identification of the antimetabolite of l-alanosine - cancer research

[CANCER RESEARCH 40, 4390-4397, December 1980]0008-5472/80/0040-OOOOS02.00

Identification of the Antimetabolite of L-Alanosine, i_-Alanosyl-5-Amino-4-

Imidazolecarboxylic Acid Ribonucleotide, in Tumors and Assessment of

Its Inhibition of Adenylosuccinate Synthetase

Anil K. Tyagi and David A. Cooney

Biochemistry Section. Laboratory of Toxicology. Division of Cancer Treatment. National Cancer Institute. NIH. Bethesda. Maryland 20205

ABSTRACT

The conjugate of L-alanosine [L-2-amino-3-(/V-hydroxy/V-ni-trosamino)propionic acid] and 5-amino-4-imidazolecarboxylic

acid ribonucleotide has been synthesized in good yield byenzymatic means, using partially purified chicken liver 5-amino-4-imidazole-A/-succinocarboxamide ribonucleotide synthetase

(EC 6.3.2.6). The Chromatographie behavior of this moleculewas characterized, as was its ability to inhibit adenylosuccinatesynthetase, an enzyme long considered to be the locus ofaction of the drug. The KÂ¡of-L-alanosyl-5-amino-4-imidazole-

carboxylic acid ribonucleotide versus a partially purified adenylosuccinate synthetase from the L5178Y/AR leukemia ofC57BL x DBA/2 F, (hereafter called BD2F,) mice was 0.228/IM, whereas the K, of L-alanosine was 57.23 mw. Administration of 50 Â¡iC\of DL-{1-t'tC]alanosine along with unlabeled L-

alanosine (500 mg/kg) to BD2Ft mice bearing s.c. nodules ofLeukemia L5178Y/AR resulted in the accumulation in tumorsof a material with properties compatible with those of L-alano-syl-5-amino-4-imidazolecarboxylic acid ribonucleotide. It coe-luted with L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide in the high-resolution Chromatographie systemused, was Bratton-Marshall positive, and inhibited adenylosuc

cinate synthetase strongly. In tumor nodules 2 hr after dosage,the concentration of this compound approximated 70 /Â¿M.Under the same circumstances, the intratumoral concentration ofL-alanosine was found to be 440 fiM. At this concentration, theantibiotic itself exerts only a marginal inhibition of leukemicadenylosuccinate synthetase. In ancillary studies, it was shownfor the first time in vivo that the parenteral administration of L-

alanosine reduces the specific activity of intratumoral adenylosuccinate synthetase by 70% and depresses the synthesis ofDNA to an equivalent or greater extent; adenine but not hypo-

xanthine (both at 250 mg/kg) was able to reverse the latterinhibition. No effect on purine salvage enzymes was exertedby L-alanosine. Viewed in concert, these experiments establishthat the adduci of L-alanosine with 5-amino-4-imidazolecarbox-

ylic acid is formed by neoplastic cells in vivo and that thisanabolite is most probably responsible for the inhibition ofadenylosuccinate synthetase and, in turn, for the diminishedsynthesis of DNA seen after a therapeutic dose of L-alanosine.

INTRODUCTION

Although it has been postulated repeatedly that the activemetabolite of antitumor agent L-alanosine [L-2-amino-3-(A/-hy-droxy, A/-nitrosamino)propionic acid] is the adduci of the anli-

biolic wilh AICOR1 (Chart 1), allempls to demonstrate this

molecule in tumors have, thus far, met with failure (10, 19).However, because its identificalion is cenlral lo any explanalionof Ihe mechanism of aclion of L-alanosine, we have undertaken

addilional, more comprehensive studies on the in vivo formationof L-alanosyl-AICOR. Leukemia L5178Y/AR, growing as s.c.

nodules in the flanks of C57BL x DBA/2 F, (hereafter calledBD2Fi) mice, has been used for these investigations becauseof its clear sensitivily lo L-alanosine.

It will be demonstraled lhat Ihe concentration of unaltered L-

alanosine in such tumors is inadequate lo explain Ihe biochemical effecls of the drug, and that, under the experimentalconditions used, a compound wilh chromalographic and chro-mogenic properties of the pulalive adduci of L-alanosine wilh

AICOR is, in fact, present in the neoplasms at a concentrationwhich ought to engender lotal inhibition of the target enzyme,adenylosuccinate synthetase. Ancillary experiments will alsobe described which fortify this conclusion and quantify theexlenl to which L-alanosine, after its anabolism, inhibits the

formation of adenylosuccinic acid in vivo, thereby restriclingthe concentralion of adenine nucleolides required for the synthesis of DNA in the actively dividing cells of this leukemia.

MATERIALS AND METHODS

L-Alanoslne (NSC 153353) was obtained from the DrugResearch and Development Branch, National Cancer Institute,Belhesda, Md. DL-[1-14C]Alanosine (specific activity, 7.1 mCi/

mmol) was supplied by Stanford Research Institute, MenloPark, Calif. This radioactive alanosine was purified before useas described earlier (1 ). L-[4-14C]Aspartic acid (specific aclivily,

19.5 mCi/mmol) was oblained from Amersham/Searle Corp.,Arlingfon Heighls, III. Adenosine, hypoxanlhine, ribose 1-phos-

phale, GTP, and IMP were purchased from Sigma ChemicalCo., St. Louis, Mo. All other reagenls were of Ihe highesl purilyavailable.

Scalable polypropylene test tubes (1600 /il), producÃs ofBrinkmann Inslrumenls, Inc., Weslbury, N. Y., were used forall enzyme assays (6).

Animals. Male BD2F, mice on an ad libitum diel of Purinamouse chow were used during the course of these studies.

Preparation of Homogenates. BD2F, mice bearing 10-day-

old L5178Y/AR s.c. tumors were killed by cervical dislocalion;

Received March 10. 1980; accepted June 3, 1980.

' The abbreviations used are: AICOR, 5-amino-4-imidazolecarboxylic acid

ribonucleotide; L-alanosyl-AICOR, L-alanosyl-5-amino-4-imidazolecarboxylicacid ribonucleotide; IMP. inosine 5'-monophosphate; DTT, dithiothreitol;TCA.trichloroacetic acid; PRPP, 5-phosphorylribose 1-pyrophosphate; HEPES,4-{2-hydroxyethyl)-1-piperazineethanesulfonic acid; SAICAR, 5-amino-4-imidaz-ole-A/-succinocarboxamide ribonucleotide.

