors E. F.Rogers and K. Pfister of the Research and De-velopment Laboratories, Merck and Company,Inc., for an array of these compounds.

phosphorus was organic in magnesium precipi-tates from concentrates of the new compound(85 per cent was organic in magnesium precipi-tates from nucleic acid fractions). Earlier workmight well have discarded a great share of thiscompound in the removal of inorganic phosphatewhen magnesium was employed. To circumventthis difficulty we used the method of Ennor andRosenberg (1952) which consists of extractingthe reduced orthophosphate-molybdate complexwith isobutanol. The extract separates the in-organic (ortho) phosphate, which then may beread colorimetrically and plated directly for themeasurement of radioactivity. However, in somecases we have found extraction of the organicphosphate into the isobutanol layer. In thesecases we applied the method of Ennor and Rosen-berg to magnesium precipitates of inorganic phos-

COOH

HC-O-POH2

CH2

H0-C-COOH

CH2

COOH

Figure 1. Formula for the new phosphoruscompound.

phate, especially in fractions containing the newcompound. The remainder of the techniquesemployed are essentially standard (Umbreit et al.,1949). Radioactivity was determined by use of athin window Geiger-MUiller counter. All sampleswere counted to an accuracy of a5 per cent, andthe appropriate corrections were made for decay,background, and coincidence. All of the experi-ments employing pn were done with the activecooperation of Dr. I. Clark, without whose facili-ties and advice the experiments would not havebeen possible.

RESULTS

Isolation and determination of the new compound.The compound isolated by Rapoport and Wagner(1951) from dog liver had the formula C7Hi1OnPand had properties corresponding to the formulashown in figure 1. This material was discoveredoriginally (Rapoport and Wagner, 1947; Rapo-

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port and Nelson, 1945) as a nitrogen-free acid materials into substances whose barium salts areresistant phosphorus contaminant of adenosine soluble. The mercury is removed and the newtriphosphate prepared by the usual barium and compound precipitated, essentially pure, withmercury precipitation methods prevalent at the barium acetate (pH 8.2). The inorganic phosphatetime. These properties provided a clue for a was separated for counting by the isobutanolmethod of isolation which is used as a method of extraction procedure described, and the total

Tissue, extracted, cold 10% trichloracetic acid

II

Suspend-0.1 N HClHeat-10 min boilingwater bath-adjust to pH 4Chill

Suspend in waterH2S ---+ HgS

II(HgS) + BaCl2 to pH 8.2

Chill

I- (When animal liver is used,the glycogen is first removed

extract by the addition of 1 volumeto pH 2-S alcohol at this point.)+ 0.05% by volumemercury reagent (2 gm HgAc2, in

I 10 ml 1% acetic acid)

HH2S ---+ HgS

+ barium acetate+ 4 volumes alcohol+ NaOH to pH 8.2Chill

IEster phosphate fractionRemove bariumMake to volume

II

Other P.Remove bariumMake to volume

IIIH2SO4 -4 BaSO4,

Frl Neutralize, make to volumeWash Determine inorganic and total phosphorus

I New compound

11(BaSO)

Figure S. Separation and determination of new compound.

determination. The method, diagrammed in fig-ure 2, is paraphrased as follows:The new compound forms an insoluble mercury

salt and an insoluble barium salt. The trichlor-acetic acid extract at pH 2 to 4 is treated withmercuric acetate. This precipitates coenzymes,adenosine triphosphate, adenosine diphosphate,adenylic acid, and the new compound. Thisprecipitate is treated with 0.1 N HCl at 100 Cfor 10 minutes which converts all of the other

counts were corrected for the counts due toinorganic phosphate before the organic phos-phorus activity was calculated.While there is no known phosphorylated com-

pound having these properties other than thenew compound illustrated in figure 1, additionalevidence of the purity and nature of the com-pound contained in the new compound fractionwas desirable. Ascending paper chromatography(in MeOH [80 vol], HCOOH [15 vol], and water

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[5 voll) of the fraction containing the new com-pound yielded two phosphorus containing spotsat R1 0.24 and R1 0.42, the last of which wasinorganic phosphate. When radioactive sampleswere chromatographed similarly, the radioactiv-ity was asociated with the same two spots. Theorganic phosphorus in this fraction thus appearsto be a single compound. From its method ofpreparation its mercury and barium salts areinsoluble. The phosphorus is bound in an acidresistant form, the hydrolysis curves resemblingthose of phosphoglyceric acid, which compoundis not present in the fractions. The fractions donot give the pentabrom acetone reaction forcitrate but give a color with Nessler's solutionwith a peak absorption in the region of 380 my.Inasmuch as all of these are properties associatedwith the compound isolated by Rapoport andWagner (1947, 1951), we assume that we aredealing with the same material.

