(1950) Gamma-Aminobutyric Acid in Brain Its Formation From Glutamic Acid

8/8/2019 (1950) Gamma-Aminobutyric Acid in Brain Its Formation From Glutamic Acid

1/9

-r-AMINOBUTYRIC iiCID IN BRAIN: ITS FORMATION FROMGLUTAMIC ACID*

BY EUGENE ROBERTS AND SAM FRANKEL(From the Division of Cancer Research, Wash ington University Scho ol of Medicine,St. Louis)

(Received for publica tion, June 3, 1950)Relatively large quantities of an unidentified ninhydrin-reactive mate-

rial were found in numerous two-dimensional paper chromatograms of pro-tein-free extracts of fresh mouse, rat, rabbit, guinea pig, human, and frogbrains. At most, only traces of this material were found in a large num-ber of extracts of many other normal and neoplastic tissues and in urineand blood. In the present experiments and in the following paper (l),the unknown compound in brain extract is conclusively identified as y-aminobutyric acid. It is shown that this compound can be formed fromglutamic acid by homogenates, washed residues, and acetone powders ofbrain. A preliminary report of some of these findings has been made (2).

EXPERIMENTALChromatography.-One-dimensional and two-dimensional chromatogramswere made by the descending method as outlined by Consden et al. (3) and

extended by Dent (4, 5).Separation of Unknown Material-Mice were killed by dislocation of thecervical vertebrae, and the brains were removed immediately and frozenin dry ice. A pooled sample weighing 7.8 gm. was homogenized in 100ml. of 75 per cent alcohol. After centrifugation, the extract was evap-orated almost to dryness, taken up in 2.6 ml. of water, and the aqueousextract was centrifuged to remove insoluble material. This extract wasthen employed for chromatography.

In Fig. 1 is shown a tracing of a chromatogram obtained from an aliquotof extract corresponding to 75 mg. of fresh mouse brain. Intense spotswere given by aspartic acid, glutamic acid, glycine, glutamine, taurine,alanine, and the unknown material. Cystine, serine, and p-alanine werepresent in smaller amounts, and traces of valine, threonine, and glutathionewere noted. Two substances which disappeared on acid hydrolysis werelocated to the left of aspartic and glutamic acids on the chromatograms.Among the substances which have been studied chromatographically (5),the unknown material corresponded most closely in its behavior to histi-

*Aided by grants from the Charles F. Kettering Foundation and the UnitedStates Pu blic Health Service.

55

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/


2/9

$6 Y-AMINO BUTYFUC ACID IN BRAIKdine, methionine sulfoxide, and y-aminobutyric acid. Its position on thechromatogram was especially favorable for separation from most of theother detectable constituents by one-dimensional chromatography withphenol, thus making possible a more adequate comparison of its proper-ties with those of the aforementioned substances.

IO-+

FIG. 1 FIG. 2FIN. 1. Tracing of a chromatogram of a peroxide-treated aliquot of brain extractcorresponding to 75 mg. of fresh tissue. The letters identify the spots and the num-bers represent visual estimates of relative intensities of color, a rating of 1 beingtaken as the color given by 1 y of amino acid. The spots delineated by a brokenline reprcscnt traces. AZ, cystine (cystcic acid); L, asps&c acid; K, glutamic acid;J, swine; I, glycine; N, taurine; Q, glutamine; H, alanine; T, ,9-alanine; 8, threonine;F, valine; X, unknown substance; R, glutathionc; P and 0, unidentified materialswhich disappew on acid hydrolysis.FIG. 2. Chromatography of unknown (X) and r-aminobutyric acid (G) in (I) col-lidinc-lutidine, (2) phenol, (3) butanol.Ten spots of 10 Al., each corresponding to 30 mg. of fresh tissue, wereplaced along the narrow edge of each of four sheets of Whatman No. 1

filter paper and run in water-saturated phenol for 24 hours. The two endstrips of each sheet were cut out and sprayed with a 0.1 per cent solutionof ninhydrin in butanol. Six bands appeared in all instances. The un-known material was located in the band furthest from the starting point.The most likely contaminant would be the small quantity of valine shownin Fig. 1. With the two end strips as reference guides, the strips of paper

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


3/9

E. ROBERTS AND S. ??RANKEL 57containing the unknown material were cut out of the four chromatograms.ill1 ninhydrin-reactive material was removed from the strips by allowingwater to flow down t.hem into beakers. Xo color was given when thesestrips were sprayed subsequently with ninhydrin after drying. The eluatewas evaporated to dryness and taken up in 2 ml. of distilled water. Sub-sequent chroma,tography of aliquots of the eluate before and after hydrol-ysis with 6 N HCl showed the presence of a large quantity of the unknownsubstance, which was stable to hydrolysis, and traces of vsline and a pep-tide material.

