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6-Azauracil inhibition of Escherichia coli B and its reversal by hadacidin
ARCHIVES
OF
BIOCHEMISTRY
6-Azauracil
AND
BIOPHYSICS
Inhibition
472-477
lo&
(19ti3)
of Escher&a
coli B and
Its Reversal
by Hadacidin’ HAROLD From the Ned
Institute
T. SHIGEURA
for Therapeutic Received
October
Research,
Rahway,
Kew Jersey
8, 1962
The inhibition of growth of Escherichia coli B by G-axauracil was found to be reversed by hadacidin. The inhibition of growth by hadacidin, however, was not affected by &azauracil. The inhibitory activity of 6-azauracil in the presence of hadacidin was restored by the addition of adenine, adenosine, deoxyadenosine, or inosine. Several other purines and pyrimidines were inactive. The inhibitory activity of 6-azauracil alone was enhanced by the addition of adenine. 6-Azauracil-resistant, strain of E. coli B was sensitive to hadacidin and 6-azauridine. The inhibition of growth by 6-azauridine was not reversed by hadacidin. The results indicated that the metabolic activity of hadacidin, namely, the suppression of formation of adenylic acid or other adenine derivatives in some manner accounted for the reversal by hadacidin of 6-azauracil inhibition of E. coli B. INTRODUCTION
effect of hadacidin and 6-azauracil on the growth of E. coli B pointed to an interesting relationship between these two compounds per se or between the formations of the essent,ial products (adenylic and uridylic acids) that are suppressed in the presence of these inhibitors. Further studies conducted on this unexpected phenomenon are described in this paper.
The antibiotic hadacidin (N-formyl hydroxyaminoacetate) has been found to suppress the conversion of inosinate to adenylosuccinate by the enzyme inosine 5’-phosphate : L-aspartate ligase (adenylosuccinate synthetase) obtained from Escherichia coli B. The inhibition was competitively reversed by L-aspartate (1). During the course of studies on the effects of hadacidin when used in combination with other known antitumor agents on the growth of E. coli B, it was observed that hadacidin appeared to reverse the potent inhibitory property of 6-azauracil (as-triazine-3,5dione). The results further indicated the presence of a competitive type of antagonism between these two compounds (2). Since 6-azauracil, in the form of the mononucleotide, has been shown to inhibit the conversion of orotidylic acid to uridylic acid (3, 4), the observation on the combined
EXPERIMENTAL
PROCEDURE
MIITSRIALS The purines, pyrimidines, and their derivatives were purchased from the California Corporation for Biochemical Research. Hadacidin was obtained from Dr. E. A. Kaczka, Merck Sharp & Dohme Research Laboratories, Rahway, N. J. CCLTIV.~TIOX
OF E'. COLI
B
The microorganism was grown at 37” in a medium consisting of the following components per liter (5) : 1lOmg. MgSOa,7HzO, 7.8 g. K?HPO,, 2.2 g. KHnPOr, 470 mg. Na citrate.2H20, 1.1 g. (NH,)zS04, and 25 g. glucose. Cells grown overnight in this medium were inoculated into 50 ml. of fresh medium (570 inoculum). After about 4 hr., the logarithmically growing cells were centrifuged,
1 This investigation was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the National Institutes of Health Contract Number SA-43.ph1886. 472
REVERSAL
OF
G-AZA4URACIL
473
ISHIBITION
malml OF HAOACIDIN
I, 0
0.5
1.0
2.0
1.5
ma/ml OF HAOACIDIN
FIG. 1. Culture medium in a total volume of 4.5 ml. contained varying concentrations of 6. azauracil or hadacidin and 0.1 ml. of cell suspension. Optical density at zero time was 0.050. The cultures were incubated at 37” for 334 hr. The dotted line indicates 700;, inhibition of growth.
nntl the pellet was fresh, st)erile medium described helow.
suspended in about. 5 ml. and used for the experiments
of
FIG. 2. Culture medium in a total volume of 4.58 ml. contained varying concent,rations of hadacidin with or without 9.7 pg./ml. 6-azauracil. The cultures were incubat’ed at 37” for 33i hr. X-X, eflect of increasing amounts of hadacidin alone; A-A, effect of constant amount of 6azauracil (9.7 pg./ml.) and increasing amounts of hadacidin; dotted line represents growth of cells in the presence of 9.7 pg./ml. R-azauracil a 1one. RESULTS
To determine the effects of the various cornpounds on the growth of E. coli B, several tubes containing t)he test compounds in a cult,ure medium of 4.5 ml. were inoculated with 0.1 ml. of cell suspension and incubated at 37” for 3353 hr. The optical density at zero time was about 0.050, and growth after R specified t,ime was estimat,ed by measurement of optical density at 600 nip in a Beckman model B spectrophotometer.
STRAIN
OF
E.
COLI
B
Cells grown in the medium described above were inoculated (55; inoculum) into fresh medium containing 5 pg./ml. 6-azaurncil and incubated at, 37” overnight. A similar inoculum from this culture was then transferred to a fresh medium containing 10 pg./ml. (i-azanracil and grown overnight,. The concentration of the drug was doubled with each successive inoculat,ion until cells resistant to 1.25 mg./ml. G-azauracil were developed. The resistant strain was maintained :it 0” on agar slant)s containing 1.25 mg./ml. 6-szarnacil.
