A SITE OF ACTION OF STREPTOMYCIN

BY W. W. UMBREIT

(From the Merck Znatitute for Therapeutic Research, Rahway, New Jersey)

(Received for publication, July 24, 1948)

Various reports (l-5) have demonstrated that streptomycin inhibits the metabolic activities of the non-proliferating cells of susceptible strains of bacteria. This paper is an attempt to trace the mode of action of strepto- mycin with the object of determining what reactions vital to the cell are interfered with. Study of the mode of action of the antibiotics of therapeu- tic value should eventualIy provide an explanation for their ability to enter the animal body and kill susceptible bacteria without killing the tissue cells. The first step toward this goal is to determine which reactions of the bacte- rium are susceptible to streptomycin inhibition at concentrations of this agent which seem to be effective in practice.

In order to investigate this problem, we began with the observations of Geiger (3), who demonstrated that, with a particular strain of Escherichiu IX& there was a marked stimulation of serine oxidation by the previous oxidation of fumarate. When streptomycin was present (at levels of 5 to 26 y per ml.) during the oxidation of fumarate, this stimulation was pre- vented. Geiger concluded that streptomycin prevents the action of some intermediate, formed during the oxidation of fumarate and a variety of other carbohydrate substances, which is necessary for the oxidation of serine. One could also interpret the data reported by Geiger as indicating that it was the jorwmtion of the intermediate which was interfered with by streptomycin. Using these observations as a starting point, we have been able to trace the mode of action of streptomycin somewhat further.

Methods

The Gratia strain of Escherichia c0.V was grown in 1 per cent Difco pep- tone, 1 per cent Difco yeast extract, 0.5 per cent K2HP04 (Medium A) at 30’ or 37” (as specified) for 16 to 18 hours. The cells were harvested by centrifugation, washed three times by resuspension in one-tenth the growth volume of distilled water, and suspended in distilled water to give a cell concentration of from 0.5 to 1.25 mg. of bacterial nitrogen per ml. Such suspensions retained the activities to be described for 3 to 5 days at refriger- ator temperatures, but thereafter the activity was gradually lost. As noted by Geiger (3), cocarboxylase was effective in restoring the lost activity for

f Sensitive in culture to 9 y per ml. of streptomycin. We are indebted to Mr. Otto Grressle for these determinations.

793

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704 SITE OF STREPTOMYCIN ACTION

a short period, but with our strain other deficiencies soon developed. The properties of the living cell suspensions vary somewhat with the growth medium (if peptone is replaced in equal amounts by N-z-amine (Medium B) or if glucose is supplied during growth), the growth temperature, the con- centration of cells, the conditions under which the measurements are made, and doubtless other factors. These variations are of a quantitative rather than a qualitative character, and are reflected in the amounts of gas ex- change observed or the degree of streptomycin inhibition. Since our object was to trace the mode of action of streptomycin rather than to study the influence of various factors on streptomycin activity, we attempted to se- lect, as the work progressed, those conditions most conducive to demon- strating the “streptomycin effect.”

Streptomycin was used as the commercial (Merck) calcium chloride complex at a level of 10 y of the free base per ml. (except where otherwise specified). Streptomycin hydrochloride was equally effective, but the cal- cium chloride complex inactivated by heat and acid or alkali was not effec- tive. Total keto acids were determined by the method of Friedemann and Haugen (6). a-Ketobutyrate was determined by the total keto acid method, standardized against its 2, 4-dinitrophenylhydrazone.2 While, in general, the specificity of this procedure could be questioned, in this par- ticular case it is reasonably valid, since the 2,4-dinitrophenylhydrazone of a-ketobutyrate is much less soluble than those of most other keto acids and actually precipitates in the initial stages of the total keto acid pro- cedure with samples containing as little as 10 y of cr-ketobutyrate. The a-ketobutyrate used in a few experiments was prepared from the N- benzoylaminocrotonic acid3 after the method of Carter et al. (7). Ammo-. nia was determined by nesslerization (8) after removing the acid-killed cells by centrifugation. Acetyl phosphate was estimated by the method of Lipmann and Tuttle (9), standardized against succinic anhydride. Standard Warburg techniques (8) were used throughout. All respiration measurements were made at 37” at pH 7.

Studies with Threonine

The studies were first directed towards the oxidation of amino acids as reported by Geiger (3). It was soon found that neither the D- nor L-

amino acid oxidases from either bacteria or animal tissues were inhibited by streptomycin, and that the Gratia chain of Escherichia coli was capa- ble of attacking only aspartate, serine, or threonine at a rapid rate. While

2 Obtained through the generosity of Dr. D. B. Sprinson, Department of Bio- chemistry, College of Physicians and Surgeons, Columbia University, New York.

3 We are indebted to Dr. E. E. Howe of the Research Laboratories of Merck and Company, Ine., for this material.

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some study was devoted to se&e and the results of Geiger were con- firmed in principle, our strain did not show the gradually increasing rate of serine oxidation found by Geiger.

The threonine system was more readily approachable. As with serine, the oxidation of threonine could be markedly increased by the previous

HOURS FIG. 1. Influence of fumarate oxidation and streptomycin upon the oxidation of

threonine. Curves A, 10 micromoles of fumarate added at zero time, 10 micromoles of DL-threonine added at 3 hours; Curves B, threonine at zero time, fumarate at 3 hours. The broken lines represent treatments contriining 20 y per ml. of strepto- mycin. The cups contain 0.25 mg. of bacterial nitrogen, 0.016 M phosphate buffer, pH 7, substrates, and water to make 3 ml.; gas phase air; temperature 37”.

oxidation of fumarate. If streptomycin was present during the oxidation of fumarate, little increased rate of threonine oxidation was observed.