IdentiJicaLion of Unknown Compound As y-Aminobutyric Acid-Micro-biological assay for hist.idine and a comparison of the chemical and chro-matographic propert.ies with those of histidine and mcthionine sulfoxideshowed that the unknown was neither of &se compounds.A known sample of -y-aminobutyric acid2 (15 y) and the unknown com-pound (52 ~1.) were then chromatographed separat,ely and in mixture inthree different. water-saturat,ed solvent systems: n-butanol, phenol, andcollidine-lutidine. The results are shown in Fig. 2. Although the R,values (3) arc widely different in the solvent systems employed, the mainconstituent of the unknown. mst,erial and the r-aminobutyric acid traveledat the same rate in each case and gave completel:r superimposable bandswhen mixed. It was therefore concluded that the unknown compound isy-aminobutyric acid. This conclusion was confirmed by application ofthe isotope derivat,ive method to the eluted material (1).Quantitative Determination of y-Aminobutyric Acid-Advantage wastaken of the fact that this amino acid could be separated from all buttraces of two of the ninhydrin-reactive materials found in brain by one-dimensional chromatography in phenol (see the previous discussion). Byusing smaller concentrations of brain extract the quantities of the inter-fering substances were reduced below a level at which positive reactionswith ninhydrin were given. Under these conditions the color given bythe y-aminobutyric acid could be used for quantitative determination.Spots corresponding to known amounts of r-aminobutyric acid and ac-curately measured amounts of unknown samples were placed along thenarrow edge of sheets of Whatman Xo. 1 filter paper and run in water-saturated phenol for 24 hours. Aft,er thorough removal of the phenol bydirecting a fan at the sheet,s or 18 to 24 hours, the sheets were sprayedon both sideswith a 0.1 per cent solution of ninhydrin in butanol. Maxi-mal color development took place in 24 to 36 hours at room temperature orin 30 minutes at 93. The developed spots were cut out, care being taken

1The microbiologicaldeterminationswere kindly performedby Dr. G. B. Rama-satma. The organism eucon ostoc mesenteroides P-60 was employed in the assay.* y-Aminobutyric acid waspurchasedrom the Department of Chemistry, Univor-eity of Illinois. It was ecrystallized from alcohol.

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


4/9

58 y-AMINOBIMYRIC ACID IN BRAINto include the same total area of paper in al l of the known and unknownsamples in any set of determinations. Suitably chosen paper blanks werealways included. The pieces of paper containing the spots were cut intosmall strips with minimal handling and were put into test-tubes. 5 ml. ofwater distilled in glass were then added and the tubes were shaken vigor-ously for aminute or two. Q uan i at t ive elutions of the color were achievedby this method. The paper fiber was then centrifuged and the color wasread at 570 rnp in the Beckman spectrophotometer. The optical densitywas proportional to the concentration in the range studied (1 to 15 7).The color was stable for at least 4 hours at room temperature when thetubes were kept out of strong light. Although the standard curves ob-tained at different times were in good agreement, a series of standards wasrun with each set of determinations in order to eliminate the influence ofpossible differences in paper and solvent on the development of the color.A similar procedure was employed in which the unknown and standardswere run on two-dimensional chromatograms in phenol and coll idine-luti-dine. Good checks were obtained when brain extracts were analyzed byboth the one-dimensional and two-dimensional methods. Three experi-mental samples of brain homogenates gave total values of 149, 202, and278 y of y-aminobutyric.acid by the one-dimensional procedure, whilevalues of 156, 200, and 295 y, respectively, were obtained for the samesamples by two-dimensional chromatography. This indicates the ade-quacy of the one-dimensional method under the conditions of the experi-ment. Recoveries of y-aminobutyric acid which had been added to brainhomogenates ranged from 92 to 100 per cent. The presence of glutamicacid and aspartic acid in concentrations 200 times as great as those of ther-aminobutyric acid did not affect the recovery.

