J. Biol. Chem.-1958-Dajani-913-24

8/3/2019 J. Biol. Chem.-1958-Dajani-913-24

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A STUDY OF THE CITRIC ACID CYCLEIN ERYTHROCYTES*

BY RASHID M. DAJANI AND JAMES M. ORTEN

(From the Department of Phyeiological Chemistry, Wayne State Uniuereity College ofMedicine, Detroit, Michigan)

(Received for publication, October 31, 1957)

It has been known for some ime that the rate of respiration in nucleatederythrocytes differs from that in non-nucleated red cells. Warburg (1)in 1909 and later Michaelis and Salomon (2), in 1930, demonstrated thatmammalian red cells have a relatively high rate of aerobic and anaerobicglycolysis but a low rate of respiration. On the other hand, nucleatedavian red cells have low aerobic and high anaerobic glycolysis with a highrate of respiration. Until recently, t,he pathway of oxidation in thesecells was almost obscure. Evidence based on enzymatic studies which

point to the possible existence of the citric acid cycle in avian red ceilsand to its absence from mammalian erythrocytes has been obtained inseveral other laboratories (3-5). However, the actual oxidative pathwaysin these cells have not as yet been conclusively demonstrated. More-over, the citric acid cycle has been implicated in the biosynthesis of por-phyrins and perhaps other substances n nucleated erythrocytes by way ofthe Shemin succinate-glycine cycle (6). It is apparent, therefore,that additional evidence is needed to determine whether a cycle in its

classical or a modified form is operative in erythrocytes. For this purpose,the present study of the separation, identification, and measurement ofthe individual acids of the cycle after incubating erythrocytes with labeledprecursors was undertaken.

EXPERIMENTAL

Determination of Citric Acid Cycle in Erythrocytes

Preparation of Erythrocyte Extract-200 to 250 ml. of fresh pooled blood

were obtained from the jugular vein of white Plymouth Rock chickens.The blood was received in a flask containing a solution of 25 mg. of heparinin 5 ml. of 0.9 per cent saline, in which 2 to 4 mg. each of penicillin and

* The data in this paper are taken from a dissertation submitted to the GraduateSchool of Wayne State University by Rashid M. Dajani, in partial fulfilment of therequirements for the degree Doctor of Philosophy, June, 1957. Supported by re-search grant No. CY-2144 from the National Institutes of Health. A preliminaryreport was presented before the American Society of Biological Chemists at Chicago,April, 1957.

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R. M. DAJASI ASD J. M. ORTEN 915

Column Chromatography-The procedure used in this study is based

upon several methods described in the literature (U-15), and modified insuch a way so as to give a higher degree of accuracy, sensitivity, and repro-ducibility.

The column was prepared by mixing thoroughly 5.5 ml. of 0.05 K H&JO4with 9 gm. of silicic acid (Mallinckrodt analytical reagent grade, 100mesh. Only that portion which passed through a 100 mesh but wasretained by a 150 mesh sieve was used. The powder was dried in vacuaat 100 before use). The hydrated silicic acid was made into a suspension

TABLE I

Per Cent Recoveries of Organic Acids from Mixtures of Pure Solution s andfrom Ac ids Added to Erythrocyte Susp ension s

Acid

Acetic.Lactic.

PyruvicCitric ,Aconitict.

Isocitric.a-Ketoglutaric.Succinic .

Fumaric..........Malic,Oxalacetic.

&Hydroxybutyric

.