4390 CANCER RESEARCH VOL. 40

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Mechanism of Action of L-Alanosine

HOOC-CP4y

\

ATP+ALANOSINE ADP+Pj

SAICAR SYNTHETASE

/NO

CH, 0I 2 II

Hâ€”Câ€”NHâ€”C-

AICOR RIBOSE-(Â¡

ALANOSYL-AICOR

GTP+ASPARTATE

ADENYLOSUCCINATE SYNTHETASE

IMP

NH

#^C/\

II CH^Â°V

IRIBOSE-Â©

ADENYLOSUCCINIC ACIDChart 1. Inhibition of adenylosuccinate synthetase by the putative active

anabolite of L-alanosine.

the tumors were removed, frozen, and homogenized (1:3, w/v)in 0.1 M Tris-HCI buffer (pH 7.6), with 0.5 HIM EDTA and 1 mMDTT. For measurement of the concentrations of L-alanosineand L-alanosyl-AICOR, the tumors were homogenized in 1 N

acetic acid (1:3, w/v). For measurement of the purine salvageenzymes, the tumors were homogenized in 0.1 M Tris-HCI

buffer (pH 7.0), containing 10 mM DTT. Homogenates werecentrifuged at 12,000 x g lor 3 min and the supernatant wasused as enzyme source.

Protein Estimation. Protein was estimated using the Bio-Rad kit for protein determination (2). Ten-/ul aliquots of a

suitably diluted supernatant solution were added to 1.0 ml ofdye solution, prepared, and diluted according to the manufacturer's directions. The absorbance at 595 nm was then meas

ured within 5 to 60 min. Aliquots of a solution of bovine albumin,Fr 5, (1 mg/ml), were used as standards.

Concentration of L-Alanosine in L5178Y/AR Tumors.L5178Y/AR s.c. tumors were transplanted in BD2F, mice. Tendays later, the animals were fasted overnight and treated withalanosine(DL-[1-"'C]alanosine, 10ftCi/mouse, along with cold

L-alanosine, 500 mg/kg). At appropriate intervals, groups of 6

animals were sacrificed, and the tumors were removed andhomogenized in 1 N acetic acid. The homogenates were centrifuged for 12 min at 12,000 x g. The concentration of L-alanosine was then measured in the supernatant by means ofhigh-resolution column chromatography (20) on an 8- x 700-mm column of Jeol AR-50 resin. Elution was performed at 37Â°

with 0.15 M lithium citrate buffer, pH 2.72. For monitoringelution times, effluents were directed through a flow cell witha 6-mm light path, and the absorbance was measured at 280nm in the Jeol UV photometer with an electronic integrator.Alanosine eluted from this column at 48 min. Fractions of 3.35ml were collected, and radioactivity of the peak fractions wascounted in a Beckman LS-230 liquid scintillation spectrometer,the concentration of L-alanosine in the tumors was calculated

on the basis of specific activity of the final injection material.Effects of L-Alanosine on DNA Synthesis. One million

L51 78Y/AR cells were implanted s.c. into BD2F! mice. After10 days, the animals were fasted overnight and divided into 6groups of 6 animals each. The first group received L-alanosine(500 mg/kg) i.p. The second and third groups received ade-

nine and hypoxanthine (250 mg/kg), respectively, in additionto L-alanosine. The fourth and fifth groups received only ade-

nine and hypoxanthine (250 mg/kg), respectively. The lastgroup received -0.25 ml of 0.9% sodium chloride solution.After 4 hr, all animals were given 5 /tCi of [2-'"C]thymidine. One

hr later, the recipients were sacrificed, and tumors were removed, frozen, and later homogenized in 5% TCA. After cen-trifugation for 3 min at 12,000 x g, the pellet was washed 3times with 1% TCA. Aliquots of 1 ml of 10% TCA were thenadded to the pellets, mixed well, and heated at 95Â°for 1 hr in

closed Eppendorf tubes. On cooling, the vessels were centrifuged at 12,000 x g for 3 min. A suitable aliquot of thesupernatant was then taken for estimation of radioactivity andmeasurement of DNA (11 ).

Hypoxanthine and Adenine Phosphoribosyltransferase.The following reagents were admixed in Eppendorf tubes: 10/Â¿Iof [8-14C]hypoxanthine (6 nmol, 332 nCi), 10 fil of PRPP and

MgCI2 (60 and 180 nmol, respectively, in 0.2 M Tris buffer, pH8.0); and 10 /il of tumor extract to initiate the reaction. Incontrol vessels, PRPP was replaced by 10 /il of 0.2 M Trisbuffer, pH 8.0. Incubation was carried out at 37Â°for 30 min;

thereafter the reaction was stopped by adding 20 fil of 2 N HCI.After a 3-min centrifugation at 12,000 x g, 5 ft! of the super

natant were spotted on Whatman 3M paper and overspottedwith 5 /Â¿Iof a mixture of IMP and hypoxanthine (0.01 M each).Ascending chromatograms were developed using 1 M ammonium acetate (pH 7.0):ethanol (30:70, v/v), as solvent for 16hr. The IMP spots were then excised and their radioactivity wasmeasured. For calculation of enzyme activity, the radioactivityin the vessels lacking PRPP was subtracted from that measuredin the vessels receiving this substrate.

In the case of adenine phosphoribosyl transferase, [8-14C]hypoxanthine was replaced by 10 /ti of [8-14C] adenine (6.0

nmol, 314 nCi), and the chromatograms were overspotted withadenine and AMP, developed, and processed as describedabove.

Adenosine Kinase. In Eppendorf test tubes were admixed 5/il of [8-14C] adenosine (7 nmol, 455 nCi), 5 fil of 0.05 M HEPES

buffer (pH 6.8), 5 fil of ATP and MgCI2 (20 and 6 nmol,respectively, in 0.05 M HEPES buffer, pH 6.8), and 5 fil ofenzyme extract. Control vessels received HEPES buffer insteadof ATP and MgCI2. The reactants were incubated for 15 min at37Â°, at which point 20 /tl of 2 N HCI containing 0.01 M AMP

were added to arrest the reaction. Five-/il aliquots of the re

sultant reaction mixtures were then spotted on Whatman 3Mpaper. Ascending chromatography was performed using iso-propyl alcohol:EDTA (saturated solution):toluene:14 M NH4OH(320:44:40:4, v/v) as solvent for 16 hr (12). The UV-absorbingspots of AMP at the origin were excised, and their radioactivitywas measured by scintillation spectrometry.

