TRANSAMINATION AND ASSOCIATED DEAMIDATION OF ASPARAGINE AND GLUTAMINE

BY ALTON MEISTER, HERBERT A. SOBER, SARAH V. TICE, AND PHYLLIS E. FRASER

(Prom the National Cancer Institute, National Znstitutes of Health, Bethesda, Maryland)

(Received for publication, February 14, 1952)

In a previous communication from this Laboratory (l), the following reaction, catalyzed by a purified rat liver preparation, was described:

Glutamine + a-keto ncid ---t a-ketoglutaric acid + a-amino acid + ammonia

With this system, no deamidation of glutamine occurred in the absence of a-keto acid, and, with all keto acids tested except two (pyruvate and a-ketobutyrate), transamination was absent or extremely slow when glu- tamate was substituted for glutamine. Experiments with W-labeled glu- tamine indicated that the ammonia evolved during this reaction was derived from t.he amide group of glutamine. Although this enzyme preparation does not catalyze either the deamidation of asparagine in the presence or absence of a-keto acids or transamination between asparagine and a- keto acids, other liver preparations have been found to catalyze an ap- parently analogous reaction with asparagine involving deamidation and transamination.

In earlier studies (2-5), it was found that asparagine deamidation oc- curred rapidly in aqueous extracts of rat liver and that virtually all of this activity was destroyed by heating the extracts at 50” for 10 minutes. Addition of pyruvate or a-ketoisocaproatc to the heated extracts resulted in remarkable acceleration of asparagine deamidation. The heat-labile activity was designated asparaginase I and the heat-stable pyruvate-acti- vated fraction as asparaginase II. Two separate systems capable of cata- lyzing the deamidat,ion of glutamine were also observed. One of these, glutaminase I, was activated by phosphate and certain other anions, while the other, glutaminase II, was activated by a-keto acids (5). The glu- tamine deamidation systems were separated, purified, and found to exhibit negligible activity toward asparagine (6).

Our findings are in accord with these results, and in addition demonstrate the occurrence of transamination reactions between asparagine and a-keto acids resulting in the formation of oxalacetic acid and the corresponding a-amino acids. The present report also describes several new transamina- tion reactions involving glutamine.

319

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320 ASPARAGINE AND GLUTAMINE

EXPERIMENTAL

Substrates--Most of the cw-keto acids were prepared as the crystalline sodium salts by enzymatic procedures (7), or synthesized as previously described (1). Sodium glyoxylate was generously furnished by Dr. D. B. Sprinson, and a-keto-&indolylpropionic acid by Dr. J. White. L-Glu- tamine was obtained from the Nutritional Biochemicals Corporation; the product yielded 96 per cent of the theoretical ammonia when boiled for 1 minute in 2 N hydrochloric acid. L-Asparagine monohydrate ([a):’ = +29.2”; 9.517 per cent solution in 3 N hydrochloric acid) was obtained from Merck. L-Aspartic acid and L-isoasparagine were donated by Dr. J. P. Greenstein.

Enzyme Preparalions-Although the keto acid-mediated deamidat.ion can be shown in homogenates or extracts after selective heat destruction of asparaginase activity, determinations of the oxalacetate formed are un- reliable, due to enzymatic degradation of this keto acid. In addition, interpretations of paper chromatographic studies with homogenates or extracts are complicated by the presence of large amounts of free amino acids (especially glutamic acid and alanine). A partial purification of the asparagine deamidation-transamination system was obtained as follows: Fresh rat liver was homogenized with 2 volumes of ice-cold distilled water in a Waring blendor for 3 minutes. The homogenate was heated rapidly to 50” in a water bath, maintained at this temperature for exactly 10 minutes, and then cooled in a -10’ bath for 5 minutes. The mixture was treated with 10 volumes of acetone (cooled to 0”) and filtered rapidly with suction. The liver powder was washed with acetone and ether, and dried in vacw at 5” for 18 hours. The powder was extracted for 4 minutes with 8 volumes of ice-cold water in a Waring blendor, and then centrifuged in a Sharples centrifuge. The clear supernatant was dialyzed at 10” against running water for 18 hours and lyophilized. The lyophilized preparation was stored at 5” and showed no loss of activity over a period of several months. The preparation was 10 to 15 times more active (on the basis of tissue nitrogen) than the original heated homogenate, and exhibited less than 10 per cent of the original oxalacetic decarboxylase and aspara- ginase activities.