Pure solutions

-_-

Mean--

99.797.5

97.0loo.8

97.0

95.895.899.8

98.399.370.0

95.5

-

---

Range Mean

85-102.0 44.09G101.5 94.8

92- 98.5 91.399-102.5 100.095- 99.0 95.8

93- 98.5 98.389- 98.0 95.497-101.0 99.5

96-W. 0 95.598-100.5 98.865- 85.0 73.5

93- 98.0 96.8

Acids added toerythrocytes

Range

35.0- 55.087.0- 99.0

89.0- 94.099.0-101.593.0- 98.0

94.0-100.090.0- 97.097.0-100.5

92.0- 98.097.0-100.066.0- 82.0

92.0- 98.0

* Average of six to twelve determinations.t The sum of cis and trans forms.

with 60 ml. of water-saturated chloroform and poured into a 12 X 360mm. glass chromatographic column to which was attached an inter-changeable sintered glass disk of medium porosity. The silicic acid waspacked with air pressure to a final height of 25 cm. A 2 ml. aliquot ofthe sample prepared from pure acids or from extracts of tissues in 20 percent tert-amyl alcohol-chloroform was transferred to the column. Thesample was then forced into the silica gel with air pressure and followedby 2 ml. of chloroform saturated with water so as to rinse the adheringacids from the sides of the tube.

The organic acids were eluted from the column with water-saturatedchloroform until ten fractions were collected and then by a gradually


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916 CITRIC ACID CYCLE IN ERYTHROCYTES

and automatically increasing concentration of the tert-amyl alcohol-

chloroform influent. The apparatus employed for this purpose permittedthe introduction of 45 per cent tert-amyl alcohol-chloroform continuouslyunder 15 mm. (Hg) pressure of Nz into a second flask containing chloro-form covered with distilled water provided to keep the system saturatedwith water. The alcohol and chloroform were mixed by a magneticstirrer before entering the column. 200 fractions of 180 drops each (4.5ml.) were collected under 2 ml. of Hz0 in every run in an automatic frac-tion collector. These fractions were titrated with a 0.0200 N NaOH by

means of a Rehburg microburette with phenol red as an indicator.Recovery Studies-With the exception of oxalacetic acid, recoveries

of the organic acids from pure solution were nearly quantitative (Table I).Recoveries from erythrocyte preparations were likewise satisfactory exceptfor acetic acid, which, in addition to the labile oxalacetic acid, was lostin part during the process of evaporation. Oxalosuccinic acid was alsonot recovered completely even from pure solution because of its extremelability. The values for aconitic acid given in Table I represent the sumof the equilibrium mixture of cis and trans forms of the acid (16, 17).

Paper Chromatography-The identity of the acids obtained by columnchromatography was further confirmed by the paper chromatographicprocedure of Ladd and Nossal (18), with use of the acidic solvent in thedescending technique. Pure acids and mixtures of pure acids with thoseobtained from the erythrocyte preparations were chromatographed simul-taneously.

RESULTS AND DISCUSSION

Incorporation of Certain Unlabeled Substances in Cycle-The first stepwas the measurement of the levels of the preformed acids in nucleatedand non-nucleated erythrocytes after they had been processed as pre-viously outlined. In Table II the results of these experiments are given.Because small amounts of the cycle acids were found in nucleated erythro-cytes, the data suggest the presence of a metabolic mechanism or mech-anisms which could produce these acids. This is merely speculative att.his stage of the investigation, for it can be argued that the acids could

have gained entrance into the cells by diffusion from the organic acidpool in plasma. Yet negligible amounts are present in non-nucleatedcells. If a citric acid cycle in nucleated erythrocytes is the main proc-ess responsible for the accumulation of these substances, a study oftheir format,ion from appropriate substrates would undoubtedly castsome light on the problem.

For this purpose 1 mg. each of unlabeled acetate, citrate, cr-ketoglutarate,or malate as a substrate was incubated with erythrocytes. Data such


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R. M. DAJANI AND J. M. ORTEN 917

as those shown in Table III were obtained. Attempts to raise the level

of biosynthesized acids further by increasing the amounts of substratelo-fold were unsuccessful.

TABLE II

Average Concentration.? of Cycle Acid8 in Washed Erythrocytes Suspendedin Krebs-Ringer Phc8P- Ihate Buffer+

Acid Non-nucleated Nucleated--

Lactic .....................................Pyruvic ..................................Citric ....................................Aconitic ...................................Isocitric ...................................a-Ketoglutaric .............................Succinic ...................................Fumaric ...................................L-Malic ...................................