It is obvious that the fraction contains organicphosphorus. It comprises between 0.05 and 0.10per cent of the organic phosphorus extractablefrom animal (and bacterial) cells with cold 10per cent trichloracetic acid and has at least someof the properties ascribed to it by Rapoport andWagner (1951). Since there is no question butthat a compound of this sort exists, the nextproblem is whether or not it is an importantmetabolic intermediate.

The activity of the new compound in vivo. Sincewe have no real knowledge of the immediateprecursors from which this compound is derived,the turnover of this compound in rat liver wastraced by way of radioactive phosphorus, ratherthan carbon. The animal provided a useful toolfor this study since the compound was known toexist in liEver and the streptomycin sensitive reac-tion has been demonstrated in animal tissue(Umbreit and Tonhazy, 1949). These studies arepresented here since they offer information, notyet obtainable on the bacteria, which serves tosupplement the bacterial studies reported later.The data apply to the phosphorus turnover onlyand tell one nothing about the carbon. One maysuppose either that the carbon of the compoundis turning over actively as well as the phosphorusor that the carbon is inert and the phosphorusis exchanged on it. There is no evidence yetavailable which will distinguish between thesepossibilities. The experiments were done by in-jecting rats with radioactive phosphorus and at

some period thereafter killing the animal andgrinding the liver in cold 10 per cent trichlor-acetic acid. (The liver from three rats usually suf-ficed for one analysis.) The trichloracetic acidextract was fractionated, and specific activity ofthe fractions compared to that of the totalextract (relative specific activity) was taken asevidence of turnover of the phosphorus in thecompound.

4.0

R.S&A

20

I.0l

3.0

RSA20

1.0

IES so

NEW *t

NEW _

COMPOUND

2462 4 6HOURS

ESTER P

46 I _ I I I

1.0

Sim=

NM CO_1 __

246

.OURS2 4 6

MEWObOUW

1 2 3 4 5 6

DAYS AFTER INJECTIONP?

Figure S. The accumulation of radioactive phos-phorus in the phosphorus compounds of rat liver.Pn injected at zero time. R.S.A. - relative specific

actvt. specific activity in compoundsactivityspecific activity of T.C.A. extract

Ester phosphate and new compound as defined intext.

Data from three separate experiments are givenin figure 3. Figure 3A shows the development ofthe relative specific activity in the ester fractionand in the new compound over the course ofseveral days. It is apparent that the phosphorusof the new compound accumulates more slowlythan in the ester fraction. Figures 3B and 3Cshow the results of relatively short term experi-ments in animals fed ad lib and starved (for 24hours prior to the injection of the radioactivephosphorus and during the 6 hour interval of

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W. W. UMBREIT

the experiment). The livers of the animals infigure 3C were almost devoid of glycogen, andaccumulation of radioactivity in the compoundsis relatively slower. The data here assembledare sufficient to establish that the new compoundis an active metabolic intermediate in the animal,

TABLE 1Phosphorus distribution and activity in

Escherichia coliCells of Escherichia coli, 120 ml (containing

6.24 g wet weight cells) + 2.5 ml p32 (ca 100MC P32) + 7.5 ml K%HPO containing 1 mg per mlas carrier. One lot received 12.0 ml water, theother 11.8 ml water + 0.2 ml streptomycin HC1solution containing 100 mg per ml of free base(amount added 20,000 jg = 100 jsg per ml finalconcentration). This was incubated for 30 minutesto allow phosphate and streptomycin to comeinto equilibrium with cells. At zero time, 41.6ml 0.05 M pyruvate (pH 7.4) and 16.6 ml 0.5 Mfumarate were added to each flask. The contentswere shaken in air, 30 C for 3 hours, stopped bythe addition of 50 ml of 10 per cent trichloraceticacid and fractionated.