The experiments with glutamine were carried out with the liver prepara- tion previously described (1, 6). This material showed no loss of activity when stored at 5” in the lyophilized state, although the freezing procedure usually resulted in a 10 to 20 per cent loss of activity.

Methods

Ammonia was determined by nesslerization after aeration into sulfuric acid traps. In several cases ammonia was determined by adsorption (at

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MEISTER, SOBER, TICE, AND FRASER 321

pH 7) and elution from Permutit (8); there were no significant differences in the results obtained with the two procedures. Oxalacetic acid was determined as described by Edson (9), and phenylalanine and tyrosine by decarboxylation with Streptowccua fatdti cells (10, 11). Glutamate and glutamine were determined by decarboxylation with Clostridium welchii (12, 13).

Paper Chromatography-The procedure consisted of a combination of ascending and descending techniques with Schleicher and Schuell (No. 598) paper (14, 15). For amino acid detection, samples of the incubated mix- tures were acidified with 3 M sodium acetate buffer (pH 4.9), placed in a boiling water bath for 3 minutes, and centrifuged to remove coagulated prot,ein.l Replicate samples were always chromatographed in five solvent mixtures: (1) phenol mix, phenol saturated with 10 per cent sodium citrate in an atmosphere of hydrocyanic acid and 0.3 per cent ammonia; (2) 77 per cent ethanol; (3) formix, tertiary butanol (70 parts), water (15 parts), and formic acid (15 parts) ; (4) lutidine mix, 2:6 lutidine (55 parts), water (25 parts), ethanol (20 parts), and diethylamine (1 part); (5) ketone mix, methylethyl ketone (16 parts), tertiary butanol (16 parts), formi’c acid (0.1 parts), and water (3.9 parts). Internal controls and chromatographic recoveries were employed as previously described (16). The solventa em- ployed for keto &id chromatography were lutidine mix, 77 per cent ethanol, and propyl alcohol and ammonia (17). The spots were visualized under ultraviolet light after spraying with 0.05 per cent o-phenylenediamine in 10 per cent trichloroacetic acid and heating at 100” for 2 minutes (17). Samples (0.01 to 0.03 cc.) of the reaction mixtures were chromatographed directly for oxalacetate detection. Pyruvate was also visualized on chro- matograms of samples containing oxalacetate. When samples containing oxalacetate were deproteinized with trichloroacetic acid or 75 per cent alcohol (followed by evaporation of the alcohol in uacuo at 26”), only pyru- vate was detected on the chromatograms.

Resu&.s

Transamination and Deamidation of Asparagine-The effect of a number of a-keto acids on asparagine deamidation is described in Table I. All but four (a-ketoisovalerate, a-ket.ophenylacetate, and d- and I-a-keto+- methylvalerate) of the keto acids studied accelerated asparagine deamida-

1 Chromstograma of thoroughly dialyzed enzyme preparations showed no amino acids. When the enzyme preparations were incubated with buffer at 37” for 2 hours, traces of ninhydrin-reacting compounds including glutamic acid and alanine were noted. Some protein breakdown probably occurs during incubation and as a result of the heat coagulation procedure. At this time it appears improbable that the minute amounts of amino acids formed in this manner play a significant rSle in the mechanism of the enzymatic reactions studied.

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322 ASPARAGINE AND GLUTAMINE

tion. The normal P-keto acids (Cd to G), r-ketovaleric acid, triacetic acid, triacetic lactone, and pyruvamide were inactive. Several a ,r-diketo acids increased deamidation; these acids were hydrolyzed to pyruvate by the enzyme preparation, L-Isoasparagine was slowly hydrolyzed by the enzyme preparation, and the rate of deamidation was not influenced by the presence of a-keto acids. The findings with asparagine are analogous to those previously observed in studies on glutamine (1) and suggested the occurrence of a transamination reaction. Under the conditions de- scribed in Table I, the formation of alanine, a-aminobutyric acid, norvaline,

TABLE I Effect of Keto Acids on Lleamidation of Asparagine

Keto acid

Pyruvic None a-Ketobutyric a-Ketovsleric a-Ketocaproic a-Ketoheptylic a-Ketoisovaleric a-Ketoisocaproic d-a-Keto-@-methylvaleric la-Keto-fi-methylvaleric Glyoxylic