Oxalacetic .................................

mmole pa 1. x 10-r

3.05.0

:t

0.5t1.ot2.0

:

mmola 9.9 1. x lW8

4.015.0

1.ot0.9t0.95t2.55.0

12.02.5

4.0* 2.0 per cent suspension.t Amount too small to be determined accurately.

TABLE III

Average Concentrations in Millimoles ver Liter X 10-aof Cwle Acid8 in NucleatedEtythwxytes Incubated wiih Certain SUbEtTaie8 foT 4 Hours

Acid Control

Lactic. .......... ......... ...... 4.0Pyruvic .......................... 15.0Citric ............................ l.O*Aconitic (cis plus trans). .... .... . 0.9*Isocitric ........................ 0.9*a-Ketoglutaric ................... 2.5Succinic ........................ 5.0Fumaric ....................... 12.5Malic ............................ 2.5Oxalacetic ..................... 4.0

-7

Xric acid,

1.0 mg.

6.5 6.240.5 41.678.0 28.7

1.8 7.01.9 1.9

18.0 16.349.6 51.058.0 56.536.5 35.730.0 28.8

* Amount too small to be determined accurately.

-

-

-

r-ketoglu-taric acid,

1.0 mg.dalic acid,

1.0 mg.

5.9 5.839.2 40.628.7 28.28.0 1.81.9 1.9

136.5 17.849.5 50.059.0 58.036.7 140.029.7 29.6

A comparison between the amounts of cycle acids in nucleated erythro-cytes after incubation with the substrates (Table III) and those of thecontrol nucleated cells (Table II) show, with no exception, that the levels


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of a11 acids are higher in t,he former. It would appear, therefore, that

an active synt.hesis of the cycle intermediat,es does exist in chicken eryth-rocytes. This, indeed, is the first direct indication that the cycle isfunctioning in these cells. Others (5, 6) have reported t.hat most of theenzymes of the citric acid cycle are present in avian erythrocytes. How-ever, t,his may be considered indirect evidence for the existence of the cyclein these cells.

The data in Table III further indicate that citrate, a-ketoglutarat,e,and malate are well utilized by the cell for the biosynthesis of the rest of

the cycle intermediates. This is also t,rue of acetate which gave rise t,osimilar quant,ities of acids when employed as a substrate. It is not.e-worthy, however, that none of the cycle components appears to accumulatein high concentrations. This is to be expected if the cycle is operat,ivebecause the average lifetime of these components, at least in the kidneyand liver, was found to be of the order of a few seconds only (19).

Furthermore, the ratio of the amounts of citrate, aconitate, and iso-citrate is in close agreement with that reported by Krebs and Eggleston(16) for the three acids in a state of equilibrium. If we accept the datain Table III at face value, it follows that an active biosynthetic mechanismfor t.he cycle acids does occur in nucleated chicken erythrocytes under theconditions of the experiment.

Incorporation of Acctate-l-C14--For t,he confirmation of the funct,ion-ing of t,he cycle in chicken red cells, radioact.ive acetate was used. 0.45mg. of sodium acetate-l-c (a&ivity, 4.11 PC. per mmole) wasdifuted with4.0 mg. of non-labeled haAc.3Hz0, which served as a carrier. Aft,erincubation of the cells in the presence of this substrate in the usual way,the acids were extracted, and chromatographed, and the fractions collectedwere titrat.ed. The aqueous layer from each fraction was t,ransferred t,o acupped aluminum planchet and dried either spontaneously in air or underan infrared light.. Each planchet was measured for radioactivity in agas flow counter. The total amount of each acid and its specific activitywere then calculated. The results are shown in Table IV. The data forthe concentration and specific activity of the respective acids leave nodoubt that al l steps of t,he cycle can occur in nucleat,ed red cells. The

am0unt.s of each acid are markedly increased by the presence of bhe varioussubstrates over and above the corresponding values of the control. Also,because each acid exhibited a considerable amount of activity, this labelingis interpreted as due to actual biosynthesis from the substrate employedvia the several stages of t,he cycle. It, is to be not.ed, however, that thespecific activit.ies of t,he individual acids are different,. By way of com-parison, all of the dicarboxylic acids, with the exception of succinic, wereof similar specific activities. The specific activity of succinate was con-


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R. M. DAJASI AND J. 111. ORTEN 919

sistently about 20 per cent higher. In contrast to this, t.he tricarboxylic

acids exhibited much higher specific activities (about 60 per cent more),while pyruvic and lactic acids, as would be expected from labeling on 1carbon at,om only, contained about half as much.

