Asparagine synthetase from γ-irradiated potatoes

ARCHIVES

OF

BIOCHEMISTRY

Asparagine

AND

BIOPHYSICS

Synthetase

133, 208-215 (1969)

from

r-Irradiated

P. MADHUSUDAEAYX Biochemistry

and Food Technology Trombay, Received

February

NAIR

Division, Bhabha Atomic Rombay 86, India 11, 1969; accepted

Potatoes

May

Research Centre,

28, 1969

r-Irradiation of potatoes at. sprout-inhibiting dose was found to enhance the aspnragine synthetase activity. This activation was dose-dependent and maximum activation was obtained between 5 and 25 krads; 10 krad being the optimum dose. Asparagine synthetase was purified to about 50-fold. L-Ssparagine was identified as the product of the reaction. The reaction was specific for I,-aspartic acid and devoid of transferase activity. Hydroxylamine at very high concentration could replace NH4+ for substrate. The react.ion showed a definite requirement for NHa+, ATP, and Mgz+. The mechanism of enzyme activity resembled that reported for other higher plants, where L-asparagine, ADP, and Pi are the products of the reaction. No AMP was detected as the product of the enzyme reaction. There was strict stoichiomet,ry between asparagine and AL)P formed and Pi liberated during the reaction. Mnzf ions could replace Mg2+ for activity. But. the pH optima shifted from 7.2 to 6.0 when Mg2+ ions were supplemented in the reaction.

Although the biosynthesis of glutamine has been studied in a wide number of organisms and its mechanism completely worked out (I), the biosynthesis of asparagine, an equally important amide, has received only scant attention. This is mainly due to the difficulty of isolating this enzyme, in a pure form, from plant sources. Glutamine synthetase does not catalyze the formation of asparagine from aspartate, NH3 , and ATP. Black and co-workers (24) have studied the formation of P-asvia @-aspartyl phosparty1 hydroxamate, phate catalyzed by aspartokinase. But this reaction did not represent true asparagine synthetase. Webster and Varner (5) showed a reaction similar to that of glutamine synthesis in the formation of asparagine from wheat germ and lupine seedling preparais tions, where P-aspartyl hydroxamate formed from L-aspartate and hydroxylamine in the presence of hIg2+ and ATP. AlDawody and Varner (6, 7) reported enzyme preparations capable of synthesizing asparagine from L-aspartate and NH, in the presence of RIg2+, Mn2+, or Co*+ ion

from a number of tissues; e.g., pig liver, pig heart, bakers yeast, dried peas, pea seedlings, and lupine seedlings. They also purified this enzyme from yeast about lOOfold. They found that the products of the reaction were asparagine, ADP, and Pi. The enzyme catalyzed an aspartate-dependent exchange of 32P orthophosphate into ATP indicating the reversibility of this reaction. Recently, Ravel et al. (8) from Lactobacillus arabinosus, and Burchall et al. (9) from Streptococcus bovis, demonstrated a new type of mechanism for the synthesis of asparagine. Here, the main difference from the other system was that the cleavage products of ATP are AMP and PPi. The present work was initiated after the observation that potatoes when given a sprout-inhibiting dose of 10 krad of y-irradiation showed an increased content of asparagine. This prompted us to search for asparagine synthetase in r-irradiated potatoes. This enzyme showed comparatively very little activity in unirradiated potatoes which was not therefore used for purification studies. This communication describes 208

ASPARAGINE

the purification of asparagine synthetase and some of its properties from r-irradiated potatoes. EXPERIMENTAL