Inosine Phosphorylase. In Eppendorf tubes, the followingwere admixed: 10 /il of [8-'"C]hypoxanthine (8 nmol, 442 nCi);

10 fil of ribose 1-phosphate (300 nmol in 0.1 M Tris buffer, pH

7.0); and 10/il of enzyme extract. Control vessels received 10/il of 0.1 M Tris buffer, pH 7.0, in place of ribose 1-phosphate.All vessels were incubated at 37Â°for 15 min, and the reaction

was stopped by adding 20 fil of 2 N HCI. For the separation ofhypoxanthine and inosine, 5-fil aliquots of these reaction mix

tures were spotted on Polygram CEL 300 polyethyleneimineplates and overspotted with 5 fil of a mixture of inosine andhypoxanthine (0.01 M each). The plates were developed indistilled water for 2 hr (14), and spots of inosine were excisedfor scintillation spectrometry.

DECEMBER 1980 4391



A. K. Tyagi and D. A. Cooney

Adenosine Phosphorylase. The following were admixed: 5/il of [8-'"C]adenosine (7 nmols, 455 nCi); 5 /il of 0.2 M HEPES

buffer, pH 7.0; 5 /il of 0.05 M potassium phosphate solution,pH 7.0, and 5 /tl of enzyme extract. Control vessels contained5 /il of HEPES buffer in place of phosphate. The vessels wereincubated at 37Â°for 15 min, and the reaction was stopped by

adding 20 /il of 2 N HCI; 5-/il aliquots of these reaction mixtures

were spotted on Whatmann 3M chromatography paper andoverspotted with 5 /il of a mixture of adenine and adenosine(0.01 M each). The chromatograms were developed in anascending manner using 1 M ammonium acetate (pH 7.0):alcohol (30:70, v/v) as solvent for 16 hr. The UV spots ofadenine were excised, and their radioactivity was measured byscintillation spectrometry.

Adenylosuccinate Synthetase Activity. Adenylosuccinatesynthetase activity was measured according to a publishedprocedure (1 ). Briefly, in a total volume of 20 /il were admixed:5 /Â¿Iof L-[4-14C]aspartic acid (6.37 nmol, 125 nCi); 5 /il of GTP:

MgCI2 (150 nmol each); 5 /tl of IMP (150 nmol); and, to initiatethe reaction, 5 jul of tumor extract. Controls were constitutedby replacing IMP with 5 /tl (200 nmol) of Tris-HCI buffer, pH8.0. Incubation was carried out for 30 min at 37Â°.The reactionwas stopped by heating the vessels at 95Â°for 2 min. Unreacted

L-aspartic acid was then decarboxylated by the addition of 50/ti of a decarboxylation reagent (17) consisting of 0.005 M a-

ketoglutarate and 0.03 M zinc sulfate in 0.66 M sodium acetatebuffer, pH 5.0; to this solution, 40 ILJ/ml of L-glutamate-oxal-

oacetate transaminase were added at the time of use. After 3hr at 37Â°,the radioactive residues in the vessels were counted

by scintillation spectrometry in a Beckman LS-230 scintillation

spectrometer.Inhibition of Adenylosuccinate Synthetase from L5178Y/

AR by L-Alanosine in Vivo. One million L5178Y/AR cells wereimplanted s.c. in the flanks of BD2F, mice. Ten days later, theanimals were fasted overnight and divided into 2 groups. Thefirst group received an i.p. dose of L-alanosine (500 mg/kg),

and the second group received an equal volume of 0.9%sodium chloride solution. At the designated time intervals, 5mice from each group were sacrificed by cervical dislocation,and tumors were dissected and frozen on solid CO2. Within 72hr, the tumors were homogenized, and adenylosuccinate synthetase activity was measured as described earlier.

Purification of Adenylosuccinate Synthetase fromL5178Y/AR Tumors. Mice bearing s.c. nodules of LeukemiaL5178Y/AR were sacrificed, and tumors were removed andhomogenized in 0.02 M potassium phosphate buffer containing0.5 mM EDTA and 2.0 mM DTT (1:3, w/v). The homogenatewas centrifuged at 20,000 x g for 30 min. The clear supernatant was then placed in a water bath and heated at 50Â°for

5 min. Thereafter, the solution was immediately cooled in anice bath. The proteins thus precipitated were removed bycentrifugation at 20,000 x g for 20 min. The resulting supernatant was dialyzed for 5 hr against 0.001 M potassium buffercontaining 0.5 mM EDTA and 2 mM DTT and loaded onto acolumn of hydroxylapatite (2.3 x 40 cm) preequilibrated with0.001 M potassium phosphate buffer containing 0.5 mM EDTAand 2.0 mM DTT. The column was first developed with 200 mlof equilibrating buffer, followed by a linear gradient of potassium phosphate buffer from 0.001 to 0.25 M, pH 7.2 (250 mleach), containing 0.5 HIM EDTA and 2 mM DTT. Fractions of10 ml each were collected. Adenylosuccinate synthetase

eluted from this column at 0.115 M potassium phosphatebuffer. The active fractions were pooled and concentrated bydialysis against Carbowax. This enzyme preparation was 15-

fold purified over the crude and exhibited a specific activity of137.47 units/mg protein (1 unit of enzyme represents theamount of protein responsible for the production of 1 nmol ofadenylosuccinic acid per hr). The heating step was especiallyuseful for removing adenylosuccinate lyase, which uses adenylosuccinic acid as one of its substrates. We also tested thispreparation for "IMP phosphatase" activity. When 3.44 nmol

(100 nCi) of IMP were incubated with 5 /il of purified enzymeat 37Â°for 30 min less than 0.034 nmol of IMP were hydrolyzed;

this corresponds to less than 1.0% decomposition.Kinetics of Inhibition of Adenylosuccinate Synthetase in