Experiments with fluoroacetate have given results which are in harmonywith the view that citrate accumulates in the presence of this inhibitor(20), thus providing further evidence t,hat the cycle is functioning in

TABLE IV

Average Concentrations and Spe ciJic Activities ofCycle Acids in NucleatedErythrocytes fncuba ted with Aceta te-l-Cl4

Acid

Lactic. ....................Pyruvic. ....................Citric. ......................Aconitic (cis plus trans) . ,Isocitric ...................a-Ketoglutsric ..............Succinic ....................Fumaric ...................

Malic .......................Oxalacetic ................

rind per 1. x 10-a

Control

4.0 6.0 8.015.0 37.1 45.01.0* 28.5 28.50.9* 1.6 1.60.9% 1.9 1.62.5 16.5 16.05.0 48.9 49.7

12.5 50.0 51.0

2.5 35.0 39.54.0 22.5 28.0

Incubated with:

Lcetde(1) f

* Amount too small to be determined accurately. (1) NaAca3Hz0,4 mg.; NaAc-l-Cl4 (activity 4.11 mc. per mmole), 0.45mg.; (2) L-malic acid, 0.05 mg.; 3) fluoroace-tic acid, 0.5 mg.

Acetnte(1) +

malate

h%~etate(3)

10.357.049.5

1.1*1.1*3.6

48.549.5

39.0135.0t-~

C.p.m.per mm & x 106

ACl?&kAcetate t m& t e

19.219.7

490.0429.0420.0265.0332.0275.0

267.0254.0

19.7 19.419.4 18.6

470.0 462.0420.0 440.0430.0 435.0270.0 265.0332.0 260.0278.0 280.0

280.0 278.0280.0 290.0

t Mixed with acetoacetate.

nucleated erythrocytes. The results obtained are given in the fourth andlast columns of Table IV. Whereas the concentration of citrate is nearlydoubled, the amounts of aconitate and isocitrate decreased somewhat(although their levels are measured at the limit of accuracy of the techniqueemployed), and that of cu-ketoglutarate decreased remarkably. It maybe noted that the specific activity of citrate remained essentially unaltered,despite the increase in its amount. In contrast, there was no significantdecrease n the levels of succinic, fumaric, and malic acids, whereas thatof oxalacetic acid was nearly five times higher.


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Table IV also gives the amounts of the intermediate acids synthesized

when acetate alone was used as compared to those obtained when the cyclewas primed with small amounts of malate (50 7) or then blocked withfluoroacetate. It will be noted that priming by the addition of malate,under the conditions of the experiment, resulted in little additional changein the amounts of the cycle acids or their specific activities.

Because the early stages in the sequence of reactions in the cycle areblocked in the presence of fluoroacetate, one would expect the acids in thelater steps to diminish and finally to disappear entirely. That this is not

the case is evident from the data in Table IV. It seems that the inhibi-tion of aconitase was not complete since minute amount,s of aconitate,isocitrate, and cr-ketoglutarate have been found in excess of the control.Doubling the quantity of fluoroacetate did not bring about any furtherdecreases in the quantity of these acids. The effect of fluoroacetate onthe levels of the dicarboxylic acids is explained on different grounds, tobe discussed shortly.

Fractions collected from duplicate runs on the same extract were usedfor paper chromatographic studies and the identity of each acid was con-firmed by this technique. The acids, as obtained from the pooled frac-tions, were homogeneous on paper and did not show any contaminationwith other substances which would result in higher counts. This leftno doubt that all the activity obtained resided in the particular acid.