PROCEI)URES

Enzyme Assays Asparagine synthetase was assayed by three procedures. Estimation of asparagine formation. With the purified enzyme, asparagine formation was estimated with a reaction mixture containing 10 mM L-aspart,ate; (neutralized to pH 7.0 with KOH); 15 rnhf, MgC12 ; 30 mM, NH&l; 5 mM, ATP; 25 m&I, Tris-hydrochloride buffer pH 7.2, and enzyme protein about 700 pg in a final volume of 1 ml. The reaction mixture was incubated at 35” for 1 hr and the reaction was stopped with 4 ml of ethanol. After removal of the precipitated protein by centrifugation, a 3-ml aliquot was taken for asparagine estimation. To this solution 20 mg of NHgOH HCl was added and the solution was evaporated to dryness on a water bath for about 1 hr. The residue was dissolved in 1 ml of water and 3 ml of FeCl, reagent (5% trichloroacetic acid and 10%; FeC13 in 0.67 N HCl) was added. The color formed was measured at MO rnp (10). A standard curve using different concentrations of asparagine and 20 mg NHzOH HCl under the same conditions was prepared. The graph was linear up to 50 mM asparagine. The validity of this method was tested by taki11g the same concentrations of synthet,ic fl-aspartyl hydroxamate, as that taken in the case of asparagine and comparing the intensity of the color formed with FeC13 reagent. In both cases, the values obt,ained for opt,ical density change at, 540 rnp was t,he same. To establish t,he validity of the estimation of asparagine with the method used, recovery of added asparagine was studied. L-asparagine was added at different concentrat.ions (0.3, 0.5, 1.0, 2.0, 3.0, and 1.0 pmoles) to reaction mixt,ures containing the same quantity of euzyme as that. used in standard assay condition. After incubation for 1 hr, asparagine was determined after converting to hydroxamate. fi-aspartyl hydroxamate was synthesized according to the met,hod of Hoper and McIlwain (11) by boiling a solution of asparagine monohydrate with hydroxylamine hydrochloride. Estimation of Pi formed. The phosphorrls liberatcd was estimated in a l-ml aliquot of the alcoholdeproteinized reaction mixture according to the method of Marsh (12). Hydroxamale formation. In crude extracts and enzyme fractions which had ATPase act,ivit,y, hydroxamate formation was estimated. In this case also the reactio11 mixture was the same except that 400 mM NHzOH HCl (neutralized with KOH)

SYNTHETSSE

209

was replaced for NH&l. The reaction was stopped with the addition of FeCIZ reagent and optical density was measured at 540 rnp. In the assays with purified enzyme, whenever possible, asparagine formation, hydroxamate formation, and Pi liberation were all measured. /IlIP dekrmination. BI)P was est,imated according to the method of Adam (13) by coupling pyruvate kinase with NASH and lactic dehydrogenase. The nucleotides w-ere purified after adsorption on activated charcoal. An 0.5.ml aliquot of the eluate was added to 2 ml of the react,ion mixt,ure containing 50 mM Tris, 7.5; MgClz ,20 mM; KCl, 160 m&f; phosphoenolpyruvate sodium salt (Sigma), 1.6 mat; NADH, 200 PM; 20 rg lactic dehydrogenase (Sigma) and 20 pg pyruvate kinase (Sigma). The OD change at 366 rnp was measured in a Beckman DB spectrophotometer. To confirm t)he formation of asparagine 10 &i of nL-aspartic acid-&X (Schwarz Bioresearch Inc) of specific activity 11.5mCi/mmole was added to standard assay reaction mixture with and without NHa+. After the incubatiou time the reaction was stopped with the addition of 0.5 ml 107,TCS. The supernatant portion, after centrifuging ollt the precipitate, was nerhtralized with KOH. The asparagine was separated from aspartic acid by chromatography on acid alumina as described by Bessman (14). An aliquot of the combined filtrate was plated and counted on a Tracerlab windonless gas flow counter SC-16. For total activity an aliquot of TCA supernatant uortion of a reaction mixture without NH&+ was used. Protein was det,ermined by hiuret procedure (15) Enzyme purijkation. 4 uuit of enzyme is defined as the amount of enzyme which catalyzes the formation of 1 j.mole of asparagine, P-aspartyl hydroxamate, or Pi per hour, under the standard assay conditions meutioned above. Specific activity refers to units per milligram of protein. .411 operations were carried out betweeu O-5’. Centrifugations were at 27,000g for 15 min. The potatoes used for these strldies were bought from the local market and stored at 0” before use. the potatoes were thawed to room temperatrlre before y-irradiation. They were t hell irradiated it! (iamma Cell 220 (Stomic Energy of Canada Ltd.) at a dose rate of 2780 rads/min. The trlbers received a 10.krad dose. After this, these potatoes were incubated at room temperature for 21 hr. Four hundred grams of irradiated potatoes were cut into small pieces and blended in a precooled Waring Blendor with 200 ml of cold distilled wat,er containing 1OV M reduced glutathione (GSH). The slurry was then squeezed through a double layer of cheesecloth and centrifuged. The srlpernata1lt flrlid was dialyzed against cold dis-

