IV. Reversal of Azaserine-induced Inhibition by Phenylalanine and Tryptophan*
A R T H U R J. TOMISEK, M A R Y R . REID, AND H O W A R D E . SKIPPER
(Kettering-Meyer Laboratory [Affiliated with Sloan-Kettering Institute], Southern Research Institute, Birmingham, Ala.)
When azaserine (O-diazoacetyl-L-serine) as- sumed its position as one of the more effective inhibitors of animal tumors (14), its mechanism of action became the object of numerous studies. Among the various approaches to the problem of inhibition mechanism was the usual one of screening for possible reversal agents by means of bacterial growth tests. The observed reversals obtained with glutamine and various purine de- rivatives eventually fitted neatly with data ob- tained by several other experimental approaches. The net result was an impressively integrated body of evidence (1, 2, 6, 16) tha t azaserine exerted its primary inhibition by blocking the de novo synthesis of purines at the step in which glutamine is used to amidinate formylglycinamide ribotide (FGARP).
Unfortunately for the proposed theory of in- hibition, reversal studies in bacteria had a major residuum of data which could not be fitted into the theory (8). More recent examples of this were the reversal of azaserine inhibition of E. colt by phenylalanine, tyrosine, and tryptophan, and, less effectively, by methionine, leucine, phen- ylpyruvic acid, and phenyllactic acid 1 (3).
While the present work concerns these reversals in E. colt, there has also been some preliminary work with mice on synthetic diets (13). Essential- ly, the effect of azaserine against Sarcoma 180 was enhanced in those mice on diets deficient in isoleucine and in some experiments in which tryp- tophan was omitted from the diet. The suggestive similarity of the effects on mice and on E. colt has persuaded us that the reversal effects in E. colt might be worthy of further study.
* This investigation was supported by grants from Parke, Davis and Company, the Alfred P. Sloan Foundation, and the Charles F. Kettering Foundation. Presented before the Bio- chemical Division of the American Chemical Society, 133(1 Meeting, San Francisco, California, April, 1958.
1 Unpublished work by Glynn P. Wheeler of this laboratory.
Received for publication November 28, 1958.
Having already applied our chromatographic- autoradiographic technic to the study of inhibition mechanisms (15, 16), we thought to try it next in a study of reversal mechanisms.
This study of reversal by phenylalanine and tryptophan of azaserine inhibition has not pro- vided a complete explanation of the reversal mech- anism, but evidence relating to several of the possible explanations has been obtained.
MATERIALS AND METHODS The experimental procedure for chromatographic studies
has been described (15, 16). It may be summarized as: (a) the preparation of E. colt (A.T.C.C. 9637) cultures growing rapidly in a glucose medium; (b) the direct addition of inhibitor and reversal agent 20 minutes before the radioformate addition; (c) the 15-minute assimilation of small amounts of radioformate of high specific activity; (d) killing of the centrifuged cells with boiling alcohol; (e) two-dimensional paper chromatog- raphy of the alcoholic extracts; (f) autoradiography of the papergrams; and (g) study of the radioactivity of each chroma- tographic spot in terms of all the other spots on the s a m e
papergram. This last step involved elution of corresponding spots
(or areas) from two or three separate papergrams, evaporation of the pooled eluates onto planchets, and measurement of radioactivity in a windowless flow counter. More often than not, insufficient chromatographic separation of the spot com- binations 19 -t- 20, 5 q- 45, 7 -t- 47, and 11 -t- 16 q- 55 re- quired planchets to be made for the combined areas rather than for the individual spots. These area counts were then apportioned to the individual components on the basis of x-ray film densitometry readings, either from additional two- dimensional papergrams or from suitable one-dimensional pa- pergrams of material recovered from the planchets. In those cases in which spots 55 and 7 (FGARP and its riboside) were included in area counts, their own radioactivity was de- termined by difference following hydrolysis N/10 HC1, 100 ~ C., I hr.) and evaporation in the presence of carrier formic acid.