It must be stressed that the above isotope experiments do not supplyinformation on the quantitative aspects of the problem. Likewise, thedegree of incorporation as well as the location of the isotope need to beascertained, a question which has not been answered in this study. More-over, the data do not indicate how much of acetate has passed over thecycle, and leave open the problem of whether there are other pathwaysin nucleated red cells which could accomplish complete oxidation of acetate.

The accompanying Fig. 1 is a typical chromatogram which shows thelevels of the organic acids when acetate was used as a substrate. Imposedon it is another chromatogram obtained from the radioactive measure-ments of the same fractions of the first chromatogram. A graph for thecitrate fraction produced by fluoroacetate inhibition is also shown. It

may be noted that the relative activities obtained on each fraction cor-responded closely to the relative titration values.

Because the total amounts and specific activities of the succinate frac-tion were higher (Table IV) than expected, it was felt necessary to check thepurity of the labeled acetate sample for possible contamination with suc-cinate. To achieve this, a 1.0 mg. sample of the sodium acetate-l-U4was mixed with 4 mg. of unlabeled sodium acetate and 0.3 mg. of succinicacid as carriers and then dissolved in 5 ml. of water. The solution was

acidified with an equivalent amount of 0.05 N H2S04 and then extracted


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R. M. DAJANI AND J. M. ORTEN 921

as was done with erythrocytes. Upon counting, only very active acetatewas found in the fractions corresponding to acetate. No activity couldbe detected in the subsequent fractions which could account for the highactivity of the succinic acid obtained from nucleated erythrocyte in-cubates. The counts in these fractions were practically the same asthat of the background.

0.70

0.60

5 0.50

s

zi0 0.4c

::

0

O 0.3c%

2

0.2c

O.IC

06

14- Titrotion curve

-* Titration curve of ,:trote-isocitrote froclionsfluoroocetote

odded____ Cl4

activity curve,-- Cl4 activity curve, 1 *

l 12

TITR ATIO N AND Cl4 ACTIVITY CURVES IOOF CYCLE ACIDS FROM ERYTHROCYTE INCUBATES

0k

8

is

60 80 loo 120 140 160 II

FRACTION NUMBERFIG. 1. Titration and Cl4 activity curves of cycle acids from erythrocyte incu-

bates.

It may be noted in Tables III and IV that the concentrations of thedicarboxylic acids are proportionally much higher than those of the tri-carboxylic acids in incubated nucleated erythrocytes. The difference couldbe explained on the premise that a mechanism other than the citric acidcycle may also be operating in these cells, possibly a dicarboxylic acidcycle or the glyoxylate cycle recently described in certain microorgan-isms (21). The concept of the existence of such a mechanism alone oralong with a tricarboxylic acid cycle, at least in certain species, s not new.This is true, for example, of Pseudomonas KBl (21), Escherichia coli andacetate-grown Aerobacter aerogenes 22, 23), and Rhizopus nigricuns (!&I).

Accordingly, succinate could arise, in addition to the citric acid cycle


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reactions, from a Yail t,o tail condensation of two acet,ate molecules,i.e. a condensation at the methyl carbon, or from the glyoxylate cycle,This possibility would then account for the comparatively high level ofsuccinate found in the present work. Because malic and furnaric acidsare in equilibrium with succinic acid, this suggestion would also furnishan explanation for the increased quantities of these acids above expecta-tions from the activity of the cycle.

More significant results in this direct.ion a.re the findings obtained fromisotopic studies with acet,ate-l-C*. It is evident that succinst.e, among

the dicarboxylic acids, has t,he highest specific activity. This also can beaccounted for if the suggested tail to tail condensation mechanism foracetate or a similar one (21) is operative. Moreover, t,he specific activitiesof citrate, malate, and isocitrate are higher than the citric acid cycle wouldprovide. This means t,hat the oxidation of acetate cannot be confinedto the condensation of oxalacetate and acetyl coenzyme A to form citrat,e.A similar conclusion was reached by Weinhouse and Millington (25),who observed that the isotope content of the carboxyl groups formed by

yeast incubat.ed with acetate-l-C4 was higher than expected if al l of t.hecitrate were formed by way of the Krebs cycle. Clearly, much remainsto be done before this question is fuIly answered. One way of attackingthe problem, of course, would be to isolate t,he isotopic succinate synthe-sized in erythrocytes and degrade it. The amount and site of labelingin the molecule would undoubtedly be decisive in this respect.