THE EFFECTS OF HAI~.~CIDIX rKAClL WEIE~ I!sl!a ALONE GROWTH OF E. COLI
The effect of increasing hadacidin on the growth organism
is shown
in
E’ig.
AND ON
B
concentrations
of the 1. A
6-AzilTHE
of micro-
concentra-
tion of 2.4 mg./ml. (1.7 X lo-? ill) of the antibiotic was required to inhibit the growth of E. co/i B by about 70%. Similar studies with (i-azauracil showed that about 10 pg.! ml. (7.7 X 1OP N) of the compound was required to attain the same per cent inhibition (Fig. l), indicat.ing that B-azauracil was about 240.fold more cffect’ive than hadacidin in suppressing the growth of fc. coli 13.
In each case, t,he amount of hadacidin or G-azauracil required to produce about 70%)
474
SHIGEURA
inhibition of growth was used, and the effect caused by the addition of increasing quantities of the other drug was examined. When the concentration of 6-azauracil was kept constant at 9.7 pg./ml., addition of increasing amounts of hadacidin resulted in a gradual rise in turbidity which was considerably above the level of that obtained by the same concentration (9.7 pg./ml.) of 6-azauracil alone (Fig. 2). Cell growth steadily increased until the concentration of hadacidin in the culture was about 300 kg./ml. At this point, further increase in hadacidin concentration lead to a gradual decrease in cell growth with the hadacidin plus 6-azauracil curve coinciding with that obtained by hadacidin alone. Since the optical density of the suspension containing 6-azauracil alone was 0.200 (represented by the dotted line in Fig. 2), the gradual decrease in optical density (increase in inhibition) of the hadacidin plus 6-azauracil curve with increasing amounts of hadacidin in excess of 300 pg./ml. appeared to be due solely to hadacidin. However, the initial increase in proliferation of the cells exposed to both drugs may be due to an antagonistic effect exerted by hadacidin, perhaps indirectly, on the inhibitory c.apacity of 6-azauracil. This reasoning was not in conflict with the results obtained when a reciprocal experiment was performed. As shown in Fig. 3, when the concentration of hadacidin was kept constant at 2 mg./ml. and the concentration of 6-azauracil was progressively increased, growth of the cells did not increase but remained constant and equal to that due solely to hadacidin (dotted line). Increasing concentration of 6-azauracil clearly did not affect the inhibition of growth caused by hadacidin. Preincubation of the bacteria with 6-azauracil for 12 min. at 25” prior to the addition of hadacidin did not alter the results shown in Fig. 2. FURTHER STUDIES ON THE EFFECTS OF HADACIDIX 6-AZAURACIL
COMBISE~ ASD
In an attempt to obtain information with respect to the mechanism by which hadacidin nullifies the inhibitory effect of 6-aza-
.6OOr x ,500 -\
>-\ I SL : ;.400-
x
\
2
i
x
t .30b s x
g .200 t0
-\ -_____
_
----
X\ _____-_--_
---______-
0
,100
0
I IO
5 M/ml
1 15
h 20
‘
of6- AZAURACIL
FIG. 3. Culture medium in a total volume of 4.6 ml. contained varying concentrations of 6aaauracil with or without 2 mg./ml. hadacidin. The cultures were incubated at 37” for 3% hr. X-X, effect. of increasing amounts of 6-aeauracil alone; O-0, effect of constant amount of hadacidin (2 mg./ml.) and increasing amounbs of 6azauracil; dotted line represents growth of cells in the presence of 2 mg./ml. hadacidin alone.
uracil, the effects of the addition of various purines, pyrimidines, and certain amino acids to a system containing both drugs were examined. The cells were grown in media containing 9.7 and 200 pg./ml. 6-aeauracil and hadacidin, respectively, in order to obtain near optimal reversal of 6-azauracil inhibition by hadacidin. (See Fig. 2, peak of curve indicated by A-A.) Various compounds listed in Table I were added to the vessels, and optical densities were measured after 4 hr. As can be seen in the table, adenine, adenosine, deoxyadenosine, and inosine were found to overcome effectively the action of hadacidin on 6-azauracil (compare vessels 5-8 with vessel 4). In each of these instances, the inhibition of growth (due apparently to restoration of 6-azauracil activity) was greater than that due to B-azauracil alone (vessel 2). It may be of significance to note here that adenine, adenosine, and deoxyadenosine are directly affected by the specific inhibitory action of hadacidin on adenylosuccinat,e synthetase, suggesting the possibility that
REVERSAL TABLE THE
EFFECTS REVERSAL
OF
VARIOUS
OF
6-AZAURACIL
I
40
COMPOUNDS
OF G-AZAURACIL BY HADACIDIN
ox
No.