The above methods have been applied successfully to the determinationof several other amino acids in pure solutions and in protein hydrolysates.

y-Aminobutyric Acid Present in Brain Chiefly in Free Form-The aver-age value of a number of determinations of y-aminobutyric acid in alco-holic extracts of mouse brain was 55 mg. per 100 gm. of fresh weight oftissue. Closely similar values were found in the supernatant solutionsafter heat coagulation of brain homogenates, in trichloroacetic acid ex-tracts, and in acid hydrolysates of whole brain. It was also found thathydrolysis with 6 N HCl did not increase the content of y-aminobutyricacid in the protein-free extracts. These results show that the y-amino-butyric acid is present in brain chiefly in the free form.

In$uence of Glutamic Acid on Content of y-Aminobutyric Acid during In-cubation of Brain Homogenates, Washed Residues, or Acetone Powders-Homogenates of freshly excised mouse brain containing 250 mg. of tissueper ml. were prepared in ice-cold 0.05 M phosphate buffer, pH 7.42. In

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


5/9

K. ROBERW AND S . FRANKEL 59several experiments the homogenates were used directly. In others thehomogenates were centrifuged in the cold at 3500 r.p.m., the supernatantsdiscarded, and the residues washed with varying volumes of phosphatebuffer one or more times. The residues were then suspended in a volumeof buffer such that the final volume of the suspension was the same as thatof the original homogenate. The acetone powders were prepared by sus-pending fresh brain in 100 to 500 volumes of acetone at - 15 in a Waringblendor, filtering rapidly with suction, resuspending in the same volume offresh acetone, filtering, and finally drying in vacua over PZOS and paraffinat 4. Suitable amounts of the powders were then suspended in phos-phate buffer with the aid of a ground glass homogenizer prior to use.Immediately after preparation of the suspensions, 2 ml. aliquots werepipetted into tubes containing 8.45 ml. of 95 per cent alcohol and 0.67 ml.of distilled water to serve as blanks. 2 ml. portions of the preparationswere also added to 0.67 ml. of water or to 0.67 ml. of a solution containinga suitable concentration of glutamic acid, previously adjusted to pH 7.4.These latter mixtures were then incubated for 2 hours at 38 in an atmos-phere of O2 in a Dubnoff incubator. The reactions were stopped by theaddition of 8.45 ml. of 95 per cent alcohol and the mixtures stirred thor-oughly and centrifuged. The clear supernatants were poured off and ali-quots were used for analysis. Whenever aliquots greater than 0.3 ml.were used, the two-dimensional chromatographic procedure was employed.The data for representative experiments are summarized in Table I. Inall of the preparations employed, the increase of r-aminobutyric acid wasaccelerated by the presence of added glutamic acid. Placing the prepara-tions in boiling water for 1 minute destroyed the activity. In several in-stances, there were increases in the content of y-aminobutyric acid in theabsence of added glutamic acid. This occurred to the largest extent inthe crude homogenates. Even in the most extensiveiy washed prepara-tions there were still easily detectable amounts of those amino acids whichwere present in the fresh tissue in the free form to the largest extent.From this it would be expected that constituents of brain, other than thosewhich react with ninhydrin, would also adhere to the washed residues oracetone powders. For this reason it was not possible to conclude fromthese experiments that glutamic acid was a precursor of y-aminobutyricacid. The possibility was not ruled out that glutamic acid was indirectlystimulating the formation of r-aminobutyric acid from some other sub-stance. The following experiments were undertaken to determine whetheror not the carbon of glutamic acid would be found in y-aminobutyric acidafter incubation with a preparation which was actively forming the lattercompound.

Formation of y-Aminobutyric Acid from Glutamic Acid by Brain Acetone

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


6/9

GO y-AMINOBUTYRIC ACID IN BRA IXPowder-Glutamic acid which was uniformly labeled with CL4was kindlyfurnished to us by Dr. Konrad Bloch. This amino acid had been isolatedfrom a hydrolysate of algae grown in C140n. The glutamic acid had aspecific activity of 27,900 c.p.m. per mg. when counted as the amino acidwith a windowless proportional counter connected to a scaling unit. Thesampleswere deposited from dilute aqueous solution on cups 10.7 sq. cm.in area. A semple containing 1 mg. of the labeled amino acid to which

TABLE IInjlxence of Glutamic Ac id on Formation of y-Aminobutyric Aci d in BrainPreparalions

Incubation conditions described in the text. In the case of the homogenatesand washed residues an amount of tissue equivalent to 500 mg. of original freshweight of tissue was employed. 50 mg. of acetone powder were used.T--Type of preparationHomogenate

Washed residue

Experiment No.