Relative activity* Keto acid Relative

activity* --

WJJ) a-Keto-r-methiolbutyric 59 8 a-Keto-y-ethiolbutyric 46

98 a-Keto-e-hydroxycaproic 65 86 ol-Keto-c-N-chloroacetylcaproic 21 68 a-Keto-S-cnrbamidovaleric 25 48 a-Ketoglutaric 30 8 a-Keto&indolylpropionic 28

58 a-Keto-fi-cyclohexylpropianic 35 10 Phenylpyruvic 101 10 p.-Hydroxyphenylpyruvic 90 85 a-Ketophenylacetic 7

* The reaction mixtures contained initially keto acid (20 PM), L-asparagine (10 PM), 0.05 M Verona1 buffer (pH 8.0), and 60 or 120 mg. of enzyme preparation in a final volume of 2.0 cc.; incubated for 90 or 120 minutes at 37”. The relative values for ammonia formation are given as per cent of the value obtained with pyruvic acid.

norleucine, a-aminoheptylic acid, leucine, glycine, methionine, ethionine, a-amino-e-hydroxycaproic acid, glutamic acid, tryptophan, @-cyclohexyl- alanine, phenylalanine, and tyrosine from the corresponding a-keto acids and asparagine was demonstrated by paper chromatography. Although l -N-chloroacetyllysine and citrulline were not detected on the chromato- grams, it is possible that small amounts of these amino acids could escape detection due to their relatively low color production with ninhydrin. No transamination was observed with the four a-keto acids which were inac- tive in increasing deamidation. The formation of oxalacetate was also demonstrated by paper chromatography, as described in the experimental section.

Comparison of l’ransaminabn with Asparagine and Aspartic Ad- Transamination between asparagine (and aspartic acid) and several a-keto

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MEISTER, SORER, TICE, AND FRASER 323

acids was followed by oxalacetate determinations, paper chromatography, and in two cases by quantitative amino acid determinations (Table II). Transamination with asparagine was significantly greater than with aspar- tic acid except with glyoxylic acid. There was no appreciable oxalacetate formed (over the control) when the enzyme preparation was incubated with aspartate and cY-ketovalerate, a-ketoisocaproate, a-keto-r-methiolbu- tyrate, cu-keto-r-ethiolbutyrate, or a-keto-c-hydroxycaproate. However, t.races of norvaline and leucine were detected on the chromatograms, in-

TABLE II

I’ransaminalion of a-Keto Acids with Asparagine and Aspartic Acid+

I

Keto acid

Tione ...................................

Pyruvic ................................. Glyoxylic ............................... a-Ketobutyric. .......................... a-Ketovaleric ........................... a-Ketoisocaproic ........................ a-Keto-r-methiolbutyric ................. a-Keto-r-ethiolbutyric. ................. a-Keto-e-hydroxycaproic ................ Phenylpyruvic .......................... p-Hydroxyphenylpyruvic. .............

Ammonia Oxalacetate Oxalacetate

PM Iry PM

0.82 0.36 0.82 10.1 7.32 3.73 9.41 6.15 6.95

10.0 7.42 4.21 9.36 6.43 0.96 6.30 4.74 1.12 6.44 5.31 0.86 4-. 98 4.08 0.92 6.50 5.02 0.90 9.82 8.46 (9.62t) 1.86 (2.OOt) 8.96 7.31 (8.40t) 2.14 (2.01t)

*The reaction mixtures contained initially keto acid (20 PM), L-aspartate or L-aaparagine (10 PM), and 60 mg. of enzyme preparation, in 1.5 cc. of 0.05 M veronal- acetate buffer (pH 8.0); incubated for 90 minutes at 37”. The formation of amino acids was observed chromatographicully as described in the text.

t Phenylalanine and tyrosine formed, aa determined with S. faecalis dc- carboxylase.