INHIBITION
6Hadacidin
Azauracil
Compounds
Tested
Optical Density at 600 mu
-1 2 3
4 5 G r s 9 10 11 12 13 1-l 15 16 17 1S l!) 20 21 2” 2.3
+ + + + + + + + + + + + + + + + + + + + +
-
,425
+ -
,147
+ + + + + + i-I+ + + + + + f + + + + I
4b
THE
Culture media in a total volume of 4.5 ml. COW tained 1.2 X 10-S X hadacidin, 6.9 X 10-j N fiazauracil, and 2 X 10-a M test compound as indicated. The optical density at zero time was 0.050. Optical density measurements were taken after incubation at 37” for 3js hr. Vessels 4-23 contained both hadacidin and 6.azauracil as indicated bj (+). Vessel 4 shows reversal by hadacidin of Gazauracil inhibition (compare with vessel 2). Restoration of 6.azauracil inhibition was shown by optical density values less than .300 (vessel 4). Vessel
475
INHIBITIOX
Adenine Adenosine Ueoxyadenosine Inosine r,-Aspartat,e Cuanine Guanosine Deoxyguanosine Hypox:rntjhine Xanthine Uracil 1Jridine Cytosine Cytidine Thymine Thymidine Orotic acid Orotidinc Glycine
,360 ,300 .I12 ,100 ,115 ,106
,200 ,278 ,260 .330 .380 ,430 ,450 ,480 ,435 ,460 .400 .480 .495 ,380 ,385
the mechanism of the process is related to the inhibition of purine biosynthesis de novo by hadacidin. Some reversal of action of hadacidin on 6-azauracil inhibition was also obtained with L-aspartate. Guanine and guanosine were weakly active. The remaining compounds were either inert or stimulated growth of the bacteria. Preincubation of the organism with hadacidin and 6-azauracil for 12 min. at 25” prior to the addition of adenine did not alter the results shown in Table I, vessel 5.
.5oc)E' 8ls ,400 .J
X-X-X
A
.500.400.300xdX
.200-'
A
\ .I00 --x+-t 1 . 0123
8 8
L ' 0123 hg/ml
; '
'
OF ADENINE
FIG. 4. (a) Culture medium in a total volume of 4.59 ml. contained 174 pg./ml. hadacidin alone in tubes indicated by A, 8.7 pg./ml. 6-azauracil alone in tubes indicated by H, and 174 ~g./ml. hadacidin plus 8.7 pg./ml. 6-azauracil in tubes C. Increasing amounts of adenine as shown were added to the tubes and growt,h was measured after incubation at 37” for 4 hr. (b) Culture medium in a tot,al volume of 4.43 ml. contained 1.13 mg./ml. hadacidin alone in tubes indicated by .4, 9.1 ~g./ml. F-azauracil in tubes indicated by B, and 1.13 mg./ml. hadacidin plus 9.1 pg. (i-azauracil in tubes indicated by C. Increasing amounts of adenine as shown were added to the tubes, and growth was measured after incubat)ion at 37” for 4 hr.
,4 more detailed investigation of t,he observation described above (the restoration of 6-azauracil inhibition in the presence of hadacidin) was made by studying the effects produced by the addition of adenine to cells incubated with either hadacidin alone, A-azauracil alone, or a combination of hadacidin and 6-azauracil at two separate phasesof the growth curves shown in Fig. 2, namely (1) the position of the curve at which reversal by hadacidin approaches maximal level (9.7 pg./ml. 6-azauracil and 200 pg./ml. hadacidin) and (9) the declining phase of the curves where t)he growth curve of hadacidin coincides with that of hadacidin plus 6-azauracil (9.7 pg./ml. 6-azauracil and 1 mg. ml. hadacidin). As shown in Figs. -la and #I, in both phasesof t’he growth curve representing inhibition by hadacidin alone (rrpresented by *-I in Figs. 4a and 4b), addition of
SHIGEURA
476 TABLE THE
EFFECT
II
,500
OF ADENINE ON THE INHIBITION GROWTH OF E. COLI B BY 6-AZAURACIL ALONE
OF
r
Culture media in a total volume of 4.45 ml. contained 5.6 pg./ml. (5 X 1OF M) or 4.5 pg./ml. (4 X 10d5 M) of 6-axauracil. Indicated amounts of adenine were added. Growth was measured after incubation for 3% hr. at 37”. 6.Azauracil
Adenine
pg./ml.
sg./ml.
O.D. at600
5.6 5.6 5.6 5.6 5.6
2.25 4.50 6.75 13.50
,550 .285 .210 .112 .087 .080
4.5 4.5 4.5
6.75 13.50
.322 ,108 .090
-
-
mp
adenine resulted in increased growth of cells. This result would be expected since hadacidin has been shown to inhibit the formation of AMP, and exogenously added adenine would be converted to AMP, thereby restoring normal growth. Addition of adenine to cells inhibited by 6-azauracil alone (indicated by B in Figs. 4a and 46) resulted in an unexpected enhancement of inhibition by 6-azauracil (seealso Table II). Qualitatively similar but more striking results were produced by the addition of adenine to cells growing in the presence of both hadacidin and 6-azauracil (indicated by C in Figs. tla and 46). It should be stressed here that in one case (Fig. 4a, C), adenine was added to cultures in which near maximal reversal by hadacidin of 6-azauracil inhibition occurred. Qualitatively similar results as above were obtained when adenosine was used instead of adenine. EFFECT OF HADACIDIX RESISTANT STRAIN
ON OF
6-AZ~UE~CILE.