210

NAIR

tilled water containing 1V M glutathione (GSH) for 4 hr. To 200 ml of dialyzed supernatant fluid, 62.6 g of solid (NH&S04 was added. Precipitated proteins were discarded after centrifugation and 42.8 g of solid (NH&S04 was stirred in to the supernatant, fluid. The solution was kept for 10 min and centrifuged. The precipitated proteins were then dissolved in 10 ml of 0.05 M Tris-hydrochloride buffer pH 7.2, containing 1(r3 M GSH and dialyzed with stirring against 2 liters of glassdistilled water containing 1OW M GSH for 3 hr, with change of water at 45.min intervals. Finally, this was dialyzed against 1 liter of 0.05 M Trishydrochloride buffer, pH 7.2. containing 5 X 1OW M GSH for 1 hr. The total volume of the dialyzed fraction was 12 ml. Ten milliliters of this dialyzed solut,ion was passed through a column of Sephadex G-100 (19 X 1.4 cm) and the enzyme elut,ed with 0.05 M Tris-hydrochloride buffer, pH 7.5, containing 5 X lo+ M GSH. Five-milliliter fractions were collected. The enzyme activity was present in fractions G to 10 These fractions were pooled and the enzyme was precipitated with (NH&S04 To 25 ml of solution, 4.4 g of solid (NH~)~SOI was added and precipit,ated proteins were separat’ed by centrifugation. To t,he supernatant fluid 6.825 g of (NH&SO4 was added. The precipitated proteins were collected by centrifugation, dissolved in about 1 ml of 0.05 M Tris buffer, pH 7.2, containing 1W3 M GSH. The solution was then passed through a column of Sephadex G-25 (34 X 0.5 cm) and t’he protein band was eluted with 0.05 M Tris buffer, pH 7.5, containing 5 X 10-d M GSH. The final volume of t.he fraction was adjusted to 4 ml with the same buffer. The enzyme thus prepared was stable when stored at -20” for about. a week. RESULTS

AND

DISCUSSION

TABLE EFFECT

OF y-IRR.~DI.ITION SYNTHISTISE;

I ON ‘PHI,; ASP.IRAGINE ACTIVITY"

Dose (krad)

Specific activity

Control 1 5 10 25 50 100 500

0.20 0.80 1.08 1.40 1.00 0.80 0.20 0.00

0 In this experiment the potatoes were given different doses of r-irradiation and incubated for 24 hr at room temperature. The enzymes used here were purified only up to the first (NH&SO.I step. Activity was determined as the amount of hydroxamate formed in the presence of NHzOH HCl. TABLE PURIFICATION

II

OF ASPARaGINIC

SYNTHETISE"

Fraction

Crude 5O-8O0/0 (NH&SO4 fraction Sephadex G-100 eluate, 30-70°j0 (NH&S04 fraction

160 144 115

.08 1.20 3.50

a Enzyme activity was estimated as pmoles hydroxamate formed per hour. Experimental conditions were the same as mentioned in Experimental Procedures except. that 400 mM NH20H KC1 was subst.it,uted for NH4+ in the reaction mixture.