Acetone powderI

L* Hrnt -inactivated.

-j-

- -----.-Final molarity ofadded glutamic acid

-

00.125000.125000.06080.00390.007800.00780.03130.12560.1250%00.12500.12500.03670.1250

-I-

i

- _._-. _Change in content ofy-aminobutyricacid per tube

74911150100-54354722698517938369840144-. ---

5 y of y-aminobutyric acid had been added was subjected to two-dimen-sional chromatography. At the same time a chromatogram was run con-taining 5 y of y-aminobutyric acid, and one containing a seriesof samplesof knonq concentrations was run to serve as a standard curve. Therewere no ninhydrin-reactive constituents other than glutamic and y-amino-butyric acids on the paper containing the radioactive glutamic acid. Onelution and estimation of the quantity of r-aminohut(yric acid, 5 y were

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


7/9

E. ROBERTS AND ti. BXANKPL 61recovered. The eluate contained no radioactivity whatsoever. Thisshows that the sample of glutamic acid employed contained no r-amino-butyric acid or other ninhydrin-reactive impurities, that no y-aminobutyricacid was formed from the glutamic acid during the chromatographic pro-cedures, and that no radioactive contaminants were present which mi-grated to the same position as the y-aminobutyric acid on the chromato-gram. The recovery from the paper containing y-aminobutyric acid alonewas 4.7 y, a value within the range of experimental error.

An experiment was then performed in which 40 mg. of fresh acetonepowder from mouse brain were incubated with 146,000 counts of glutamicacid (5.25 mg.) in a final volume of 1 ml. under the conditions previouslydescribed; 3.16 ml. of 95 per cent alcohol were then added. Samples of

TABLE IIRadioactivity Found in Eluates of Ninhydrin-Positive Areas frost Two-Dimensional

Chromalograms ajfer Incubation of @Labeled Glutamic Acid with AcetonePowder of Mouse Brain

146,OfXl counts of glutamic acid were added originally to th incubation mixture.IAmino acid *rota10untsnsamp~e-- --. ~..-_-_.-_ -. -.~.~- ..- ._ _. s.J%n.

Glutamic acid. 127,800y-Aminobutyric acid 354Glutamine. 20Glycine............ ,... ,....._.,...i 0Leucines............ ,,.,... _...___...i 0Valine.... ,... ,... ,,,. .... . ,. . . . . 0Alanine..... ,.._.. ..... ,.... . ,. ., 0Taurine. . . . . . . . . . . . .._.._.... ......_.. ., ,. 0_._ ._._ . ..___~._ .___~.--. _...~ ..-.the clear supernatant (0.75 ml.) were subjected to two-dimensional chro-matography. It was found that the total content of y-aminobutyric acidafter incubation exceeded by 40 y that found in the zero time control.The spots corresponding to glutamic acid and to all of the detectableconstituent,s which were clearly separated from the large glutamic acidspot were eluted with suitable volumes of water, plated, and count,ed. Inthis manner it was possible to obtain results for glutamic acid, y-amino-butyric acid, glutamine, glycine, the leucines, valine! alanine, and taurine.The counts are shown in Table II.Of the constituents other than glutamic acid, by far the largest, act.ivitywas found in the y-aminobut,yric acid. Glutamine was the only ot,her con-stituent having a significant numbrr of counts. The act,ivit,y rceovcrcd inthe glutamic a&1 spot (127,801)a.p.m.) was somewhaBt8igher than would

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


8/9

62 y-AMINOBUTYRIC ACID IN BRAINhave been expected if 1 mole of COZ had been liberated per mole of glu-tamic acid present (117,000 c.p.m.). The reaction with ninhydrin prob-ably does not go to completion on paper when such large quantities ofamino acid are present.