WITHOUT WITHBTRHPTOHY~N pSTREPTOHYCIN

Inorganic P, as % oftotal ............... 17.3 17.8

Organic P, as % oftotal ............... 82.7 82.2Fractionation of organic phosphorus-as %

total organicFound in medium..... 40.2 40.5In trichloracetic acidExtract .......... 12.1 12.8New compound ... 0.089 (1.65) 0.11 (0.24)Ester. 7.0 (2.6) 6.6 (1.4)Adenylic acid....... 1.27 (0.25) 0.7 (0.20)Other.......... 2.6 2.1

Nucleic acid.......... 16.8 (0.13) 14.3 (0.17)Cell residue.......... 31.2 32.5

Figures in parentheses are specific activity offractions.

which is the question these experiments weredesigned to determine.While the new compound is involved in the

metabolism of the animal, as evidenced by theincorporation of radioactive phosphorus into it,the data presented here indicate that this incor-poration is slower than that into the ester frac-

tion, and that, therefore, the material is not asactive a part of metabolism as the phosphorylatedesters. We have had two experiments of shortterm duration in which the incorporation ofphosphorus into the new compound was fasterthan incorporation into the ester phosphate. Wetherefore feel that there are unknown conditionsin which the new compound may play a moreactive part in metabolism, but at present we arenot in a position to specify what these conditionsmay be.

Relation of the new compound to streptomycininhibiton. After some preliminary studies whichindicated that the same kind of compound couldbe obtained from bacterial cells, attempts weremade to determine whether this compound wasrelated to streptomycin action. Accordingly, ex-periments were done with a streptomycin sensi-tive strain of E. coli. Considerable quantities ofcells were needed for these experiments whichmade it difficult to obtain sufficient numbers inthe most sensitive state. A compromise was madetherefore; the cells were grown on 1 per centtryptone, 1 per cent yeast extract, 0.5 per centK2HPO4 medium with 1 per cent glucose (ratherthan 0.1 per cent) and the streptomycin concen-tration was increased to 20 to 100 ,ug per ml(the strain is sensitive to 10 ,ug per ml).

Details of an experiment are given in table 1,from which it is apparent that there is no signifi-cant difference in the amount of phosphorus ap-pearing as inorganic or organic forms in the me-dium, cells, trichloracetic acid extracts, or nucleicacids due to the presence of streptomycin, i.e.,the antibiotic is not permitting phosphorus toleak out of the cell or altering in any major wayany of the other fractions. The trichloraceticacid extract would contain presumably the meta-bolically active intermediates. This was separatedtherefore into fractions containing the new com-pound, the adenylic acid derived from adenosinetriphosphate, the phosphorus associated with thecompounds of the Meyerhof-Embden system("ester phosphorus"), and a fraction containingunknown materials (barium soluble, alcohol sol-uble). From the amount of phosphorus in eachfraction there is again no major change due tothe presence of streptomycin. The new compoundis present in streptomycin treated cells (in factit seems to be somewhat higher than in untreatedcells). However, presence is not evidence that ithas been formed during the incubation period.

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For this kind of evidence one must examine theradioactivity of these fractions, data on which aregiven in parentheses. It will be noted that thespecific activity of the new compound fraction ismuch lower in the presence of streptomycin thanthat found in the untreated sample, to such anextent, indeed, that there has been an 86 per centinhibition of the incorporation of radioactivephosphorus into it.There is also a lowered specific activity in the

ester phosphorus fraction (amounting to 40 percent inhibition of phosphorus incorporation).

TABLE 2Formation of the new compound by

Escherichia coliNineteen ml of a suspension of Escherichia coli

containing 13.4 mg dry weight of cells per ml(2.55 g dry weight of cells) at pH 7, supplied toeach flask below:

Streptomycin, 10 mg/ml(final concentration20 pg/ml)

After 30 minutes, add 10P" (in ml)

After 10 minutes, add 40H,O (in ml)

0.2 M pyruvate-pH 7 -(in ml)

0.5 M fumarate-pH 7(in ml)

2 3

.10.11 -

4

0.1

S 6

0.1

) lo l olholo140120

20

201 3 3

20 120 120

-1 -1 - -117 117

Shaken in air 30 minutes. Reaction stopped,trichloracetic acid, new compound fractionisolated.