When the enzyme was isolated from unirradiated potatoes, the activity was comparatively low. With a sprout-inhibiting dose of 10 krad of r-irradiation, when given to potatoes, the increase in specific activity was about seven times that of control. The data given in Table I show that 10 krad is the most effective dose in the activation of the enzyme and the activity gradually decreases as the dose absorbed is increased. At 500 krad, there was no enzyme activity.

synthesis of the enzyme. It is more probable to assume that the enzyme is released from binding by breaking of the hydrogen bonds or reducing the disulfide groups by utilizing the energy of radiation. However, further experiments are necessary to clarify this point. The recovery of the enzyme and increase in total activity during a typical purification are given in Table II. The specific activity was increased about 47-fold, with a recovery of 70% of the total activity. Reaction products. The product of the

This

reaction

Isolation

increase

and purification

in activity

upon

of the enzyme.

r-irradiation

may either be due to a release of the enzyme from proenzyme, to hydrolysis of proteins induced by radiation, unmasking of the active center of the enzyme, or a de novo

was

established

as

L-asparagine

after confirmation by paper chromatography of the reaction mixture. The experiment in which NH, was omitted did not show any brown ninhydrin-reacting spot (Table III).

ASPARAGINE

The mechanism of asparagine synthetase has been controversial. The mechanism of its synthesis in higher organisms reported by Webster and Varner (5) represents a reaction similar to glutamine synthesis where ATP gave rise toADP and Pi. But two recent reports on the enzyme from bacterial sources showed that the reaction is a pyrophosphate cleavage of ATP; AMP, and PPi being the products of the reaction. In the present case, with the potato enzyme, ADP was the only product detected after chromatography of the reaction mixtures. On Whatman No. 3 paper, the chromatography of the reaction mixture with and without aspartic acid (solvent: isobutyric acid, 100 ml; water, 55.8 ml, NHdOH (O.SSO),4.2 ml; and versene (0.1 M) 1.6 ml; at pH 4.6) showed no spot corresponding to AMP. The RF values of TABLE

Substance

chromstographed

III

Mean RF value (What-

Complete

reaction

mixture

Reaction NH,

mixture

minus

SYNTHETGE

Ni;Fo$in

No.3)

1G 42

L-aspartate L-asparagine

jl6 \40 16

Blue Brown (Blue ‘IBrown Blue

a The solvent used was phenol NHdOH and water; (phenol solvent 160 g phenol + 400 ml water), 200 ml; ammonia (O.SSO) 1 ml. The reaction mixture was precipitated with 4 ml of et,hanol and concentrated on water bath. TABLE RUXJIR~MENTS

Complete Aspartate NHb+ MgClz ATP

IV

FOR ASPARAGINI”

Omission of reagent’l

system

Asparagine formed

Time

(mini

0 10 30 GO

HYNTHEBIS Pi;20bez;yd

Gmoles)

2.2 0.0 0.0 0.0 0.0

2.54 0.40 0.35 0.29 0.00

ci Experimental conditions were same as standard assay except that omission of these reagents was made in the react,ion mixtr~re.

V

OF ASPMLIGINI~:

SYNTHI~XISE~

Asparz2agresfrmed

Pi formed (~moles)

0.0 1.18 1.78 2.00

0.0 1.24 1.85 2.10

I‘ In these experiments the ATPase activity; i.e., Pi liberated in the absence of aspartate, was subtracted from the esperimetlt,al vallle to arrive at the results of phosphate formation. The asparagine formed was estimated as described under Experiment,al Procedure. TABLE

VI

RIXOVISRY OF ADDKD ASP~RLGIXE ENZYME KFLXTION MIXTURIV Amount of L-asparagine

man

Authentic Authentic

TABLE STOICHIOMETRY

taken

CHRONATOGR.~PHY OF ASP~R:IGINE REXTION PILODUCT~

211

SYNTHETASE

(pmoles)

0.30 0.50 1.00 2.00 3.00 4.00

monr

Amount of asyaragine recoxred 'umdesj

0.28 0.48 0.9G 2.04 2.9G 3.sci

93 9G YG 102 98.5 99

n Asparagine at different concentrations given above, was added into 1 ml of reaction mixture containing 25 mM Tris buffer, pH 7.2, and 700 pg of enzyme protein. This was incubated for 1 hr at 35”. The reaction was stopped with 4 ml of ethanol. A 3-ml aliquot of the deproteinized supernatant portion was taken for estimation of asparagine as described under Experimental ProcedLLre.