Asparagine Aspartate

dicating the occurrence of some transamination. On the other hand, trans- amination of these keto acids with asparagine proceeded readily as ob- served chromatographically and by oxalacetic acid formation. There was measurable transamination between aspartate and phenylpyruvate and p-hydroxyphenylpyruvate as determined by oxalacetate and amino acid formation, although considerably more transaminat.ion occurred between these keto acids and asparagine. About twice as much transamination occurred with pyruvate and cr-ketobutyrate and asparagine as with these keto acids and aspartate, while glyoxylate transaminated to about an equal extent with both aspartate and asparagine. The observed formation of oxalacetic acid was 65 to 85 per cent of the value obtained for ammonia

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324 ASPARAGINE AND GLUTAMINE

formation, suggesting that some decarboxylation of oxalacetate occurred during the reaction. Only 20 to 40 per cent of the expected oxalacetate could be detected at the end of the reaction in experiments with heated liver homogenates. When 5 to 15 /IM of oxalacetic acid were incubated with the enzyme preparation under the conditions described in Table II, 70 to 80 per cent of the added oxalacetate was present at the end of in- cubation.

A diagrammatic representation of a chromatogram developed with for- mix is presented in Fig. 1. The chromatogram demonstrates that there

DIASRAWIATIC CHROYATOGRAYS OF ASPARACINE-KY0 ACID TRANSAYlNATlON

LEUCINE

t ALANINE

ETHIONINE ALANINE

SLYCINE

:K’c ASPARACINE USPCN 1 ii

AWN ASP .-

CLYO~YLIC

ASPSN ASP

PYRtvlc

‘i AWN ASP -- AWN ASP .l_.

.-&O- ISOCAPROIC

No To 60 50 -7 40

30

20 ASI’M ASP --z-

%Ky RUTYRIC -

Fro. 1. Diagrammatic representation of chromatogram demonstrating aspara- gine-keto acid and aspartic-keto acid transaminat.ion. The solvent was formix. Experimental details are given in the text. Although phenylelanine and methionine were not separated by this solvent mixture, resolution of these amino acids was readily obtained with three other solvent mixtures (lutidine mix, phenol mix, and ketone mix).

was greater formation of alanine, methionine, leucine, and phenylalanine with asparagine than with aspartic acid, and that the formation of glycine was about the, same with asparagine and aspartic acid. The formation of alanine may be ascribed to amination of pyruvate formed by oxalacetate decarboxylation. No alanine was formed when the ensyme preparation was incubated with asparagine alone or with inactive keto acids. How- ever, traces of alanine were formed when aspartate was incubated with the enzyme. The mechanism of this slight formation of alanine from aspartate is not known. The formation of some aspartate in the experiments with asparagine occurred to about the same extent in the presence and absence of keto acids, and is compatible with the known occurrence of residual asparaginase in the ensyme preparation.

Isoasparagine transaminated with a-keto-n-valeric acid and phenylpy-

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MEISTER, SOBER, TICE, AND FRASER 325

ruvic acid to about the same extent as did aspartic acid. The very weak transaminase activity observed with the a-amide may be attributed to its conversion to aspartate by prior enzymatic hydrolysis.

I’ransamination Reactions Involving Glutamine--Previous studies on the glutamine-a-keto acid reaction have been extended to several additional a-keto acids (Table III). With this enzyme preparation there was no ammonia formed from glutamine in the absence of a-keto acids. Trans- amination was followed by paper chromatography and determinations of the disappearance of glutamine or glutamic acid by decarboxylation with

TABLE III

Transamination Reactions Znvolving Glutamine’

I Glutamine

a-Keto acid

Sane ........................... Pyruvic. ........................ Glyoxylic ....................... a-Keto-c-hydroxycaproic ........ a-Keto-t-N-chloroacetylcaproie a-Keto-r-ethiolbutyric .......... a-Keto-,?-cyclohexylpropionic. ... a-Keto-fl-indolylpropionic .......

.

T Disa pearance of g utamine P

PM

0 10.2 8.11 9.66 2.60 7.80 1.26 4.19

- I

fix

0

10.6 8.70

10.2 2.75 8.16 1.46

Glutamate

Disa pearnnce of IJ utamate P

-~.

Iry

0

10.9 7.92 0.10 0 0 0

4.42 / 0

* The reaction mixtures contained initially keto acid (40 PM), glutamine or glu- tamate (‘20 PM), and 30 mg. of enzyme in 4 cc. of 0.05 M veronal-acetate buffer (pH 7.1). Incubated at 37” for 90 minutes. The formation of the corresponding amino acids was observed chromatographically as deecribed in the text.