The above experiment shows conclusively that the carbon chain of glu-tamic acid can be converted to that of y-aminobutyric acid. It is notpossible at the present time to deal with the stoichiometry of the reactionbecause of the relatively low specific activity of the glutamic acid and theweak activity of the enzyme preparation employed. It appears highlyprobable, however, that the formation of r-aminobutyric acid from glu-tamic acid in brain takes place by a! decarboxylation.

Toxicity of y-Aminobutyric Acid-Two mice, weighing approximately20 gm., were given a single intraperitoneal injection of 100 mg. of y-amino-butyric acid in 0.5 ml. of water, and two other mice were given 0.25 ml.of the same solution. There were no signs of toxicity and the mice ap-peared to be completely normal thereafter. This indicates that the acutetoxicity of y-aminobutyric acid is low.

DISCUSSIONAn extensive survey of the free amino acids in many normal and malig-

nant mouse tissues, a part of which has been reported (6, 7), revealed thepresence of considerable quantities of a ninhydrin-reactive substance in ex-tracts of brain which was present, at most, in traces in the other tissuesexamined. This substance was also detected in the brains of other species.The experiments reported in this paper and in the following one (1) estab-lished with certainty that this compound is y-aminobutyric acid. Experi-ments with homogenates, washed residues, and acetone powders of mousebrain invariably showed increases in the content of y-aminobutyric acidupon incubation with glutamic acid. With isotopically labeled glutamicacid, it was found that glutamic acid could serve as a precursor of y-amino-butyric acid. Recent unpublished experiments in our laboratory withhomogenates and sterile autolysates of liver, muscle, and tumors haveshown increases of a substance which corresponds closely in its propertiesto y-aminobutyric acid after incubation for varying periods of time. Itmay thus be that the formation of y-aminobutyric is of wide occurrencein mammalian tissues. The uniquely high concentrations of this substancein brain may possibly be a result of a greater rate of formation than inother tissues, a slower rate of removal, or a combination of both. A studyis being made of the metabolism of y-aminobutyric acid.

y-Aminobutyric acid has recently been reported to be present in bac-teria (8), plants (9), human blood and urine (5), adult ox and embryo calfmuscle (lo), and yeast (11). In bacteria and plants, this compound can

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom


9/9

E. ROBERTS AND S. FRANKEL 63arise as a result of the action of glutamic acid decarboxylase (12, 13).The presence of this enzyme has not yet been demonstrated in any of theother sources in which y-aminobutyric acid has been reported. The ex-periments reported in this paper indicate that such an enzyme may bepresent in brain tissue.

SUMMARY1. y-Aminobutyric acid has been shown to be a constituent of brain in

which it exists preponderantly in the free form.2. Experiments with various crude enzyme preparations from mousebrain showed a net increase of y-aminobutyric acid, which was accelerated

by the addition of glutamic acid.3. It was shown by the use of isotopically labeled glutamic acid that

the latter amino acid can serve as a precursor for the y-aminobutyric acid.4. It is suggested that the formation of this compound in brain takes

place by the Q! decarboxylation of glutamic acid.BIBLIOGRAPHY

1. Udenfriend, S., J. Biol. Chem., 187, 65 (1950).2. Robe rts, E., and Frankel, S., Federation Proc., 9, 219 (1950).3. Cons den, R., Gordon, A. H., and Martin, A. J. P., Bioc hem . J., 38,224 (1944).4. Dent, C. E., Bioche m. J., 41, 240 (1947).5. Dent, C. E., Bioche m. J., 43, 169 (1948).6. Robe rts, E., and Frankel, S., Cancer Res., 9, 645 (1949).7. Robe rts, E., Frankel, S., and Harm an, P. J., Proc . Sot. Exp. Biol. and Med., 74,

383 (1959).8. Work, E., Bioc him. et biophys. acta, 3, 400 (1949).9. Steward, F. C., Thom pson, J. F.;and Dent, C. E., Scie nce, 110, 439 (1949).10. Gordon, A. H., Biochem . J., 100, 99 (1949).

Il. Reed, L. J., J. Bio l. Chem., 183, 451 (1949).12. Gale, E. F., Adva nces in Enzymo l., 6, 1 (1946).13. &ha les, O., Mims, V., and Sch ales, S. S., Arch. Bioche m., 10, 455 (1946).

byguest,onJun

e7,2010

w

ww.jbc.org

Downloadedfrom

Documents

(1950) Gamma-Aminobutyric Acid in Brain Its Formation From Glutamic Acid