ATP, ADP, and AMP were 47, 56, and 67 respectively. The experiment in which aspartic acid was present gave a spot with an RF value of 57. There n-as no destruction of AMP, when AMP \vns incubated, with and without ATP with reaction mixture and assayed at 260 rnk after separation of nucleotide with paper chromatography. The final purified enzyme was devoid of any pyrophosphatase activity. The asparagine formation required the presence of ATP, ibIg*+, and NHh+ and aspartic acid. The enzyme had some ATI’ase activity. So there was some Pi liberation in the case when ATP alone was present (Table IV). The enzyme reaction showed a

212

NAIR

one-to-one mole stoichiometry of the products, i.e., asparagine and Pi formation (Table V). The results of the recovery (Table VI) of added asparagine from an enzymereaction mixture, show that there is 100% recovery of added asparagine. This confirms the validity of this assay method. From these observations, it was concluded that cleavage of ATP in the case of potato enzyme is to ADP and Pi. ATP

+ I,-Aspartate + NH, + L-asparagine + AI>P + Pi

Cl?

To substantiate the mechanism of potato asparagine synthetase, as shown in Eq. (l), quantitative determination of ADP formed was carried out. The data given in Table VII show that there is an equivalent amount of ADP formed per mole of asparagine synthesized. Since the enzyme showed some ATPase activity, there was some ADP formed when NHh+ was omitted from the reaction mixture. This is equivalent to the amount of Pi liberated (Table IV) when aspartate or NHa+ was omitted from the reaction mixture. The hydroxamate formation and asparagine formation also showed good agreement. To check the intermediate formation of P-aspartyl phosphate (4) the enzyme reaction was conducted with and without hydroxylamine. In the case where hydroxylamine was omitted the reaction was stopped with the addition of P-chloromercuriphenyl sulfonic acid and hydroxylamine was then TABLE STOICHIOMETRY

VII

OF ADP Hydnxaate_

FORMATION” ADP formed (pm&s)

Time (min)

Asfya$ne (fimoles)

(qnoles)

With NHa+

w:IPt a+

30 GO

1.58 2.2

1.42 2.0

1.G 2.39

0.36

ClThe reaction mixtllre employed was the same as described under Experimental. NHzOH HCl 400 rnM (neutralized with KOH) was used instead of NH&l in the case when hydroxamate formation was determined. ADP was determined in an aliquot of the reaction mixture purified after act)ivated-charcoal chromatography of nucleotides by coupling with pyruvate kinase, NADH, and LDH.

TABLE FORMATION

VIII

OF RADIOACTIVE ASPARSGINE DL-ASPARTIC AcID-G~Q

Experiment

Total activity

in the reaction

mixture

(dpm)

Activity in asparagine (dpm)

FROM

Amount of asparagine formed bnoles)

2.8 X lo6 With NH*+ 1 2 Without NH4+ 1 2

2.8 x 105 3.4 x 105

2.2 2.3

842 942

a To t,he reaction, same as described under Experimental Procedure, 10 &i of oL-aspartic acid-4-l% was added. NHd+ was omitted in some cases. The asparagine formed was separated from aspartic acid by chromatography of the TCAprecipitated and neutralized reaction mixture on acid alumina column. The radioactivity of the combined eluate was determined.