Vitro by t-Alanosine. For this study, partially purified adenylo

succinate synthetase was used. The reaction mixture in a totalvolume of 25 /il contained: 5 /il of L-[4-14C]aspartic acid (0.78

to 12.5 mM); 5 /il of GTP:MgCI2 (0.1 to 10 mM each) in 0.05M Tris-HCI, pH 8.0; 5 /il of IMP (0.1 to 10 mM) in 0.05 M Tris-HCI; pH 8.0; or 5 /il of buffer and 5 /il of L-alanosine ( 15 to 60mw) in 0.05 M Tris-HCI, pH 8.0; or 5 /il of partially purified

adenylosuccinate synthetase from L51 78Y/AR (3.45 units).When IMP was the variable substrate (0.1 to 10 mM), the

concentration of GTP and L-aspartic acid were kept constant

at 5.0 and 10.0 mM, respectively. Similarly, the concentrationsof IMP and L-aspartic acid were fixed at 5.0 and 10.0 mM,

respectively, when GTP was the variable substrate (0.1 to 10.0mM). The reactants were mixed by a brief centrifugation at12,000 x g and incubated at 37Â°for 30 min. The reaction wasterminated by heating at 95Â°for 2 min, and incubation mixtures

were processed as detailed earlier. The kinetic constants werederived from Lineweaver-Burk plots.

SAICAR Synthetase. SAICAR synthetase was measured asfollows. In a total volume of 25 /il were admixed: 10 /il of L-[4-14C]aspartic acid (12.74 nmol, 250 nCi; 5 /il of ATP-MgCI?

(75 nmol each); 5 /il of AICOR (30 nmol in 0.1 M KHCO3) or 5/il of 0.05 M Tris-HCI buffer, pH 8.0; and, to initiate the reactions, 5 /il of enzyme extract. The reactants were mixed by abrief centrifugation of 12,000 x g, and incubated at 37Â°for 30min; thereafter, the reaction was stopped by heating at 95Â°for

2 min. The vessels were opened, 50 /il of decarboxylationreagent were added, and the assemblies were incubated at37Â°for 3 hr to dissipate residual L-[4-14C]aspartic acid. Radio

activity remaining in the vessels was then counted by scintillation spectrometry. For calculations, radioactivity in vesselslacking AICOR was subtracted from radioactivity in vesselsreceiving this additive, and the difference was taken as ameasure of enzyme activity.

Purification of SAICAR Synthetase from Chicken Liver. Apartially purified preparation of this enzyme was obtained by amodification of the method of Buchanan ef al. (4). Thus, 10 gof an acetone powder prepared from fresh chicken liver wereextracted at 2Â°by stirring for 30 min with 100 ml of 0.05 M

potassium phosphate buffer at pH 7.3. The crude extract wastreated with pancreatic RNase, 0.2 mg of a crystalline preparation being added per ml of enzyme solution. The mixture wasallowed to come to 37Â° over a period of 10 to 15 min in a

constant temperature bath. The small amount of precipitatebrought about by this procedure was centrifuged at 4Â°.Suffi

cient solid ammonium sulfate was then added to the supernatant solution to bring the salt concentration to 55% of satura-




Mechanism of Action of L -Alanosine

tion. The precipitate, which contained all the activity, wascollected by centrifugation, dissolved in 100 ml of 0.05 Mpotassium phosphate buffer, (pH 7.3), and refractionated withammonium sulfate. The protein precipitating between 25 and45% of saturation was collected by centrifugation and dissolved in 20 ml of 0.02 M phosphate buffer (pH 7.3). Thisenzyme solution was dialyzed against 2 changes of 4 literseach of 0.01 M phosphate buffer (pH 7.3) containing 1 rriMDTT. The dialyzed material was diluted 2-fold with cold water

and loaded onto a column (2.5 x 40 cm) of hydroxylapatite.The column was developed with a linear gradient of 0.05 to0.35 M phosphate buffer (pH 7.0) containing 1 rriM DTT. Theenzyme eluted at 0.18 M phosphate. Active fractions werepooled and frozen. The enzyme was purified 35.0-fold and

exhibited a specific activity of 955.0 nmol per mg protein perhr.

The synthesis of AICOR was carried out as described earlier(18).

Preparation and Purification of L-Alanosyl-AICOR. L-Alan-osyl-AICOR was prepared by condensing L-alanosine with AI

COR via the action of SAICAR synthetase from chicken liver.The following were dissolved in 200 fil of water: 6 mg ATP; 2mg MgCI2; 8.0 mg KHCO3, 3.0 mg DL-[1-'"CJalanosine (specific

activity, 7.4 mCi/mmol); and 4.0 mg AICOR. To this mixturewere added 2.8 ml of purified chicken liver enzyme, and thereactants were incubated at 37Â° for 6 hr. The incubation

mixture was then deproteinized with 0.1 N HCI and centrifugedat 12,000 x g for 12 min. A portion of this supernatant (0.8ml) was then loaded on a 0.8- x 15-cm Hamilton HA-X4 column

equilibrated with 0.015 M lithium citrate buffer, pH 2.65 (20),and operated at a flow rate of 0.67 ml/min. The column wasdeveloped with the equilibrating buffer for 90 min at 37Â°,

followed by 0.0375, 0.075, 0.15, and 0.225 M lithium citratebuffers (pH 2.72), applied for periods of 60, 60, 75, and 60min each (20). u-Alanosyl-AICOR eluted from this column as asingle symmetrical peak at ~260 min. For desalting, the peak

fractions from this chromatography were collected, lyophilized,reconstituted in 1 ml, and loaded on an 0.8- x 15-cm columnof Aminex A-14 resin in the bicarbonate form. The column was

developed with a linear gradient of 0 to 1.0 M ammoniumbicarbonate. The L-alanosyl-AICOR eluted from this column at

240 min; it was collected, frozen, lyophilized repeatedly, andstored at â€”70Â°.The yield of L-alanosyl-AICOR from this procedure was ~5%.