Except for 5-amino-l-(St-phosphoribosyl)-4-imidazolecar- boxamide (AICRP) (proved only for amethopterin-treated runs) and for glutamine (not enough C 14 to permit further work), the known spots on the papergrams from control runs without inhibitor have been characterized by fairly rigid criteria (16). In experiments involving inhibition by azaserine, identities of the purine derivative spots were generally not verlfied--on the grounds that these spots were tending to disap- pear; but 2-formamido-N-ribosylacetamide (FGAR), FGARP, alanine, glutamic acid, aspartic acid, and glutamine w e r e
conclusively identified. In addition, succinic and fumaric acids from these latter experiments were identified by two-dimen- sional co-chromatography. In experiments with added phenyl- alanine or tryptophan, with or without azaserine, the spot identities were, for the most part, assumed to be the same as those in the absence of phenylalanine or tryptophan.
The experiments with cell-free systems were patterned after those of Love (10) except as noted. The E. coli strain in these cases was the B-96 mutan t (4) supplied by Dr. Joseph Gots. The nonproliferating cells equivalent to about 0.35 gin. dry weight were harvested from a nonaerated culture grown on a salts-glucose-adenine medium and sonieally dis- rupted in ~5 ml. of the phosphate buffer. The soluble fraction of this sonicate was used without dialysis.
RESULTS Effect of phenylalanine and tryptophan on the
metabolism of formate.--For practical purposes it can be said that L-phenylalanine and ~t ryptophan had no effect on the metabolism of formate-C 14 in E. coli.
There were some small differences which can be gleaned from Table 1. Phenylalanine and trypto- phan both appeared to cause a slight reduction in the radioactivity of nucleotides (spots 34 A- 17-k 29, 11, 12, and 16) and a corresponding increase in the radioactivity of certain nucleotide degradation products such as guanine, guanosine, hypoxanthine, and inosine (spots 4, 45, 8, and 5). Among the amino acid metabolic pools there ap- peared to be a slight decrease in the radioactivity of glutamic acid (spot 8) and an increase in that for alanine (spot 6). However, it is well to em- phasize that these differences were not of a mag- nitude to have any effect on the conclusions con- cerning azaserine inhibition and reversal. I t is even probable that these minor effects were en- tirely unrelated to the capacity of the phenyl- alanine and tryptophan to reverse the azaserine inhibition.
Effect of azaserine on the metabolism of formate.-- In confirmation of the earlier work from this laboratory (16), this quantitative study (sum of all purine derivatives, Table 1) illustrates the az- aserine inhibition of purine synthesis and the con- comitant intracellular accumulation of FGARP and FGAR.
Azaserine caused an increase in the radioactivity and also the amount (shown by ninhydrin color) of glutamine. I t also caused an increase in the radioactivity of alanine and a decrease in the radioactivity of glutamic acid.
Effect of phenylalanine and tryptophan on the inhibition of E. coli cells by azaserine.--While purine synthesis in E. coli was reduced very drasti- cally by 1.5 t~g/ml of azaserine and only slightly less drastically by 0.5 ~g/ml of azaserine, the addi- tion of 100 ~g/ml of L-phenylalanine along with
1.0 ~g/ml of azaserine permitted almost unin- hibited entry of radioformate into the purine- derivative metabolic pools. The same amount of phenylalanine added with 2.0 ~g/ml of azaserine was less effective in reversing the azaserine inhibi- tion, although the radioactivity in the total purines (Table 1) was still 6 times greater in Run 70 than in Run 32--despite the fourfold greater az- aserine concentration.
~Tryp tophan at 124 ~g/ml (equimolar to the phenylalanine concentration used above) was not quite as effective a reversal agent for azaserine inhibition as was phenylalanine, the total purine synthesis for 0.5 ~g/ml of azaserine being ninefold larger in the presence of tryptophan (compare Runs 32 and 83 of Table 1).