The high level of oxalacetate in experiments with fluoroacetate aboveexpectation is merely due to contamination with greater amounts ofacetoacetic acid, which was identified by paper chromatography. Thisis in harmony with the view that fluoroacet,ate not only gives rise to ac-cumulation of citrate but also diverts acetate or pyruvate or both intochannels other than the citric acid cycle wit,h the formation of acetoacetateand probably of fatty acids (26-28).

Cycle in Arm-Nucleated ErythrocytesThe isotopic technique was furt.herextended t,o t,he st,udy of t,he cycle in non-nucleated red cells. Beef or dogblood was used in this experiment. The erythrocytes were separated asusual and suspended in Krebs-Ringer phosphate. The substrate was

identical t,o t,hat used with nucleated cells, and the conditions under whicht,he experiment, was carried were also the same. The am0unt.s of the acidseluted are recorded in Table T. The data show clearly the fact that thetricarboxylic acid cycle is almost entirely inactive in t,he non-nucleatederythrocyte as compared with the nucleated red cell. This is in agree-ment with the opinion of the earlier workers (29-431) that the cycle isincomplete in mammalian erythrocyt,es and is in accord with the findingsof Rubinstein and Denstedt (4) that the mature non-nucleated red cell

cannot utilize pyruvate. The present findings likewise indicate that t,he


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Acid

Lactic. ..........................Pyruvic ...........................Citric, ............................

Aco nitic (cis plus trans). ..........Isocitric .........................a-Ketoglutaric ....................Succinic ..........................Fumaric .........................Malic .............................Oxalacetic .......................

R. M. DAJASI AS11 J. M. ORTES 923

citric acid cycle may indeed serve as a source of succinat,e for the forma-

tion of porphyrins in nucleated erythrocytes by way of the succinate-glycine cycle (6).

TABLE V

Amounts and Spe cific Activities of Cycle Acids RiosynthesizedfromAcetate -l-Cl4 by Non-Nucleated Erythrocytes

Non-nucleated bovine erythrocytes

Control Cells incubated with substrate*

.I__~mmole per 1. x lo-= mm& pn 1. x lo--a c.p.?n. perm?nole x 106

3.0 0.45t 0.1t5.0 1.7 0.1t

:

t

f:

t t0.5t 1.4 0.5

1.ot2.0

2.12 ; 0.02.57 i 4.0

: :

* NaAcs3Hz0, 4.0 mg.; NaAc-l-Cl4 (4.11 mc. per mmole), 0.45 mg.; L-malic acid,

0.05 mg.t Amount,5 too sma ll to be determined accurately.

SUMMARY

A modified column chromatographic technique which employs hydratedsilicic acid and is adaptable for the complete separation of the citric acidcycle components and of certain other relat,ed acids from extracts of eryth-rocytes has been described. The efficiency and validity of the methodwere established by recovery studies of the acids from pure solutions aswell as from mixtures of the acids added to erythrocyte suspensions. Inconjunction, paper chromatography was employed as a confirmatory meansfor the characterization of each acid eluted from the column.

Effect,ive procedures for obtaining red cells free from leukocytes, for

incubation with va.rious substrates, and for extraction of the organic acidsfrom these preparations were presented.

The levels of t,he citric acid cycle members were shown to be very low inboth nucleated and non-nucleated washed erythrocytes, although theywere much lower in the non-nucleated washed erythrocytes.

The amounts of the acids of the cycle were increased substantially innucleated chicken red cells incubated with certain substrates. On theot.her hand, mammalian non-nucleated erythrocytes did not utilize these

substrates.