C. welchii. There was no detectable transamination of glutamate with a-keto-c-N-chloroacet,ylcaproic, a-keto-r-ethiolbutyric, a-keto-fl-cyclohex- ylpropionic, a-keto+indolylpropionic, and a-keto-s-hydroxycaproic acids.* Glyoxylic acid catalyzed the deamidation of glutamine and transaminated

* It was previously noted that, with the exception of pyruvic and a-ketobutyrie acids, the purified enzyme failed to catalyze appreciable transamination between glutamate and a-keto acids. On the other hand, with liver homogenates these keto acids transaminate with glutamine and glutamate at rates of about the same order of magnitude. Most of this glutamic-keto acid transaminaee activity is removed in the initial centrifugation step in the preparation. Fractionation of liver homoge- nates by the procedure of Schneider and Hogeboom (18) revealed that most of the glutamine-keto acid activity (transaminase and deamidase) was present in the supernatant fraction. The glutamic-phenylpyruvic system was found chiefly in the mitochondrial fraction (cf. Hird and Itowsell (19)), while almost all of the glutamic- pyruvic system remained in the supernatant.

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326 ASPARAGINE AND GLUTAMINE

with both glutamate and glutamine yielding glycine. a-Keto-a-carbami- dovalcric acid catalyzed only slight ammonia formation under the condi- tions described in Table III. With a larger amount of enzyme (90 mg.) and a longer incubation period (4 hours), chromatographic findings com- patible with the formation of citrulline were observed. a-Ketophenyl- acetic acid was inactive in deamidation and transamination.

Thus far attempts to separate or alter the relative rates of the deamida- tion and transaminat,ion reactions of glutamine have not been fruitful. It was reported that a-keto acid-catalyzed deamidation of glutamine was

TABLE IV Efect of Keto Acid Concentration on Transamination and Deamidation of Glutamine*

Keto acid concentration

ef 5

10 20 30 40 60 80

100 200 400

- I

L

Glutamine plus pyruvate

Deamidation

IVY NEi

4.01 5.85 7.82

7.40 7.12 5.90

Transamina- tion

PM

4.30 6.00 7.30

7.65 7.01 5.65

1 Deamidation

Glutamine plus phenyl- pyruvate

~__ fix NBa

3.96 5.96 4.43 3.23 2.62 1.94 1.62 1.18

--. __

~~ - I ’ rransamina-

tion

PM

3.68 6.31 4.71 3.37 2.82 2.21 2.08 0.94

-

.- I

-

Glutamine plus a-ketoisocaproate

>eamidation

PY NHr

Transamina- tion

3.22 3.01 4.94 5.12

5.36 5.11

4.41 4.70 2.26 2.03

- l The reaction mixtures contained initially 10 PM of glutamine, a-keto acid as

indicated, 30 mg. of enzyme, in 4 cc. of 0.05 M veronal-acetate buffer (pH 7.1); incu- bated for QO minutes at 37”. Transamination is expressed in terms of glutamine disappearance, as determined by decarboxylation with C. welchii.

inhibited by high concentrations of cu-keto acids (5, 6). It seemed of im- portance to determine the effect of keto acid concentration on the gluta- mine-transamination reaction. A marked decrease of both deamidation and transamination occurred with high concentrations of phenylpyruvate and cr-ketoisocaproate, while somewhat less inhibition was observed with pyruvate (Table IV). Within experimental error the ratio of deamidation to transamination remained constant, a result compatible with the close association of these reactions.

DISCUSSION

The asparagine-cy-keto acid reaction appears analogous to the glutamine- or-keto acid reaction previously studied. In both systems, appreciable

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MEISTER, SOBER, TICE, AND FRASER 327

deamidation occurred only in the presence of a-keto acids, and deamidation was invariably associated with transamination. The relative activity of the various keto acids was approximately the same for both systems, al- though the rates have been found to vary somewhat, depending upon the pH and the keto acid concentration. cY-Ketoisovaleric, d- and I-cr-keto- p-methylvaleric, and a-ketophenylacetic acids were not active in either system. In considering the structural requirements for activity in the cY-keto acid series, it may be noted that, with the exception of glyoxylic acid, all of the active cr-keto acids possess 2 hydrogen atoms on the &carbon atom. Except in three cases (pyruvic, a-ketobutyric, and glyoxylic acids), transamination between glutamine and a-keto acids was considerably more rapid t.han the corresponding reaction with glutamate. Comparison of asparagine with aspartate revealed that the former transaminated more rapidly with cr-keto acids than did the latter except with glyoxylic acid. In many instances transamination between aspartate or glutamate and a-keto acids either was not observed or was very slow, while the analogous reactions with the w-amides occurred readily. These findings suggest that aspart.ate and glutamate are not intermediates in these transamination- deamidation syst.ems, and that deamidation is probably not the initial step in the reaction.