This preparation of L-Aalanosyl-AICOR gave a single spot on

paper electrophoresis (at 25 V/cm in 50 mvi sodium phosphatebuffer, pH 7.2) and thin-layer chromatography, using BakerFlex Silica Gel IB-F precoated plates with n-butyl alcohohaceticacid:water (5:3:2) as solvent. The compound was Bratton-Marshall2 positive (3, 16) and a very strong inhibitor of ade-

nylosuccinate synthetase. At neutral pH, it gave an absorptionmaximum at 254 nm (Chart 2). When the solution was acidified

2 The Bratton-Marshall reaction was performed as follows: To 10 pi of sample

on ice were added 20 pi OH 0 N H2SO4 and 10 pi of 0.1 % sodium nitrite. After 1min, 10 pi of 0.5% ammonium sulfamate were added to remove excess sodiumnitrite. Three min later, 10 pi of 1.0% naphthyl ethylenediamine HCI were added.L-Alanosyl-AICOR yielded a purplish color which was stable if samples weremaintained in the cold. For determining the sensitivity of this procedure, variousconcentrations of L-alanosyl-AICOR were used; it was observed that 20 nmol ofL-alanosyl-AICOR was the lower limit of detection under these experimentalconditions.

0.3

0.2

0.1

0.3

0.2

0.1

254 nm

L-Alanosyl-AICOR

269 nm

SAICAR

210 250 290 330

WAVE LENGTH Inm)

Chart 2. UV spectra of L-alanosyl-AICOR and SAICAR. The spectra wererecorded in 0.025 M Tris-HCI buffer. (pH 7.0) and a 1-cm light path.

to pH 2 with HCI, there was a 30% loss in absorbance eventhough it retained the same Amax.Based on the specific activityof the radioactive alanosine used to synthesize it, this compound had a molar extinction coefficient of 5.75 x 10".

Identification and Concentration of u-Alanosyl-AICOR inL5178Y/AR Tumors. Mice bearing Leukemia L5178Y/ARwere treated with DL-[1-'4C]alanosine, 50 ftCi/mouse, along

with cold L-alanosine, 500 mg/kg. After 2, 4, and 8 hr the

animals were sacrificed, and tumors were removed, frozen,homogenized in 1 N acetic acid, and centrifuged for 12 min at12,000 x g. A portion of the resulting supernatant (0.8 ml) wasloaded on a 0.8- x 15.0-cm of column of Hamilton HA-X4 resin,

and elution was performed as described above. The concentration of L-alanosyl-AICOR was calculated on the basis of the

specific radioactivity of the injection material.Lithium citrate, used for eluting the above column, inhibits

adenylosuccinate synthetase. Hence, for preparing L-alanosyl-

AICOR from tumors to test on adenylosuccinate synthetase, a0 to 1.0 M ammonium bicarbonate gradient was utilized. Thus,tumors were homogenized in 1 N acetic acid, the pH wasadjusted back to 7.0 with HCI, the extracts were clarified bycentrifugation, and a portion of the supernatant (0.8 ml) wasloaded on a Hamilton HA-X4 column (0.8 x 15.0 cm; HCO3

form) preequilibrated with distilled water. The column wasdeveloped with a 0 to 0.1 M ammonium bicarbonate gradient(100 ml each). L-Alanosine and L-alanosyl-AICOR eluted in thissystem at 75 and 240 min, respectively. Fractions of 3.35 mlwere collected. A 500-/Â¿laliquot from each fraction was taken

for the measurement of radioactivity, and the remainder waslyophilized. The products of lyophilization were reconstitutedin 1.0 ml of water and relyophilized to remove traces of ammonium bicarbonate. All fractions were then separately reconstituted in 25 /il of water. To test these fractions for the inhibitionof adenylosuccinate synthetase, the following were admixed ina total volume of 25 p.\:5 Â¡i\of L-[4-14C]aspartic acid (6.37 nmol,

125 nCi); 5 jul of GTP-MgCI2 (1 25 nmol each); 5 /il of IMP (125

DECEMBER 1980 4393




nmol) or 5 /il of 0.05 M Tris buffer, pH 8.0); 5-/tl aliquot from

each reconstituted fraction; and, to initiate the reaction, 5 /il(3.45 units) of adenylosuccinate synthetase partially purifiedfrom Leukemia L5178Y/AR. The reaction was carried outexactly as described earlier. Inasmuch as the concentration ofL-alanosyl-AICOR was low, the remaining 20-jal aliquot of every

4 consecutive fractions were pooled, relyophilized, reconstituted in 10 /Â¿Iof water, and subjected to the Bratton-Marshall

reaction.Kinetics of Inhibition of Adenylosuccinate Synthetase in

Vitro by L-Alanosyl-AICOR. Kinetic studies were performed bymixing the following reagents in sequence at 4Â° in a totalvolume of 25 /il: 5 /il of L-{4-14C]aspartic acid (0.78 to 12.5

HIM); 5 /il of GTP-MgCI2 (0.1 to 10 rtiM each) in 0.05 M Tris-HCI(pH 8.0); 5 /il of IMP (0.1 to 10 mwi) in 0.05 M Tris-HCI (pH 8.0)or 5 /il of buffer alone; 5 /il of alanosyl-AICOR ( 2.4 to 19.5 /ÃŒM)in 0.05 M Tris-HCI (pH 8.0) or 5 /il of buffer alone; and 5 /il of

partially purified adenylosuccinate synthetase from L5178Y/AR (3.45 units). The reaction was started by brief centrifugationat 12,000 x g for 20 sec, and the vessels were incubated at37Â°for 30 min. The reaction was terminated by heating at 95Â°

for 2 min, and the product was analyzed as described earlier.When the concentration of IMP was varied (0.1 to 10.0 mw), L-

aspartic acid and GTP were fixed at 10.0 and 5.0 rriM, respectively. Similarly, at variable concentrations of GTP (0.1 to 10.0Ã•TIM),the concentrations of L-aspartic acid and IMP were fixed

at 10.0 and 5.0 HIM, respectively. The reaction was carried outas described earlier, and the kinetic constants were derivedfrom Lineweaver-Burk plots.

RESULTS

Inhibition of DNA Synthesis by u-Alanosine. Previous workers using cells in culture have established that L-alanosineinhibits the incorporation of [14C]formate into adenine but not

into guanine (7). In addition, Novikoff rat hepatoma cells treatedwith L-alanosine accumulate IMP (8). These findings strongly

suggest that adenylosuccinate synthetase is the primary site ofaction of L-alanosine. Inasmuch as the concentration of ATP isdepressed by L-alanosine both in normal liver and in tumors

(1), it would be expected that dATP and DNA synthesis wouldbe affected in turn. As Table 1 documents, a single dose of L-

alanosine (500 mg/kg) given to mice bearing s.c. nodules ofLeukemia L51 78Y/AR inhibited the synthesis of DNA to aprominent degree. To extend these studies, we next determined whether the influence of L-alanosine on DNA synthesiscould be reversed by adenine and hypoxanthine; the formerbase can be converted directly to AMP via the action of adeninephosphoribosyltransferase and bypass the adenylosuccinatesynthetase step, while hypoxanthine cannot. As Table 1 shows,when hypoxanthine (250 mg/kg) was given along with L-alan

osine, no reversal of the inhibition of DNA synthesis wasobserved; whereas the same dose of adenine almost completely reversed the inhibition of DNA synthesis produced byadministration of the drug.