COLI
B
The 6-azauracil-resistant strain was found to be sensitive to hadacidin and also to 6-azauridine. About 2 mg./ml. hadacidin and 900 wg./ml. 6-azauridine were required to inhibit the growth of cells by about 70 %. A go-fold larger concentration of B-azauri-
FIG. 5. Culture medium in a total volume of 4.50 ml. contained varying amounts of hadacidin alone (X-X), and 445 gg./ml. 6-azauridine and varying concent’rations of hadacidin (h-A). Dotted line represents growth of cells in t.he presence of 445 pg./ml. 6-axauridine alone.
dine was necessary as compared to the amount of 6-azauracil required to inhibit the parent strain of E. coli B. When the effect of increasing concentrations of hadacidin on the inhibition of growth caused by 6-azauridine was examined, it was observed that hadacidin did not reverse the inhibition. On the contrary, hadacidin enhanced the action of 6-azauridine in inhibiting the growth of the 6-azauracil-resistant strain (Fig. 5). DISCUSSION
Although the metabolic activities of hadacidin and 6-azauracil may not be completely known at present, the two principal biosynthetic processesknown to be inhibited by these two drugs are shown in Scheme I. 6-Azauracil may presumably be converted to the active form, 6-azauridine 5’-phosphate, via the pyrophosphorylase (Step 1) or the phosphorylase and kinase steps (Steps 2 and 3). The immediate product prevented from being formed by this inhibitor is uridylic acid. The inhibition by 6-azauridine 5’.phosphate of orotidylic acid decarboxylase obtained from rat liver and yeast has been shown (4). Although no comparable data from microorganism are available, the accumulation of erotic acid, orotidylic acid, and uridylic acid in cultures of E. cobi B subjected to inhibitory concentrations of 6-azauracil has been observed (6, 7). The conversion of 6-azauracil to K-azauridine in similar cultures of E. coli B (6, 8) and to the
REVERSAL
OF
6-AZAURACIL
Orotidylate
Inosinate
orot,idylic acid decarboxylnse
\ 1
1
Step
2
6-azauridine J
----_-
L-aspartxte GTP Mg++ adenylosuccinate thetase
6-azauracil Step
___-
4X
INHIBITION
/
Step
syn-
3 ---
blocked by 6azauridine 5’.phosphate
----
-
I
Uridylate
blocked
by hadacidin
Adenylosuccinate Scheme
di- and triphosphates and trace amount of monophosphate of azauridine in Streptococcus faecalis cultures have also been reported (9). These results suggested that the inhibition of growth of E. cobi B by 6-azauracil may be attributed to the suppression of uridylate formation. The block exerted by hadacidin on the pathway de nouo of purine biosynthesis (1) will ult,imately affert, diverse synthetic processes such as the formation of adeninecont’aining nucleotides and nucleic acids. Although the precise nature of the metabolic relationship between hadacidin and G-azauracil cannot be delineated from the observations reported in this paper, certain deductions may be made. The experiments clearly demonstrated that the inhibition of growl’th of E. coli B by 6-azauracil was reversed by small amounts of hadacidin. This effect by hadacidin on 6-azauracil inhibition was overcome by the addition of adenine or derivatives of adenine, indicating that the inhibitory capacity of 6-azauracil was restored by exogenously added adenine. The latter observation rules out the possibility that the effect of hadacidin on 6-azauracil inhibition may be due to a direct reaction between hadacidin and 6-azauracil or due to t#he format,ion of a permeability barrier. Furthermore, the effect caused by adenine and adenine derivatives was specific since other purines and pyrimidines were essentially wit,hout effect. It should also be noted that exogenously added adenine enhanced the inhibitory capacit,y of 6-azauracil alone, suggesting that R-azauracil is con-
I
verted to the active form more readily in the presence of added adenine or one of it*s derivatives. These results indicated that the inhibition due to hadacidin alone, namely, the suppression of formation of AMP or other adenine derivatives in some mamier accounted for the reversal of 6-azauracil inhibition by hadacidin. The observation that 6-azauracil-resistant, strains of 13. coli B was sensitive to 6-azauridine and not adversely affected by hadacidin indicated that the conversion of the nucleoside to the active nucleotide was not affected by hadacidin. It may also be concluded that the step inhibited by 6-azauridine 5’-phosphate, namely, the conversion of orotidylic acid to uridylic acid was also insensitive to hadacidin. REFERENCES 1. SHIGEURA,
Chem.
H. T., AND &IRDON, 237, 1937 (1962).
C. N.,
J. Biol.
2. fHIGEURA, H. T., AND GORDON, c. x., C'ance,' Res. 22, 1356 (19G2). C. -4., AND HASDWHUMACHER, I?. 3. PASTERNAK, E., J. Biol. Chem. 234, 2992 (1959). 4. HANDSCH~NACHER, R. E., J. Riol. C’hem. 236, 2917 (1960). 5. YATES, H. A., AND PARDEE, A. B., J. Biol. Chem. 221, 743 (1956). 6. SKODA, J., .4xD SORM, F., Biochim. Biophys. ncta 28, 059 (1958). 7. HhNDSCHUM4CHER, It. E., ~~:atuw 182, 1099 (1958). J., HESS, V. F., AND SORM, F., E.c8. SKODA, perientia 13, 150 (1957). 9. HANDSCHUMACHER, R. E., J. Biol. Chem. 236, 764 (1960).