added. There was no hydroxamate formation in this case. This eliminated P-aspartyl phosphate as an intermediate in the reaction. Further confirmation of asparagine as the reaction product was obtained from the formation of radioactive asparagine from m-aspartic acid-4-14C. The data in Table VIII clearly show that only L-aspartic acid took part in the reaction. The yield of asparagine calculated, by taking into consideration that only L-aspartic acid underwent reaction, was 22%. This agreed well with the determination of asparagine formed in the reaction also. Substrates. Under the experimental conditions described with Fig. 1, the KM value for L-aspartic acid was calculated to be 2.6 X 10e2 M. This enzyme did not show any glutamine synthetase activity. When 10 pmoles of L-glutamic acid was replaced in the reaction mixture, it failed to show hydroxamate formation or glutamate-dependent Pi release. In the presence of 10 pmoles of L-aspartate, the KM value for ATP and RIg2+ ion were calculated to be 1 X 1O-2‘M and 4 X lo-” M, respectively (Fig. 2a, b). The KM value for NH4 determined from Lineweaver-Burk plot was found to be 4 X 1OW M. (Fig. 3) NH2OH could replace r\‘Hd+. Figure 4 shows that

ASP.41:A(:INE

‘18

SYNTHETASE

nature. The potato enzyme was also inhibited by asparagine to a lesser degree (Table IX) compared to bacterial enzymes. Since this reaction resembled that of glutamine synthesis, the transferase activity of this enzyme was tested. When aspartic acid was replaced with 10 pmoles of Lasparagine, in the presence of either ATP or 5 pmoles of ADP and 25 pmoles of inorganic phosphate and 400 pmoles of hydroxylamine HCl, no hydroxamate formation was observed. Metal requirements. The effects of ?tIg2+ ions on the reaction at various pH values FIG. 1. The double reciprocal plot for the effect of L-aspartate concentration on the reaction. The reaction mixture was the same as described under ExperimeJJtal Procedure except that various coJJcentrations of aspartate were added to the reaction mixture. Both asparagine formation and phosphate liberation were tested for activity determirlations.

I”----- -7

/‘j

i

I.Oi

,’

/

FIG. 3. Liueweaver-Burk plot for the elect of NH*+ coJJcerJtration on asparagine synthetase.

0

0 ‘1s

0 I

02

0.3

0.4

0.5

‘1s

J

FIG. 2. a, b The double reciprocal plots for the effect of ATP (a) and MgClz(b) on asparagine FYJJt.hesis. maximum activity was obtained only at 400 ml4 NH,OH concentration. Here the activity was expressed as the percentage of the activity observed with 30 lllM NH,+; increasing concentrations of lYH,OH inhibited the reaction. E$ect of asparagine on the reaction. The bacterial enzymes (S, 9) were inhibited by aspa.ragine. Burchall et al. (9) reported 55 5% inhibition with aspartate/asparagine ratio of 50 with the Stre$ococcus enzyme. They have also shown that the inhibition is of the competitive type whereas in the Lactobacillus arabinosus enzyme, the inhibition was found to be noncompetitive in

!I .&20” Ii dLL2LL-J 200

400

mM

600

800

NH,OH

FIG. 4. The effect of differelit coJJceJJtration of NH&H HCl on the reaction. 111 the startdard assay mixture various corJceJJtratioJJs of NH&H were added instead of NH,+. In this case, the activity was determined as hydroxamate formed. The activit,y was expressed as the percentage of activity observed with starJdard assay mixture containiJJg 30 my NH4+.

TABLE INHIBITION

IX

TABLE

OF ASPMUGINE SYNTHETMG WITH ASPAH~GINE~

L-asparagine nlM

Aspartate Asparagine

None 0.2 0.5 1.0 5.0 10.0 20.0

50 20 10 2 1 0.5

EFFECT

Supplement

O/oInhibition

14 18 27 34 41 64

a The asparagine was incubated with t,he enzyme for 5 min before the addition of aspartate. Standard assay mixture was used. Pi formed was only estimated in this case.