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Cl* from acetate-l-Cl* was incorporated by nucleated erythrocytes into

all of the acids of the cycle, although to varying degrees. Fluoroacetateincreased the quantity of citrate and acetoacetate from acetate and itlowered the levels of aconitate, isocitrate, and cY-ketoglutarate to a con-siderable extent.

Negligible amounts of acetate-l-P were incorporated into the cycleacids by non-nucleated red cells.

In view of the foregoing results, it is concluded that the citric acid cycleis active in nucleated chicken erythrocytes and is essentially inactive in

non-nucleated bovine or canine red cells.BIBLIOGRAPHY

1. Warburg, O., 2. physiol. Chem., 69, 112 (1999).2. Michaelis, L., and Salomon, K., J. Gen. Physiol., 13, 683 (1930).3. Ashwell, C., and Dische, 8., Biochim. et biophys. acta, 4, 276 (1950).4. Rubinstein, D., and Denstedt, 0. F., J. Biol. Chem., 204,623 (1953).5. Hunter, F. B., J. Cell. and Camp. Physiol., in press.6. Shemin, D., Russell, C. S., and Abramsky, T., J. Biol. Chem., 216, 613 (1955).7. Barron, E. S. G., and Harrop, G. A., Jr., J. Biol. Chem., 84, 89 (1929).8. Beck, W., and Valentine, W. H., Cancer Res., 13,309 (1953).9. Martin, S. P., McKinney, G. R., and Green, R., Ann. New York Acad. SC., 69,

996 (1955).10. Vallee, B. L., Hughes, W. L., Jr., and Gibson, J. G., Blood, 1, 82 (1947).11. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and

tissue metabolism, Minneapolis, 119 (1951).12. Marvel, C. S., and Rands, R. D., Jr., J. Am. Chem. Sot., 73,2642 (1950).13. Bulen, W. A., Varner, J. E., and Burell, R. C., Anal. Chem., 24, 187 (1952).14. Donaldson, K. O., Tulane, V. J., and Marshall, L. M., Anal. Chem., 24.185 (1952).15. Kinnory, D. S., Takeda, Y., and Greenberg, D. M., J. BioZ. Chem., 212,379 (1955).16. Krebs, H. A., and Eggleston, L. V., Biochem. J., 38, 426 (1944).17. Lees, H., and Kuyper, A. C., J. BioZ. Chem., 226, 641 (1957).18. Ladd, J. N., and Nossal, P. M., Australian J. Exp. BioZ. and Med. SC., 32, 523

(1954).19. Krebs, H. A., in Greenberg, D. M., Chemical pathways of metabolism, New York,

1, 109 (1954).20. Kalnitsky, G., and Barron, E. S. G., Arch. Biochem., 19, 75 (1948).21. Kornberg, H. L., and Krebs, H. A., Nature, 179, 988 (1957).22. Ajl, S. J., Bact. Reo., 16, 211 (1951).23. Ajl, S. J., and Kamen, M. D., J. BioZ. Chem., 189,845 (1951).24. Foster, J. W., Carson, S. F., Anthony, D. S., Davis, J. B., Jefferson, W. E., and

Long, M. V., Proc. Nat. Acad. SC., 36, 663 (1949).25. Weinhouse, S., and Millington, R. H., J. Am. Chem. Sot., 69, 3089 (1947).26. St&ten, D. W., Jr., and Boxer, C. E., J. BioZ. Chem., 166, 231 (1944).27. Crandall, D. I., Gurin, S., and Wilson, D. W., Federation Proc., 6, 246 (1947).28. Elliot, W. B., and Kalnitsky, G., J. BioZ. Chem., 136, 487 (1950).29. Quastel, J. H., and Wheatley, A. H. M., Biochem. J., 26, 117 (1931).30. Quastel, J. H., and Whealtey, A. H. M., Biochem. J., 32, 936 (1938).31. Nossal, P. M., Australian J. Ezp. Biol. and Med. SC., 26, 123 (1948).

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J. Biol. Chem.-1958-Dajani-913-24