New compound-organic 112 61 17.8 1.79.914.0P found, pg

Specific activity 0 0 0 0 3.51O

There is evident no inhibition of the incorporationof phosphorus into the adenylic acid (derivedfrom adenosine triphosphate) or into the nucleicacid.A further experiment estimated the incorpora-

tion of phosphorus into the new compound in theshort interval of 30 minutes. The details of theexperiment and the results are given in table 2.It is first noted that there is much more of thenew compound in cells without substrate than inthose in the presence of substrate. This is notnewly formed compound since it contains noradioactivity. In the presence of pyruvate (Smith

et al., 1949; Umbreit and Tonhazy, 1949) thismaterial apparently decreases in amount, but nonew compound is synthesized since no radioactivephosphorus appears in it. In the presence ofpyruvate plus a dicarboxy acid (fumarate) thereis a similar decrease in the amount of the com-pound as is seen with pyruvate alone, but thepresence of the fumarate now has permitted theincorporation of radioactive phosphorus into thecompound. Streptomycin has inhibited this in-corporation.

In order to obtain the quantities of cells re-quired for the analytical experiments, air grownE. coli cells were employed. These cells possess asystem capable of oxidizing acetate which is not

TABLE 3Formation of the new compound by Escherichia

coli, air grown cells

SPCIFICACTIV-

SUBSTATE ADDED STREPTOtMYCICOM'-POUND

None.........0 3.9Pyruvate (1mm)..... 0 3.8Pyruvate (1 mM) ... 505g/ml 3.3Pyruvate (1 mM) +Oxalacetate (2 mm) 0 17.3

Pyruvate (1 mm) +Oxalacetate (2 mM). 50 sg/mI 5.0

Pyruvate (1 mm) +Oxalacetate (2 mm). 25 pg/ml 1.7

Pyruvate (1 mM) +Oxalacetate (2 mM). 50pg/ml (2 hours) 3.0

sensitive to streptomycin, and the effect of strep-tomycin on such cells is not demonstrable readilyin manometric experiments (cf Umbreit et al.,1951). Aliquots of such cells containing 5.25 dryweight of cells were incubated for 30 minuteswith 50 ,uC p" contained in 0.01 M phosphatein order to promote equilibrium between externaland internal phosphate. Streptomycin was sup-plied at several concentrations as indicated (table3), and the systems were incubated for another30 minutes to allow the streptomycin to penetrate.At this point pyruvate or pyruvate + oxalacetatewas added, and the suspension was incubated inair with shaking for 60 minutes when the systemswere stopped by the addition of sufficient tri-chloracetic acid to render the final concentration

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10 per cent. The extraction was carried out atroom temperature for 3 days, the fraction con-taining the new compound isolated from thetrichloracetic acid extract, and the specificactivity of the organic phosphorus determined.The data obtained are given in table 3. Appar-ently during the hour of incubation before thesubstrates were added, some radioactive phos-phorus was incorporated into the new compoundresulting in a specific activity of 3 to 4. Subse-quent incubation with pyruvate (with or withoutstreptomycin) did not alter this activity signifi-cantly. Incubation with oxalacetate and pyruvate,however, increased the specific activity aboutfourfold. In the presence of streptomycin thesesubstrates did not permit the increased radio-activity. The streptomycin was not merely slow-ing the rate at which radioactivity was incorpo-rated into this fraction since incubation for anadditional hour (see table 3) with streptomycinresulted in no increase in activity. The specificactivities of the streptomycin containing samples(varying from 1.7 to 5.0) are not significantlydifferent, largely due to the relatively smallamount of phosphorus found with these samples(of the order of 25 ,ug) which make precise esti-mation of the specific activity difficult.Using the same cellular suspension, but in one-

third the amount, a series of isolations was madeafter varied incubation periods with oxalacetateand pyruvate. The precision of measurement wassomewhat less in the data of table 3, but theactivity of the fraction containing the new com-pound rose steadily reaching its peak at 90 min-utes, and thereafter losing activity so that by 5hours it was virtually the same as the endogenous.The amount of the compound per cell had atendency to drop during the rapid oxidationperiod and to return to the endogenous values(or above) when oxidation slowed.