FIG. 5. The effect of pH on asparagine thetase in the presence of Mg2+ (15 rmoles) MnZ+ (15 /Imoles).

sy11-

and

were studied. The pH optimum for the enzyme varied with respect to the metal ion present in the medium. With Mg2+ the pH optimum is 7.2 whereas with Mn2+ the optimum is shifted to pH 6.0 (Fig. 5). At pH 7.2 the activity with Rln2+ is only 60% of that at 6.0. The activity decreases sharply at high pH level; this may be due in part to precipitation of ;\/In2+ as Mn(OH):! . With Mg2+ there was excellent activity even at pH 9.0. EDTA completely inhibited the reaction. Fluoride and molybdate were other potent inhibitors. ADP, which was competitive inhibitor for glutamine synthesis, did not inhibit the reaction even after preincubation with enzyme and Mg2+ ions. Sulfhydryl requirement for the reaction was shown with p-chloromercuribenzoate-inhibition of the enzyme activity. This is also inferred from the observation that GSH

X

OF DIFFERENT ADDITIONS ASP.\R.IGINE SYNTHICT.UIP Concentration (mu)

None cu so4 Zn SO4 EDTA Na fluoride benzoate P-chloromucri Na molybdate Disodium hydrogen arsenate ADP

2 2 5 2 5 2 2 5

ON

% Inhibition

20 40 100 GO 90 80 20 0

a Enzyme assay procedure was same as described under Experimental Procedure except that these substances were added at above-mentioned concentrations.

protected the enzyme from inactivation during purification (Table X) . The asparagine synthetase of potato showed characteristics of the synthetase reaction similar to those reported for higher plants like peas, lupine seedlings, etc., in that this resembled also the glutamine synthetase, but was devoid of any transferase activity. It differed from the bacterial asparagine synthetase, which represented an unique reaction of ATP activation. Usually when ATP activation involves pyrophosphate cleavage, the product is an adenylate substrate complex; however, in bacterial asparagine synthetase, there is no indication of the formation of adenylate derivative of aspartic acid as an intermediate. In the case of the potato asparagine synthetase, no AMP or pyrophosphate was formed. On the other hand, there was good stoichiometry between asparagine formed and inorganic phosphate liberated. The fact that the enzyme activity was increased by r-irradiation posed two possibilities, either a radiation-induced synthesis of new enzyme protein or a hydrolysis or release of bound protein from an inactive complex. Further work to examine these possibilities is in progress. ACKNOWLEDGMENTS The author thanks Dr. A. Rreenivasan for his helpful suggestions and criticisms. Mr. K. K. Ussuf provided valuable assistance in this study.

ASPAKAGINE

21.5

SYNTHETASE S., .\su

SHIVI,:.

W.,

J. Kiol.

Chem. 237, 2815

(1962). of Amino 1. Ml~:rs’rlzll, A., in “Biochemistry Acids,” II ed, Vol. I p. 457. Academic Press, New York, (1965). 2. BL.\cK, S., .\ND GRAY, K;. N., .J. Am. Chem. Sot. 75, 2271 (1953). :(. BL.\c~Ii,

s.,

.\ND WRIGHT,

N. c;.,

d. AnL.

Chena.

?j. (i.,

J. Hid.

Chen~.

Sot. 75, 57% (1953). 4. BLACK,

S., .\x‘D WRIGHT,

213, 27; 39; 51 (1955). 5. WI.;IISTI
9, BURCHALL, J. J., Rr:lcxu,T, E. C., AND WOHIN, M. J., J. Hiol. Chem. 239,1794 (1964). E’., \ND TIJ.~TI.I.;, I,. C., ,I. nio/. 10. LIPM.\NN, Chenz. 159,21 (1945). 11. 12. &kRSIi, B. B., Hiorhim. Biophgs. Acta 32, 357 (1959). 13. Au.\?n, H., in “Methods of Enzymatic Analgsis” (H. U. Bergmeyer, ed.), p. 573. Academic Press, Sew York (1963). in Enzymology” 14. BI:SSSL~X, S. P., in “Methods (S. P. Colowick and N. 0. Kaplan? eds.), Vol. III, 1~. 575. Academic Press, New York (1957). A. (;., T~a~v.i\\~lCl~~,, c. J., AND 15. ~:OIIN.\I,I,, I).\wD, M. M., J. Riol. Chew 177, 751 (1949).