The Influence of L-Alanosine on the Enzymes of PurineSalvage. The foregoing studies suggested that the enzymesresponsible for the salvage of adenine might modulate thetherapeutic activity of L-alanosine by catalyzing the reutilization

of this base, which as documented is an efficacious antidoteagainst the toxicity of the antibiotic. In addition, the possibility

was entertained that the state of adenine deprivation producedby the administration of L-alanosine might induce the enzymesof adenine salvage; such induction would be of obvious therapeutic concern. However, as Table 2 documents, no differences in these systems were discerned either basally or afterprovocation with L-alanosine given at a dose of 500 mg/kg.

Inhibition of Adenylosuccinate Synthetase by L-Alanosinein Vitro and in Vivo. Clark and Rudolph (5) reported that L-

alanosine itself is a rather weak inhibitor of Novikoff ascitestumor adenylosuccinate synthetase. Hurlbert ef al. (10) suggested that the antitumor effect of L-alanosine was exerted by

an anabolite of the drug and not by the drug itself. Our firststep to confirm this contention sought to compare the magnitude of inhibition of adenylosuccinate synthetase produced byL-alanosine in vitro with that exerted by the drug in vivo.

Adenylosuccinate synthetase partially purified from Leukemia L5178Y/AR was used for kinetic studies in vitro. Inasmuchas L-alanosine is an analog of L-aspartic acid, we first studiedthe inhibition of this enzyme by L-alanosine using L-aspartic

acid as the variable substrate; the inhibition observed wasnoncompetitive and weak, with a Ki of 57.23 HIM (Chart 3). Inthe interest of completeness, we further studied the inhibitionof adenylosuccinate synthetase by L-alanosine using GTP and

IMP as variable substrates. Inhibition was noncompetitiveagainst these substrates also, with a KÂ¡of 30.42 HIM for GTPand 37.10 mm for IMP (Table 3).

Table 4 presents the effect of L-alanosine on adenylosucci-

Table 1Influence of adenine and hypoxanthine on the inhibition of DNA synthesis by L-

alanosineBD2F, mice bearing 10-day-old L5178Y/AR tumors were given injections of

L-alanosine. adenine, or hypoxanthine separately or in combinations thereof.After 4 hr, all the groups were given injections of [2-'"C]thymidine (5 fiCi/animal).

One hr after administration of thymidine. the animals were sacrificed, and thetumors were removed and frozen. Measurements of the incorporation of thymidine into DNA were carried out as described in the text.

Treatment0.9%

NaCI solutionL-AlanosineL-Alanosine + adenineL-Alanosine + hypoxanthineHypoxanthineAdenine[2-14C]Thymidine

incorporated(nCi/mgDNA)4.63

Â±0.29a0.69 Â±0.1 9b

4.10 Â±0.400.71 Â±0.15to

4.72 Â±0.884.49 Â±0.41%

ofinhibition085.1

11.4584.67

03.30

a Mean Â±S.D.0 Significantly different from the control group (p < 0.002).

Table 2Effect of L-alanosine treatment on the purine salvage enzymes in L5178Y/AR

tumorsAnimals bearing 10-day-old L5178Y/AR tumors were given injections of L-

alanosine (500 mg/kg). Control group received the same volume of 0.9% NaCIsolution. After 4 hr. the animals were sacrificed and the tumors were removed.Measurements of purine salvage enzymes were then conducted as described in"Materials and Methods."

Specific activity (nmol/mgprotein/hr)EnzymeAdenine

phosphoribosyltransferase

Hypoxanthine phosphoribosyltransferase

Adenosine kinaseAdenosine phosphorylaseInosine phosphorylase0.9%

NaCIsolution25.83Â± 2.09a

30.98 Â±2.5627.63

Â± 3.8411.33Â± 2.4890.18 Â±10.17L-Alanosine24.28

Â± 2.18

27.51 Â±2.7224.26

Â± 1.148.14 Â± 1.60

81.21 Â±14.62"Mean Â±S.D.





0.75

- 0.50

0.25

1.0 3.0 5.0 7.0

â€¢L̂-Aspartic acid [mM]

Chart 3. Inhibition of adenylosuccinate synthetase of Leukemia L5178Y/ARby L-alanosine. Adenylosuccinate synthetase activity was measured accordingto the radiometrie method given in "Materials and Methods", with L-aspartic acid

as the variable substrate in the absence (O) or presence of 30.0 mM (A) or 60.0mM (â€¢)L-alanosine. The Km for L-aspartic acid was 1.18 mM, and the K, for L-alanosine was 57.23 mM.

Table 3Summary of the effect of L-alanosine and L-alanosyl-AICOR in vitro on

adenylosuccinate synthetase from L51 78Y/ARThe assay techniques for enzyme analysis are given in "Materials and Meth

ods."

SubstrateL-Aspartic

acidGTPIMPL-Aspartic

acidGTPIMPKm

(/IM)1180.0480.0300.01190.0526.3345.0InhibitorL-AlanosineL-AlanosineL-AlanosineL-Alanosyl-AICORL-Alanosyl-AICORL-Alanosyl-AICORK,(JIM)57,230.030.420.037,105.011.68.80.228Typeof in

hibitionNCaNCNCNCNCC

" NC, noncompetitive; C, competitive.

Table 4Effect of L-alanosine on adenylosuccinate synthetase in vivo in L51 78Y/AR

tumorsBD2F, mice bearing 10-day-old s.c. L5178Y/AR tumors were given a single

i.p. dose of L-alanosine (500 mg/kg). Animals in control groups received thesame volume of 0.9% NaCI solution. At designated time periods, five L-alanosine-treated and five 0.9% NaCI solution-treated animals were sacrificed, and thetumors were removed, frozen, and homogenized. Adenylosuccinate synthetaseactivity was then measured as described in "Materials and Methods."