For either the phenylalanine or the tryptophan experiments, the inhibition of de novo purine syn- thesis remaining after the addition of reversal agents was of the same nature as the azaserine inhibition without reversal agent. An autoradio- gram from the run with 1.0 ~g/ml of azaserine plus 100 ~g/ml of phenylalanine (Fig. 2) differed from a typical control run (Fig. 1) only in the small increase of radioactivity in FGARP and FGAR (spots 55 and 7). This result of a slight blockade in the amidination of FGARP was indistinguish- able from that observed previously for the slight blockade due to 0.2 ~g/ml of azaserine (Figs. 1 and 2 of Ref. 16). Remarkably illustrated in the quantitative data of Table 1 is the conclusion: to the extent that radioactivity failed to be in- corporated into purine derivatives, it could be ob- served as an intracellular accumulation of FGARP and FGAR. This conclusion holds even at azaser- ine concentrations sufficient to stop almost all growth, and it is not negated by the presence of the reversal agents phenylalanine or trypto- phan.
Effect of phenylalanine on the azaserine-induced inhibition of "purine" synthesis in soluble extracts of E. coli.--The reversal of azaserine inhibition by phenylalanine was also demonstrated for ~the cell-free extracts of an E. coli mutant whose ge- netic defect permitted the accumulation of AICRP to serve as an index of synthesis rate over the de novo purine synthesis pathway up to tha t point (Table 2).
DISCUSSION
Mechanism of azaserine action.--The earlier study of azaserine inhibition (16) showed that, under the conditions of these experiments, 2.0 ~g/ml of azaserine stopped all growth (as measured by turbidity); and at 0.1 ~g/ml there was no apparent inhibition. The drastic inhibition of pu-
TOMISEK et al.--Reversal of Azaserine Inhibition by Phenylalanine 491
rine synthesis shown with 0.5 #g/ml of azaserine (Run 3~, Table 1) was thus obtained under con- ditions of only about 10 per cent inhibition of growth. These data illustrate in quantitative fash- ion the previous evidence (16) tha t a primary site of azaserine inhibition in E. coli is in the amidination of FGARP (or possibly a closely sub- sequent reaction).
The increase in size of the metabolic pool of glutamine as a result of azaserine inhibition has been shown previously by Barker et al. (1) for the alga Scenedesmus, when bicarbonate-C TM was used as the radioactive substrate. A similar in- crease is noted here for E. coli with formate-C TM
used as the radioactive substrate. Mechanism of phenylalanine and tryptophan re-
TABLE 1
THE EFFECT OF PHENYLALANINE (100 pG/ML) AND TRYPTOPHAN (124 #G/ML) ON THE LABELED METABOLIC POOLS OF AZASERINE-INHIBITED E. coli FED RADIOFORMATE
versal of azaserine inhibition.--For both phenyl- alanine and t ryptophan the chromatographic ex- periments indicate tha t a high level of azaserine in the presence of the reversal agent has the same effect on the metabolism of formate by E. coli as does a much lower level of azaserine in the absence of reversal agent. In terms of reversal mechanism, these experiments have provided good negative evidence on alternative metabolic path- way hypotheses, since the chromatographic pat- terns did not indicate the presence of compounds which might be a par t of some novel route of
TABLE
EFFECT OF PHENYLALANINE ON THE AZASERINE-INDUCED INHIBITION OF THE SYNTHESIS OF AMINOIMIDAZOLE-
CARBOXAMIDE DERIVATIVES BY CELL-FREE EXTRACTS
FROM E. coli
Optical System* denJities t
Control system, zero incubation Control system:
Plus phenylalanine Plus azaserine (1 mg/ml) Plus azaserine (1 mg/ml) and phenylalanlne Plus azaserine (~ mg/ml) Plus azsserine (~ mg/ml) and phenylalanine
0 . 0 5 0.19 0.19 0.11 0.~20 0.07 0.15
* Each tube contained 1.0 ml. of soluble sonicate, substrates in the amounts used by Love (10) (including glut, amine, ribose- 5-phosphate, 3-phosphoglyceric acid, and adenosine-5'-triphos- phate), and a final volume of ~.5 ml. L-Phenylalanine, when present, was used at a level of 0.~1 mg/ml. Incubation was for 4 hours.
t The optical densities are for the color produced in the Bratton-Marshall test in a typical experiment.
purine synthesis. At the same time, these chroma- tographic experiments show tha t the decrease in degree of azaserine blockade was adequate to explain the reversal. Halvorson reached about the same conclusion in his work on the azaserine inhibition of yeast, in which the most effective reversal agent was leucine (5). He noted tha t serine was released into the medium as azaserine disappeared from the medium and tha t leucine served to prevent both the utilization of azaserine and the appearance of serine in the medium.