OF
BIOCHEMISTRY
6-Azauracil
AND
BIOPHYSICS
Inhibition
472-477
lo&
(19ti3)
of Escher&a
coli B and
Its Reversal
by Hadacidin’ HAROLD From the Ned
Institute
T. SHIGEURA
for Therapeutic Received
October
Research,
Rahway,
Kew Jersey
8, 1962
The inhibition of growth of Escherichia coli B by G-axauracil was found to be reversed by hadacidin. The inhibition of growth by hadacidin, however, was not affected by &azauracil. The inhibitory activity of 6-azauracil in the presence of hadacidin was restored by the addition of adenine, adenosine, deoxyadenosine, or inosine. Several other purines and pyrimidines were inactive. The inhibitory activity of 6-azauracil alone was enhanced by the addition of adenine. 6-Azauracil-resistant, strain of E. coli B was sensitive to hadacidin and 6-azauridine. The inhibition of growth by 6-azauridine was not reversed by hadacidin. The results indicated that the metabolic activity of hadacidin, namely, the suppression of formation of adenylic acid or other adenine derivatives in some manner accounted for the reversal by hadacidin of 6-azauracil inhibition of E. coli B. INTRODUCTION
effect of hadacidin and 6-azauracil on the growth of E. coli B pointed to an interesting relationship between these two compounds per se or between the formations of the essent,ial products (adenylic and uridylic acids) that are suppressed in the presence of these inhibitors. Further studies conducted on this unexpected phenomenon are described in this paper.
The antibiotic hadacidin (N-formyl hydroxyaminoacetate) has been found to suppress the conversion of inosinate to adenylosuccinate by the enzyme inosine 5’-phosphate : L-aspartate ligase (adenylosuccinate synthetase) obtained from Escherichia coli B. The inhibition was competitively reversed by L-aspartate (1). During the course of studies on the effects of hadacidin when used in combination with other known antitumor agents on the growth of E. coli B, it was observed that hadacidin appeared to reverse the potent inhibitory property of 6-azauracil (as-triazine-3,5dione). The results further indicated the presence of a competitive type of antagonism between these two compounds (2). Since 6-azauracil, in the form of the mononucleotide, has been shown to inhibit the conversion of orotidylic acid to uridylic acid (3, 4), the observation on the combined
EXPERIMENTAL
PROCEDURE
MIITSRIALS The purines, pyrimidines, and their derivatives were purchased from the California Corporation for Biochemical Research. Hadacidin was obtained from Dr. E. A. Kaczka, Merck Sharp & Dohme Research Laboratories, Rahway, N. J. CCLTIV.~TIOX
OF E'. COLI
B
The microorganism was grown at 37” in a medium consisting of the following components per liter (5) : 1lOmg. MgSOa,7HzO, 7.8 g. K?HPO,, 2.2 g. KHnPOr, 470 mg. Na citrate.2H20, 1.1 g. (NH,)zS04, and 25 g. glucose. Cells grown overnight in this medium were inoculated into 50 ml. of fresh medium (570 inoculum). After about 4 hr., the logarithmically growing cells were centrifuged,
1 This investigation was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the National Institutes of Health Contract Number SA-43.ph1886. 472
REVERSAL
OF
G-AZA4URACIL
473
ISHIBITION
malml OF HAOACIDIN
I, 0
0.5
1.0
2.0
1.5
ma/ml OF HAOACIDIN
FIG. 1. Culture medium in a total volume of 4.5 ml. contained varying concentrations of 6. azauracil or hadacidin and 0.1 ml. of cell suspension. Optical density at zero time was 0.050. The cultures were incubated at 37” for 334 hr. The dotted line indicates 700;, inhibition of growth.
nntl the pellet was fresh, st)erile medium described helow.
suspended in about. 5 ml. and used for the experiments
of
FIG. 2. Culture medium in a total volume of 4.58 ml. contained varying concent,rations of hadacidin with or without 9.7 pg./ml. 6-azauracil. The cultures were incubat’ed at 37” for 33i hr. X-X, eflect of increasing amounts of hadacidin alone; A-A, effect of constant amount of 6azauracil (9.7 pg./ml.) and increasing amounts of hadacidin; dotted line represents growth of cells in the presence of 9.7 pg./ml. R-azauracil a 1one. RESULTS
To determine the effects of the various cornpounds on the growth of E. coli B, several tubes containing t)he test compounds in a cult,ure medium of 4.5 ml. were inoculated with 0.1 ml. of cell suspension and incubated at 37” for 3353 hr. The optical density at zero time was about 0.050, and growth after R specified t,ime was estimat,ed by measurement of optical density at 600 nip in a Beckman model B spectrophotometer.
STRAIN
OF
E.
COLI
B
Cells grown in the medium described above were inoculated (55; inoculum) into fresh medium containing 5 pg./ml. 6-azaurncil and incubated at, 37” overnight. A similar inoculum from this culture was then transferred to a fresh medium containing 10 pg./ml. (i-azanracil and grown overnight,. The concentration of the drug was doubled with each successive inoculat,ion until cells resistant to 1.25 mg./ml. G-azauracil were developed. The resistant strain was maintained :it 0” on agar slant)s containing 1.25 mg./ml. 6-szarnacil.