Adenylosuccinate synthetase activity (nmol/hr/mg protein)

Time(hr)0.5124824480.9% NaCIsolution9.25Â±0.65Â°10.80

Â±0.9011.60Â±0.4010.10Â±0.909.80Â±0.4510.65Â±0.309.65Â±0.40L-Alanosine2.85

Â±0.122.85Â±0.143.30Â±0.272.65Â±0.182.70Â±0.345.65Â±0.285.40Â±0.29%

ofinhibition69.2073.6071.5073.8072.5047.0044.00

Mean Â±S.D.; p< 0.001.

nate synthetase in vivo. When a therapeutic dose of the drug(500 mg/kg) was given to mice bearing nodules of LeukemiaL5178Y/AR and the inhibition of adenylosuccinate synthetasewas followed over time, it was observed that from 30 min to 8

hr the antibiotic inhibited tumoral adenylosuccinate synthetaseby about 70%. Thereafter, gradual restitution was observed.Greater than 90% of the inhibition observed was reversible bydialysis.

Concentration of L-Alanosine in L5178Y/AR Tumors. SinceL-Alanosine itself can inhibit adenylosuccinate synthetase, it

became important to determine whether the nodules of Leukemia L51 78Y/AR used in the studies described earlier contained the antibiotic in vivo at a concentration commensuratewith the kinetics of inhibition measured in vitro. Hence, as anext step, we measured the concentration of L-alanosine in

these tumors. It was found that the concentration of unalteredL-alanosine fell to 440 Â¡IMwithin 2 hr (Table 5) after the

administration of the drug. By 4 and 8 hr, the concentration ofthe drug decreased further despite the fact that the inhibitionof adenylosuccinate synthetase had been found to persist at-70% over this time span. These concentrations of L-alanosine

were incapable of exerting the magnitude of enzyme inhibitionobserved, a finding which ruled out the possibility that L-alan

osine itself could be functioning in vivo as the proximateinhibitor of adenylosuccinate synthetase.

Condensation of L-Alanosine with AICOR. Since the concentration of L-alanosine in tumors was inadequate to explain

the strong inhibition of adenylosuccinate synthetase producedby it, support was sought for the hypothesis that an anaboliteof the drug, possibly L-alanosyl-AICOR, was responsible for

the action of the antibiotic in vivo.Preparatory to these studies, "authentic" L-alanosyl-AICOR

was prepared from L-alanosine and AICOR by the catalytic

action of a preparation of SAICAR synthetase partially purifiedfrom avian liver. This compound was homogeneous on electro-phoresis and thin-layer chromatography, a strong inhibitor ofadenylosuccinate synthetase, and Bratton-Marshall positive. Iteluted as a symmetrical peak at 260 min from a high-resolutioncolumn of Hamilton HA-X4 resin developed as described pre

viously (20). Thus, although strict chemical identity was established, the material thus isolated was utilized as a provisionalbut plausible standard in the work to be described.

Inhibition of Adenylosuccinate Synthetase in Vitro by L-Alanosyl-AICOR. In order to determine the inhibitory potencyof L-alanosyl-AICOR, we next studied in detail the effect of thisantimetabolite on adenylosuccinate synthetase, partially purified from Leukemia L5178Y/AR. When L-aspartic acid andGTP were used as variable substrates, L-alanosyl-AICOR in

hibited adenylosuccinate synthetase in a noncompetitive manner with apparent K/s of 11.6 and 8.8 JUM,respectively (Table

Table 5Concentration of L-alanosine and L-alanosyl-AICOR in L51 78Y/AR tumors

L5178Y/AR tumors were transplanted in BD2F2 mice. Ten days later, theanimals were fasted overnight and treated with alanosine [50 fiCi of DL-[1-"*C]alanosine + L-alanosine (500 mg/kg)]. At appropriate times, groups of 6

mice were sacrificed. Tumors were homogenized in 1 N acetic acid and centri-fuged at 12,000 x g for 12 min. The concentrations of L-alanosine and L-alanosyl-AICOR were measured as described in the text.

Time after L-alanosine administration

(hr)24

8Concentration

of L-alanosine(Â»Ml442

Â±39.2a330

Â±24.8170 Â±15.2Concentration

of L-alanosyl-AICOROÃ•M)70.2

Â± 9.553.1 Â±12.220.3 Â±3.6â€¢

Mean Â±S.D.

DECEMBER 1980 4395




3). However, as shown in Chart 4, the anabolite was a formallycompetitive inhibitor versus IMP with an apparent K of 0.228

Presence and Concentration of L-Alanosyl-AICOR in

L5178Y/AR Tumors. Inasmuch as it was possible to condenseL-alanosine with AICOR in vitro and the resulting antimetabolite,L-alanosyl-AICOR, was a very potent inhibitor of adenylosuccinate synthetase, a search was mounted for L-alanosyl-AICOR

in tumors. Mice bearing L5178Y/AR nodules were given 10juCi of DL-[1-14C]alanosine along with L-alanosine, 500 mg/kg,

and the tumors were removed and analyzed for L-alanosyl-

AICOR. In another approach, radioactive alanosine was givento mice along with 5-amino-4-imidazolecarboxamide ribosideas a precursor for AICOR. L-Alanosine-sensitive P388 and

neuroblastoma cells in tissue culture were also carried throughsimilar experimental approaches. Nevertheless, all of our initialefforts to identify this anabolite in vivo met with failure (19).However, when a 5-fold greater dose of radioactive DL-[1-14C]alanosine (50 /Â¿Ci/animal) was given, along with a thera

peutic dose of L-alanosine (500 mg/kg), to mice bearing nod

ules of Leukemia L51 78Y/AR, and the chromatography was

0.2 -

â€¢1IMP[mM]B

Chart 4. Inhibition of adenylosuccinate synthetase of Leukemia L5178Y/ARby L-alanosyl-AICOR. The activity of the enzyme was measured as detailed in"Materials and Methods" with IMP as the variable substrate in the absence (â€¢)

presence of 0.97 UM (O), 1.94 UM (A), or 3.89 ^M (â€¢)L-alanosyl-AICOR The Kmfor IMP was 0.345 mM. and the apparent K, for L-alanosyl-AICOR was 0.228 UM.