The “asparagine” enzyme preparation exhibited both asparagine-keto acid and glutamine-keto acid activities, while the more purified “glu- tarnine” enzyme did not exhibit significant asparagine-keto acid activity. Although the data are compatible with the existence of separate glutamine- keto acid and asparagine-keto acid systems, there is as yet no conclusive evidence to justify such designations. The presence of aspartic-alanine transaminase activity in muscle preparations has been interpreted to repre- sent the combined action of the glutamic-alanine and the glutamic-aspartic transaminases (20, 21). Although this may also be true for hepatic sys- tems, evidence compatible with the existence of an aspartic-alanine system in pigeon liver has been obtained by Moulder et al. (22), and a specific aspattic-alanine transaminase system in pigeon and hen liver has been described by Kritsmann and Samarina (23). The possibility of pyruvate- activated p-decarboxylation of aspartate to alanine must also be con- sidered (cf. Meister et al. (16)).

The relatively rapid transamination of glyoxylic acid with aspartate, glutamate, asparagine, and glutamine to yield glycine is of interest and is in accord with the findings of Weinhouse and Friedmann (24) on the rapid conversion of injected glyoxylic and glycolic acids to glycine in intact rats. The catalysis of a number of transamination reactions by liver and other biological material suggests the existence of at least several separat.e transaminases different from the highly specific glutamic-alanine and glu-

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323 ABPARAQINE AND GLUTAMINE

tamic-aspartic systems. Attempts to resolve this problem by further frac- tionation of liver are in progress. Efforts in this direction with the more stable systems of Escherichia coli have led to the isolation of separate glutamic-(phenylalanine, tyrosine) and glutamic-(isoleucine, valine, leu- tine) transaminase systems and the demonstration of other transaminase systems not involving dicarboxylic amino or keto acids (25).

It was previously suggested (1) either that the glut,amine-a-keto acid re- action involved simultaneous transamination and deamidation or that the transamination step preceded deamidation. The latter mechanism would lead to the intermediate formation of a-ketoglutaramic acid. The ar-keto analogues of glutamine (a-ketoglutaramic acid) and asparagine (cr-keto- succinamic acid) were prepared in pure form by a procedure similar to that described in the previous paper (7), and it was found that both compounds were only slowly deamidated by preparations capable of catalyzing rapid transamination (and associated deamidation) between glutamine or aspara- gine and various cr-keto acids. In addition, the y-methylamide of a-keto- glutaric acid was not appreciably hydrolyzed under conditions whereby active transamination and methylamine formation occurred with the y-methylamide of glutamic acid (26). The evidence suggests that neither the free dicarboxylic acids nor the or-keto acids corresponding to glutamine and asparagine are intermediates in the reactions. It seems probable that transamination and deamidation occur simultaneously or as closely linked reactions. Although cu-ketoglutaramic acid and a-ketosuccinamic acid were not significantly attacked by the transaminase preparations, these keto amides were rapidly hydrolyzed to ammonia and the corresponding dicarboxylic acids by other liver fractions and by homogenates of a number of other rat tissues. The metabolism of these compounds will be described in a subsequent publication.

Although the mechanisms of the glutamine-keto acid and asparagine- keto acid transamination-deamidation reactions require further investi- gation, the evidence suggests that both glutamine and asparagine play a significant role in transamination in liver.a

SUMMARY

1. A number of cr-keto acids were found to catalyze the deamidation of asparagine by a rat liver preparation.

2. The a-keto acid-stimulated deamidation of asparagine appears analo- gous to the a-keto acid-glutamine reaction previously described, in that

J The glutamine-keto acid transamintltion-deamidation system has thus far been found chiefly in liver. After selec’tive heat destruction of glutaminaae, Home activity was observed with homogenates of rat kidney. No activity was noted with homoge- nates of rat brain and skeletal muscle or of hog heart.

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MEISTER, SOBER, TICE, AND FRASER 329

deamidation of asparagine is associated with transamination leading to the formation of the corresponding a-amino acid and oxalacetic acid.