THE EFFECTS OF HAI~.~CIDIX rKAClL WEIE~ I!sl!a ALONE GROWTH OF E. COLI
The effect of increasing hadacidin on the growth organism
is shown
in
E’ig.
AND ON
B
concentrations
of the 1. A
6-AzilTHE
of micro-
concentra-
tion of 2.4 mg./ml. (1.7 X lo-? ill) of the antibiotic was required to inhibit the growth of E. co/i B by about 70%. Similar studies with (i-azauracil showed that about 10 pg.! ml. (7.7 X 1OP N) of the compound was required to attain the same per cent inhibition (Fig. l), indicat.ing that B-azauracil was about 240.fold more cffect’ive than hadacidin in suppressing the growth of fc. coli 13.
In each case, t,he amount of hadacidin or G-azauracil required to produce about 70%)
474
SHIGEURA
inhibition of growth was used, and the effect caused by the addition of increasing quantities of the other drug was examined. When the concentration of 6-azauracil was kept constant at 9.7 pg./ml., addition of increasing amounts of hadacidin resulted in a gradual rise in turbidity which was considerably above the level of that obtained by the same concentration (9.7 pg./ml.) of 6-azauracil alone (Fig. 2). Cell growth steadily increased until the concentration of hadacidin in the culture was about 300 kg./ml. At this point, further increase in hadacidin concentration lead to a gradual decrease in cell growth with the hadacidin plus 6-azauracil curve coinciding with that obtained by hadacidin alone. Since the optical density of the suspension containing 6-azauracil alone was 0.200 (represented by the dotted line in Fig. 2), the gradual decrease in optical density (increase in inhibition) of the hadacidin plus 6-azauracil curve with increasing amounts of hadacidin in excess of 300 pg./ml. appeared to be due solely to hadacidin. However, the initial increase in proliferation of the cells exposed to both drugs may be due to an antagonistic effect exerted by hadacidin, perhaps indirectly, on the inhibitory c.apacity of 6-azauracil. This reasoning was not in conflict with the results obtained when a reciprocal experiment was performed. As shown in Fig. 3, when the concentration of hadacidin was kept constant at 2 mg./ml. and the concentration of 6-azauracil was progressively increased, growth of the cells did not increase but remained constant and equal to that due solely to hadacidin (dotted line). Increasing concentration of 6-azauracil clearly did not affect the inhibition of growth caused by hadacidin. Preincubation of the bacteria with 6-azauracil for 12 min. at 25” prior to the addition of hadacidin did not alter the results shown in Fig. 2. FURTHER STUDIES ON THE EFFECTS OF HADACIDIX 6-AZAURACIL
COMBISE~ ASD
In an attempt to obtain information with respect to the mechanism by which hadacidin nullifies the inhibitory effect of 6-aza-
.6OOr x ,500 -\
>-\ I SL : ;.400-
x
\
2
i
x
t .30b s x
g .200 t0
-\ -_____
_
----
X\ _____-_--_
---______-
0
,100
0
I IO
5 M/ml
1 15
h 20
‘
of6- AZAURACIL
FIG. 3. Culture medium in a total volume of 4.6 ml. contained varying concentrations of 6aaauracil with or without 2 mg./ml. hadacidin. The cultures were incubated at 37” for 3% hr. X-X, effect. of increasing amounts of 6-aeauracil alone; O-0, effect of constant amount of hadacidin (2 mg./ml.) and increasing amounbs of 6azauracil; dotted line represents growth of cells in the presence of 2 mg./ml. hadacidin alone.
uracil, the effects of the addition of various purines, pyrimidines, and certain amino acids to a system containing both drugs were examined. The cells were grown in media containing 9.7 and 200 pg./ml. 6-aeauracil and hadacidin, respectively, in order to obtain near optimal reversal of 6-azauracil inhibition by hadacidin. (See Fig. 2, peak of curve indicated by A-A.) Various compounds listed in Table I were added to the vessels, and optical densities were measured after 4 hr. As can be seen in the table, adenine, adenosine, deoxyadenosine, and inosine were found to overcome effectively the action of hadacidin on 6-azauracil (compare vessels 5-8 with vessel 4). In each of these instances, the inhibition of growth (due apparently to restoration of 6-azauracil activity) was greater than that due to B-azauracil alone (vessel 2). It may be of significance to note here that adenine, adenosine, and deoxyadenosine are directly affected by the specific inhibitory action of hadacidin on adenylosuccinat,e synthetase, suggesting the possibility that
REVERSAL TABLE THE
EFFECTS REVERSAL
OF
VARIOUS
OF
6-AZAURACIL
I
40
COMPOUNDS
OF G-AZAURACIL BY HADACIDIN
ox
No.