DISCUSSION

It seems apparent from the data of theseexperiments that the compound is formed whenpyruvate and fumarate or oxalacetate are presentand is not formed when only pyruvate is supplied.However, it is also apparent that the retentionor breakdown of the compound within the cell isinfluenced by the presence of both external sub-strate and streptomycin so that the situation isfar from simple. We therefore feel that it is safe

only to conclude that streptomycin inhibits theformation of the new compound, but that it isnot apparent what role the new compound playsin bacterial or animal metabolism. At least twopossibilities are available from present data. First,the compound might be the actual intermediateentering the terminal respiration system, com-parable to citrate in the Krebs cycle. Second, thecompound might be a "coenzyme" whose presenceis necessary for other reactions to proceed. Agreat deal of study will be required before therole of this compound in metabolism can beevaluated. At present it is of interest that suchan intermediate exists and that its formation isinhibited by streptomycin.Some attempts have been made to isolate the

compound in sufficient quantity to perform meta-bolic experiments with it. It can be isolated read-ily from rat liver, but the small quantity present(approximately 0.3 Mg P per g wet weight) pre-cludes this source as a starting material. Driedhorse liver and fresh beef liver also contain thecompound, but fractions containing it are con-taminated by a brown waxy material which inter-feres with metabolism and which we do not knowyet how to remove.

SUMMRY

A seven-carbon phosphorylated compound,2-phospho4-hydroxy4-carboxy-adipic acid, isshown to be a metabolic intermediate as deter-mined by the incorporation of radioactive phos-phorus. In Escherichia coli it is formed on theaddition of pyruvate and oxalacetate or fumarate,and its formation is inhibited markedly by strep-tomycin.

REFERENCES

BARUuus, I. L. 1951 Inhibition of the anaero-bic pyruvate dissimilation in Escherichia coliby dihydrostreptomycin. J. Bact., 61, 375-376.

ENNOR, A. H., AND ROSENBERG, H. 1952 Ob-servations on the determination of the specificactivity of the inorganic phosphate fractionof TCA extracts of liver. Biochem. J., 50,524-530.

MARTIUS, C. 1943 Der mechanismus der citro-nensAurebildung im Tierk6rper im susam-menhang mit dem abbau der Brentstrau-bensAure. Z. physiol. Chem., 279, 96-104.

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OGINSKY, E. L., SMITH, P. H., AND UMBRBIT, W.W. 1949 The action of streptomycin.I. The nature of the reaction inhibited.J. Bact., 58, 747-759.

RAPOPORT, S., AND NZLSON, N. 1945 Isolationof acid soluble nucleotides of liver. J. Biol.Chem., 161, 421-427.

RAPOPORT, S., AND WAGNER, R. H. 1947 Isola-tion of a-phospho-trihydroxyglutaric acidfrom dog liver. J. Biol. Chem., 167, 621-622.

RAPOPORT, S., AND WAGNER, R. H. 1951 Aphosphate ester of a tricarboxylic acid inliver. Nature, 168, 295-296.

SlOTH, P. H., OGINBSKY, E. L., AND UMBREIT, W.W. 1949 The action of streptomycin. II.The metabolic properties of resistant and de-pendent strains. J. Bact., 58, 761-767.

UMBREIT, W. W. 1949 A site of action of strep-tomycin. J. Biol. Chem., 177, 703-714.

UMBREIT, W. W. 1952a The mode of action ofstreptomycin. Proc. 2nd Intern. Congr. Bio-chem., in press.

UJBRsIT, W. W. 1952b Respiratory cycles. J.Cellular Comp. Physiol., 41, Supplement 1,March 1953.

UJBiIT, W. W., AND TONHAZY, N. E. 1949The action of streptomycin. III. The actionof streptomycin in tissue homogenates. J.Bact., 58, 769-776.

UMBEIT, W. W., Buzus, R. H., AND STAUFFEB,J. F. 1949 Manometric techniques and re-

lated methods for the study of tissue metabolism.Second Edition. Burgess Publishing Co.,Minneapolis, Minn.

UMBIEIT, W. W., SMTH, P. H., AND OGINSKY, E.L. 1951 The action of streptomycin. V.The formation of citrate. J. Bact., 61, 595-

604.

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