Separate experiments involving the preincuba- tion of azaserine with phenylalanine eliminated the possibility tha t phenylalanine might have a
strictly chemical effect on the stability of az- aserine.
A hypothesis of lessened cell permeability to- ward azaserine in the presence of reversal agent was shown to be inapplicable, since phenylalanine reversed the inhibition of A I C R P synthesis by azaserine in a cell-free extract of a purine-requiring mu tan t of E. coli. This mu tan t (of a parent strain different from tha t used in the chromato- graphic experiments) has its genetic defect con- veniently located to permit the study of only the early steps of purine synthesis. I t was with whole cells of this mutan t tha t Gots and Gollub (3) studied several aspects of the phenylalanine reversal of azaserine inhibition. Their data in- cluded some evidence against the cell-permeability hypothesis, in that the reversing action of phenyl- alanine was almost as effective after 1 hour of preincubation with azaserine as when the inhibitor and reversal agent were added simultaneously. The level of azaserine required for inhibition in our undialyzed cell-free extract was very high; but it must be noted tha t the test system contained added glutamine (0.91 mg/ml) , which in itself is a reversal agent (3, 11). In addition, the active transport of azaserine observed for whole cells by Jacquez (7) and Pine (1~) would not be opera- tive in increasing the effective concentration of inhibitor. The high azaserine levels required for the inhibition of pigeon liver fractions (9) are of the same order of magnitude as those used in the present study.
Another hypothesis is tha t phenylalanine and other possible nitrogen donors increase the met- abolic pool of glutamine, thereby causing a mass- action type of reversal. Our papergrams showed that the glutamine pool was increased as a result of azaserine inhibition; but there was no evidence of either (a) the large further increase which this hypothesis would require as a result of phenyl- alanine reversal or (b) an increase due to reversal agent without inhibitor. The reversals observed with thienylalanine (3) and phenylpyruvic acid, 1 and also the greater effectiveness of phenylalanine as a reversal agent as compared with glutamine (11), afford additional evidence against this hy- pothesis.
Despite the elimination of several possibilities,
1~o. 1.--Autoradiogram of an E. coli extract, wild type, fed formate-C 14. No inhibitor or reversal agent.
I~G. ~.--Autoradiogram of an E. coli extract, wild type, fed formate-C 14. Azaserine, 1.0 ~g/ml; L-phenylalanine, 100 /~g/ml.
TOMISEK et al.--Reversal of Azaserine Inhibition by Phenylalanine 493
an actual explanation of reversal mechanism is not available. At the present time, the most prob- able hypothesis would seem to be that the reversal agents cause the affected enzyme to have a de- creased affinity for the inhibitor.
SUMMARY
Phenylalanine and tryptophan both appear to act by "preventing" the inhibitory action of az- aserine on E. coli rather than by compensating for the inhibition. This preventive action has been demonstrated for both whole cells and soluble sonicate fractions.
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15. TOmSEK, A. J.; KELLr, H. J.; REIn, M. R.; and SKIPPER, H. E. Chromatographic Studies of Purine Metabolism. II. The Mechanism of E. coli Inhibition by A-Methopterin. Arch. Biochem. & Biophys., 76:45-55, 1958.
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1959;19:489-493. Cancer Res Arthur J. Tomisek, Mary R. Reid and Howard E. Skipper Tryptophanof Azaserine-induced Inhibition by Phenylalanine and Chromatographic Studies of Purine Metabolism IV. Reversal