INHIBITION
6Hadacidin
Azauracil
Compounds
Tested
Optical Density at 600 mu
-1 2 3
4 5 G r s 9 10 11 12 13 1-l 15 16 17 1S l!) 20 21 2” 2.3
+ + + + + + + + + + + + + + + + + + + + +
-
,425
+ -
,147
+ + + + + + i-I+ + + + + + f + + + + I
4b
THE
Culture media in a total volume of 4.5 ml. COW tained 1.2 X 10-S X hadacidin, 6.9 X 10-j N fiazauracil, and 2 X 10-a M test compound as indicated. The optical density at zero time was 0.050. Optical density measurements were taken after incubation at 37” for 3js hr. Vessels 4-23 contained both hadacidin and 6.azauracil as indicated bj (+). Vessel 4 shows reversal by hadacidin of Gazauracil inhibition (compare with vessel 2). Restoration of 6.azauracil inhibition was shown by optical density values less than .300 (vessel 4). Vessel
475
INHIBITIOX
Adenine Adenosine Ueoxyadenosine Inosine r,-Aspartat,e Cuanine Guanosine Deoxyguanosine Hypox:rntjhine Xanthine Uracil 1Jridine Cytosine Cytidine Thymine Thymidine Orotic acid Orotidinc Glycine
,360 ,300 .I12 ,100 ,115 ,106
,200 ,278 ,260 .330 .380 ,430 ,450 ,480 ,435 ,460 .400 .480 .495 ,380 ,385
the mechanism of the process is related to the inhibition of purine biosynthesis de novo by hadacidin. Some reversal of action of hadacidin on 6-azauracil inhibition was also obtained with L-aspartate. Guanine and guanosine were weakly active. The remaining compounds were either inert or stimulated growth of the bacteria. Preincubation of the organism with hadacidin and 6-azauracil for 12 min. at 25” prior to the addition of adenine did not alter the results shown in Table I, vessel 5.
.5oc)E' 8ls ,400 .J
X-X-X
A
.500.400.300xdX
.200-'
A
\ .I00 --x+-t 1 . 0123
8 8
L ' 0123 hg/ml
; '
'
OF ADENINE
FIG. 4. (a) Culture medium in a total volume of 4.59 ml. contained 174 pg./ml. hadacidin alone in tubes indicated by A, 8.7 pg./ml. 6-azauracil alone in tubes indicated by H, and 174 ~g./ml. hadacidin plus 8.7 pg./ml. 6-azauracil in tubes C. Increasing amounts of adenine as shown were added to the tubes and growt,h was measured after incubation at 37” for 4 hr. (b) Culture medium in a tot,al volume of 4.43 ml. contained 1.13 mg./ml. hadacidin alone in tubes indicated by .4, 9.1 ~g./ml. F-azauracil in tubes indicated by B, and 1.13 mg./ml. hadacidin plus 9.1 pg. (i-azauracil in tubes indicated by C. Increasing amounts of adenine as shown were added to the tubes, and growth was measured after incubat)ion at 37” for 4 hr.
,4 more detailed investigation of t,he observation described above (the restoration of 6-azauracil inhibition in the presence of hadacidin) was made by studying the effects produced by the addition of adenine to cells incubated with either hadacidin alone, A-azauracil alone, or a combination of hadacidin and 6-azauracil at two separate phasesof the growth curves shown in Fig. 2, namely (1) the position of the curve at which reversal by hadacidin approaches maximal level (9.7 pg./ml. 6-azauracil and 200 pg./ml. hadacidin) and (9) the declining phase of the curves where t)he growth curve of hadacidin coincides with that of hadacidin plus 6-azauracil (9.7 pg./ml. 6-azauracil and 1 mg. ml. hadacidin). As shown in Figs. -la and #I, in both phasesof t’he growth curve representing inhibition by hadacidin alone (rrpresented by *-I in Figs. 4a and 4b), addition of
SHIGEURA
476 TABLE THE
EFFECT
II
,500
OF ADENINE ON THE INHIBITION GROWTH OF E. COLI B BY 6-AZAURACIL ALONE
OF
r
Culture media in a total volume of 4.45 ml. contained 5.6 pg./ml. (5 X 1OF M) or 4.5 pg./ml. (4 X 10d5 M) of 6-axauracil. Indicated amounts of adenine were added. Growth was measured after incubation for 3% hr. at 37”. 6.Azauracil
Adenine
pg./ml.
sg./ml.
O.D. at600
5.6 5.6 5.6 5.6 5.6
2.25 4.50 6.75 13.50
,550 .285 .210 .112 .087 .080
4.5 4.5 4.5
6.75 13.50
.322 ,108 .090
-
-
mp
adenine resulted in increased growth of cells. This result would be expected since hadacidin has been shown to inhibit the formation of AMP, and exogenously added adenine would be converted to AMP, thereby restoring normal growth. Addition of adenine to cells inhibited by 6-azauracil alone (indicated by B in Figs. 4a and 46) resulted in an unexpected enhancement of inhibition by 6-azauracil (seealso Table II). Qualitatively similar but more striking results were produced by the addition of adenine to cells growing in the presence of both hadacidin and 6-azauracil (indicated by C in Figs. tla and 46). It should be stressed here that in one case (Fig. 4a, C), adenine was added to cultures in which near maximal reversal by hadacidin of 6-azauracil inhibition occurred. Qualitatively similar results as above were obtained when adenosine was used instead of adenine. EFFECT OF HADACIDIX RESISTANT STRAIN
ON OF
6-AZ~UE~CILE.