performed on a HA-X4 column with lithium citrate buffers, aprominent radioactive peak, coeluting with L-alanosyl-AICOR,was detectable in the tumors of the recipients at concentrationsof 70, 53, and 20 /ÃŒMat 2, 4, and 8 hr, respectively (Table 5).As described in "Materials and Methods" for preparing L-

alanosyl-AICOR from tumors to test on adenylosuccinate syn

thetase, a 0 to 1.0 M ammonium bicarbonate gradient wasutilized, and all Chromatographie fractions from these studieswere tested for inhibition of partially purified adenylosuccinatesynthetase from L5178Y/AR and Bratton-Marshall positivity.Fractions correspondng to the alanosine peak had no effect onadenylosuccinate synthetase (Chart 5); however, those corresponding to the peak coeluting with alanosyl-AICOR stronglyinhibited the partially purified preparation of adenylosuccinatesynthetase (Chart 5). Only the fractions corresponding to thispeak wer jserved to be Bratton-Marshall positive (Chart 5).The presence of a radioactive peak, coeluting with L-alanosyl-

AICOR, and its remarkable capability to inhibit adenylosuccinate synthetase and Bratton-Marshall positivity (Chart 5)strongly suggest that the compound is L-alanosyl-AICOR.

DISCUSSION

In its capacity as a structural and functional analog of thedicarboxylic amino acids, L-alanosine is metabolized extensively by several of the enzymes which ordinarily operate on L-

aspartic acid (1, 13, 15). Therapeutically speaking, the mostimportant such enzyme is thought to be SAICAR synthetasebecause it uses L-alanosine as a fraudulent substrate andgenerates L-alanosyl-AICOR, an antimetabolite known to inhibit

adenylosuccinate synthetase with notable potency (Refs. 9,10, and 19; Chart 4).

Up to the present, attempts to determine whether this chainof events was operative in vivo have met with failure (19).However, using a number of novel approaches, we have nowsucceeded in demonstrating that administration of L-alanosine

does inhibit adenylosuccinate acid synthetase in s.c. tumornodules and that, as a consequence of this inhibition, DMAsynthesis is reduced strongly. It is especially relevant in thisregard that adenine, but not hypoxanthine, reverses the depression of nucleic acid biosynthesis engendered by parenteraldoses of L-alanosine. Inasmuch as hypoxanthine alone of thispair of purines must be metabolized via adenylosuccinate syn-

Chart 5. Chromatography of an extract ofL5178Y/AR tumor on a column of HamiltonHA-X4 resin. Mice bearing L5178Y/AR tu

mors were given alanosine Â¡.p.at a dose of500 mg/kg + 50 Â¡uCi/mouse and sacrificed2 hr later. Tumors were removed, homogenized in 1 N acetic acid, adjusted back to pH7.0. and centrifuged at 12,000 x g for 5 min.A portion of the resulting supernatant (0.8 ml)was loaded on a 0.8- x 15-cm column ofHamilton HA-X4 resin, which then was devel

oped with a gradient of 0 to 1 M ammoniumbicarbonate (100 ml each) as detailed in thetext. Adenylosuccinate synthetase was assayed as in "Materials and Methods". â€¢.ra

dioactivity; O, percentage of inhibition of theactivity of adenylosuccinate synthetase. D,Bratton-Marshall negative; â€¢ Bratton-Marshall positive.

Câ€¢B24.0oELLÂ°

18.0

x5Q_(J.=

12.0oÂ§TJto

* 6.00-Alanosine|A\ii^mm1

Â»'Â°\/

Â°^-I01

Hydroxy Alanosine|iâ€¢'

*\~^>^-&Q-OO-U^^O-O-Q<>QOOOu3Ox<^QgJfo^0

50 100 150oAlanosyl-AMf

**\IGORo<WyvrtWÃ¯WÃ¯Â»*/*Â»aufiou9f^t3-eri100

180

gâ€¢<5"VI60

Â£Ci3'Ã›)e

SynthetaseÂ§R,u200

250 300Time (minutes)





thetase, its inertness as an antidote to the effects of u-alanosineprovides a cogent indication of the locus of action of the drugin vivo.

Experiments recounted in the present paper also establishthat L-alanosine is present in tumors at concentrations inade

quate to explain the protracted inhibition if adenylosuccinatesynthetase measured (Table 4). Conversely, a strongly acidicanabolite of L-alanosine is detectable in tumors if appropriate

doses of radiolabeled drug are administered; this anabolite,extracted from tumor nodules, coelutes with L-alanosyl-AICORon high-resolution chromatography, is Bratton-Marshall posi

tive, and inhibits adenylosuccinic acid synthetase fromL5178Y/AR tumor cells with an apparent KÂ¡of 0.228 /Â¿M.Onthe basis of these properties, it is concluded to be L-alanosyl-AICOR. Moreover, because the concentration of this anabolitein tumors approximates 70 ,UM,a level ~307 times greater than

its apparent KÂ¡,it is a very plausible candidate for the role ofproximate antimetabolite of L-alanosine in vivo.

At present L-alanosine is undergoing Phase I and II trials in

a panel of human cancers. In view of the pivotal roles thatSAICAR synthetase and L-alanosyl-AICOR play in determining

the efficacy of this antibiotic versus experimental tumors, efforts will be mounted to measure them in specimens of humanneoplasms. It is hoped that such measurements will havepredictive value. Additionally, in view of the potency of L-alanosyl-AICOR, synthesis of this anabolite will be attempted

in anticipation of its use as a novel oncolytic drug in its ownright.

ACKNOWLEDGMENTS

We are grateful to Dr. H. N. Jayaram for helpful suggestions during the courseof this work. The authors are also grateful to Shirley Swindell and Ruth Davis forhelp with the typing of this manuscript.

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2. Bradford, M. M. A Rapid and Sensitive Method for the Quantitation ofMicrogram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem., 72: 248-254, 1976.

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DECEMBER 1980 4397



1980;40:4390-4397. Cancer Res Anil K. Tyagi and David A. Cooney Synthetasein Tumors and Assessment of Its Inhibition of Adenylosuccinatel-Alanosyl-5-Amino-4-Imidazolecarboxylic Acid Ribonucleotide, Identification of the Antimetabolite of l-Alanosine,

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identification of the antimetabolite of l-alanosine - cancer research

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