3. Transamination between asparagine and pyruvate, a-ketoisocaproate, phenylpyruvate, p-hydroxyphenylpyruvate, cu-keto-r-methiolbutyrate, CY- ketobutyrate, and seven other a-keto acids occurred much more rapidly than transamination between aspartic acid and these keto acids. Iso- asparagine was only about as active as aspartic acid in transamination with a-keto-n-valerate and phenylpyruvate. Glyoxylic acid catalysed the deamidation of asparagine and transaminated at similar rates with both asparagine and aspartic acid, yielding glycine.

4. Several new transamination reactions between glutamine and a-keto acids have been described. These include reactions leading to tryptophan and glycine formation.

5. Equivalent inhibition of deamidation and transamination was ob- served in the glutamine system with high concentrations of a-ketoiso- caproate, phenylpyruvate, and pyruvate.

6. cu-Ketoisovaleric, d- and Z-a-keto-p-methylvaleric, and a-ketophenyl- acetic acids were inactive in the deamidation and transamination reactions of asparagine and glutamine.

7. The evidence suggests that neither t.he free dicarboxylic amino acids nor the a-keto acids, corresponding to asparagine and glutamine, are inter- mediates in the transamination-deamidation reactions, and that t.rans- amination and deamidation may occur simultaneously or as closely linked reactions.

BIBLIOGRAPHY

1. Meister, A., and Tice, S. V., J. Biol. Chem., 187, 173 (1950). 2. Errera, M., and Greenstein, J. I’., J. Nut. Cancer Inst., 7, 437 (1947). 3. Greenstein, J. P., and Carter, C. E., J. Nat. Cancer Inst., 7.57 (1947). 4. Price, V. E., and Greenstein, J. P., .I. Nat. Cancer Insl., 7, 275 (1947). 5. Greenstein, J. P., and Price, V. E., .I. BioZ. Chem., 178,695 (1949). 6. Errera, M., J. Biol. Chem., 178, 483 (1949). 7. Meister, A., J. Biol. Chem., 197, 309 (1952). 8. Folin, O., and Bell, R. D., J. Biol. Chem., 19, 329 (1917). 9. Edson, S. L., Biochem. J., aS, 2082 (1935).

10. McGilvery, R. W., and Cohen, P. P., J. Biol. Chem., 174,813 (1948). 11. Gale, E. F., Biochem. J., 41, p. vii (1947). 12. Krebs, H. A., B&hem. J., 43, 51 (1948). 13. Meister, A., Sober, H. A., and Tice, S. V., J. Biol. Chem., 189, 591 (1951). 14. Block, R. J., Anal. Chem., 22, 1327 (1950). 15. Block, R. J., and Sober, H. A., in Alexander, J., Colloid chemistry, theoretical

and applied, New York, 7, 181 (1950). 16. Meister, A., Sober, A., and Tice, S. V., J. Biol. Chem., 189, 577 (1951). 17. Wieland, T., and Fisher, E., Naturwissenschajten, 36, 219 (1949). 18. Schneider, W. C., and Hogeboom, G. H., J. Biol. Chem., 183, 123 (1950).

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330 ASPARAGINE AND GLUTAMINE

19. Hird, F. J. R., and Rowsell, E. V., Nature, 166, 517 (1950). 29. Green, D. E., Leloir, L. F., and Kocito, V., J. Biol. Chem., 161,559 (1945). 21. O’Kane, D., and Gun&us, I. C., J. Biol. C&m., 170, 433 (1947). 22. Moulder, J. W., Vennesland, B., and Evans, E. A., Jr., J. Biol. Chem., 160. 395

(1945). 23. Kritsmann, M. G., and &marina, 0. P., Doklady Akad. Nauk. S. S. S. ht., 63, 171

(1948); Chem. Abstr., 43, 2252 (1949). 24. Weinhouse, S., and Friedmann, B., J. Biol. Chem., 191, 707 (1951). 25. Rudman, D., and Meister, A., Abstracts, American Chemical Society, 121st meet-

ing, Milwaukee (1952). 26. Meieter, A., Sober, H. A., Tice, S. V., and Fraser, P. E., Abstracts, American

Chemical Society, 121st meeting, Milwaukee (1952).

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and Phyllis E. FraserAlton Meister, Herbert A. Sober, Sarah V. Tice

GLUTAMINEDEAMIDATION OF ASPARAGINE AND TRANSAMINATION AND ASSOCIATED

1952, 197:319-330.J. Biol. Chem. 

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