COLI
B
The 6-azauracil-resistant strain was found to be sensitive to hadacidin and also to 6-azauridine. About 2 mg./ml. hadacidin and 900 wg./ml. 6-azauridine were required to inhibit the growth of cells by about 70 %. A go-fold larger concentration of B-azauri-
FIG. 5. Culture medium in a total volume of 4.50 ml. contained varying amounts of hadacidin alone (X-X), and 445 gg./ml. 6-azauridine and varying concent’rations of hadacidin (h-A). Dotted line represents growth of cells in t.he presence of 445 pg./ml. 6-axauridine alone.
dine was necessary as compared to the amount of 6-azauracil required to inhibit the parent strain of E. coli B. When the effect of increasing concentrations of hadacidin on the inhibition of growth caused by 6-azauridine was examined, it was observed that hadacidin did not reverse the inhibition. On the contrary, hadacidin enhanced the action of 6-azauridine in inhibiting the growth of the 6-azauracil-resistant strain (Fig. 5). DISCUSSION
Although the metabolic activities of hadacidin and 6-azauracil may not be completely known at present, the two principal biosynthetic processesknown to be inhibited by these two drugs are shown in Scheme I. 6-Azauracil may presumably be converted to the active form, 6-azauridine 5’-phosphate, via the pyrophosphorylase (Step 1) or the phosphorylase and kinase steps (Steps 2 and 3). The immediate product prevented from being formed by this inhibitor is uridylic acid. The inhibition by 6-azauridine 5’.phosphate of orotidylic acid decarboxylase obtained from rat liver and yeast has been shown (4). Although no comparable data from microorganism are available, the accumulation of erotic acid, orotidylic acid, and uridylic acid in cultures of E. cobi B subjected to inhibitory concentrations of 6-azauracil has been observed (6, 7). The conversion of 6-azauracil to K-azauridine in similar cultures of E. coli B (6, 8) and to the
REVERSAL
OF
6-AZAURACIL
Orotidylate
Inosinate
orot,idylic acid decarboxylnse
\ 1
1
Step
2
6-azauridine J
----_-
L-aspartxte GTP Mg++ adenylosuccinate thetase
6-azauracil Step
___-
4X
INHIBITION
/
Step
syn-
3 ---
blocked by 6azauridine 5’.phosphate
----
-
I
Uridylate
blocked
by hadacidin
Adenylosuccinate Scheme
di- and triphosphates and trace amount of monophosphate of azauridine in Streptococcus faecalis cultures have also been reported (9). These results suggested that the inhibition of growth of E. cobi B by 6-azauracil may be attributed to the suppression of uridylate formation. The block exerted by hadacidin on the pathway de nouo of purine biosynthesis (1) will ult,imately affert, diverse synthetic processes such as the formation of adeninecont’aining nucleotides and nucleic acids. Although the precise nature of the metabolic relationship between hadacidin and G-azauracil cannot be delineated from the observations reported in this paper, certain deductions may be made. The experiments clearly demonstrated that the inhibition of growl’th of E. coli B by 6-azauracil was reversed by small amounts of hadacidin. This effect by hadacidin on 6-azauracil inhibition was overcome by the addition of adenine or derivatives of adenine, indicating that the inhibitory capacity of 6-azauracil was restored by exogenously added adenine. The latter observation rules out the possibility that the effect of hadacidin on 6-azauracil inhibition may be due to a direct reaction between hadacidin and 6-azauracil or due to t#he format,ion of a permeability barrier. Furthermore, the effect caused by adenine and adenine derivatives was specific since other purines and pyrimidines were essentially wit,hout effect. It should also be noted that exogenously added adenine enhanced the inhibitory capacit,y of 6-azauracil alone, suggesting that R-azauracil is con-
I
verted to the active form more readily in the presence of added adenine or one of it*s derivatives. These results indicated that the inhibition due to hadacidin alone, namely, the suppression of formation of AMP or other adenine derivatives in some mamier accounted for the reversal of 6-azauracil inhibition by hadacidin. The observation that 6-azauracil-resistant, strains of 13. coli B was sensitive to 6-azauridine and not adversely affected by hadacidin indicated that the conversion of the nucleoside to the active nucleotide was not affected by hadacidin. It may also be concluded that the step inhibited by 6-azauridine 5’-phosphate, namely, the conversion of orotidylic acid to uridylic acid was also insensitive to hadacidin. REFERENCES 1. SHIGEURA,
Chem.
H. T., AND &IRDON, 237, 1937 (1962).
C. N.,
J. Biol.
2. fHIGEURA, H. T., AND GORDON, c. x., C'ance,' Res. 22, 1356 (19G2). C. -4., AND HASDWHUMACHER, I?. 3. PASTERNAK, E., J. Biol. Chem. 234, 2992 (1959). 4. HANDSCH~NACHER, R. E., J. Riol. C’hem. 236, 2917 (1960). 5. YATES, H. A., AND PARDEE, A. B., J. Biol. Chem. 221, 743 (1956). 6. SKODA, J., .4xD SORM, F., Biochim. Biophys. ncta 28, 059 (1958). 7. HhNDSCHUM4CHER, It. E., ~~:atuw 182, 1099 (1958). J., HESS, V. F., AND SORM, F., E.c8. SKODA, perientia 13, 150 (1957). 9. HANDSCHUMACHER, R. E., J. Biol. Chem. 236, 764 (1960).