A typical experiment is illustrated in Fig. 1. First it may be noted from Curves C that a small amount of oxygen is consumed in the absence of added substrate, which may or may not show a streptomycin inhibition as in this case. For simplicity in presentation, the curves in this and subse- quent figures have been corrected for the respiration in the absence of sub- strhte. From Curves B it will be noted that there is a relatively slow oxidation of threonine. Fumarate is oxidized more slowly if it is added 3

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706 SITE OF STRBPTOMYCIN ACTION

hours after threonine than if it is added at zero time, and the oxidation of fumarate added after threonine is inhibited by streptomycin. From Curves A the initial addition of 10 micromoles of fumarate causes a rapid oxygen uptake for 2 hours, which is not altered in rate by the presence of streptomycin. After 2 hours the rate slows and this rate (between the 2nd and 3rd hours) is, in this case, slightly inhibited by streptomycin. The in- hibition over this period is variable and is greatly dependent upon the salt concentration in which the reaction is run. At the 3rd hour, 10 micromoles of nL-threonine were added. It is evident that the oxidation of fumarate has promoted the subsequent oxidation of threonine and that streptomycin, when present during the fumarate oxidation, has prevented this phenom- enon.

It first appeared that, if one could determine by what pathway the threo- nine was oxidized, one could determine the mode of action of streptomycin. This strain of Escherichia coli, grown under the conditions described, pos- sesses a threonine deaminase which,’ as has been shown by Chargaff and Sprinson (lo), produces ammonia and cr-ketobutyric acid. In the.case of data comparable to those given in Fig. 1, per micromole of threonine added, we found 0.9 to 1.1 micromoles of ammonia, with or without pre- vious fumarate oxidation or in the presence or absence of streptomycin. Further D-, L-, or nn-threonine gave comparable results, characteristic of threonine deaminase (10). The other product of deamination is oxidized by most suspensions, but if oxidation is stopped before the reaction has used 0.5 mole of O2 per mole of threonine, keto acid accumulation with the characteristics of cY-ketobutyrate can be detected. We have, there- fore, concluded that the first step in the oxidation of threonine is deami- nation to ru-ketobutyrate and ammonia. This is independent of previous fumarate oxidation in the presence or absence of streptomycin; hence the streptomycin effect noted is concerned with the oxidation of Lu-keto- butyrate. The most direct way of establishing this fact would be to substitute cY-ketobutyrate for threonine, but unfortunately added ru-keto- butyrate is but slowly oxidized (with or without the simultaneous addi- tion of ammonia), even after intensive fumarate oxidation, possibly be- cause at pH 7 it does not readily penetrate the cell.

The data of Fig. 2 summarize the effects of previous oxidation of fumarate and the presence or absence of streptomycin upon the oxidation of threo- nine. Threonine added to cells which have not previously oxidized fuma- rate take up close to 0.5 mole of 02 per mole of threonine and simultane- ously release close to 1.0 mole of CO2 (Figs. 2, A, 2, B). These data are in accordance with Reaction 1 which is similar to that reported by Long

(1) Threonine --t NH8 + a-ketobutyrate 0.5 “propionate” + CO,

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W. W. UMRREIT 707

and Peters (11) for the oxidation of ar-ketobutyrate by brain tissue. Re- action 1, as is evident from Fig. 2, is not influenced by streptomycin. Further, since the same results are obtained whether the threonine is added at zero time or after 3 hours of endogenous respiration, the respi- ration in the absence of added substrate under these conditions does not either increase the toxicity of streptomycin or generate the ability to oxi- dize threonine further. Occasional suspensions, as might be expected, do

100

HL”RS2 HOURS HOURS FIG. 2. Oxidation of threonine. In A, 10 micromoles of threonine were added at

zero time; in B, added after 3 hours respiration without substrate; in C, added after 3 hours respiration on 10 micromoles of fumarate. 1 mg. of bacterial nitrogen per cup in 0.003 M phosphate, pH 7. The broken lines represent treatments containing 10 7 of streptomycin per ml.

show increased respiration with threonine after 3 hours of endogenous respiration. This effect we have attributed to the occasional presence of oxidizable substrates in the cell. In the presence of streptomycin the ad- dition of threoniue after 3 hours of fumarate oxidation leads to the same result (0.5 mole of 02, 1.0 mole of CO2 per mole of threonine) as does the addition of threonine at zero time or after 3 hours of endogenous respira- tion. However, if streptomycin is not present, the oxidation of fumarate permits the oxidation of threonine to proceed much further (Fig. 2, C). While the reaction written in Reaction 1 probably represents the course of threonine oxidation when added without previous fumarate oxidation (or with fumarate oxidation in the presence of streptomycin), it does not

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708 SITE OF STREPTOMYCIN ACTION

necessarily represent the first stage of the reactions when fumarate has been oxidized. Here, as will be noted from Fig. 2, 1 mole of CO2 and 1 mole of ammonia are formed; yet the product of fumarate oxidation may react with the cr-ketobutyrate before the latter is oxidized. Thus, the oxidation of added propionate may not be stimulated by previous fuma- rate oxidation. This is indeed the case; added propionate is virtually in- ert, either because of the reasons outlined above, or because it does not enter the cell.

HOURS HOURS

FIG. 3. Oxidation of threonine after fumarate oxidation. 1.0 mg. of bacterial nitrogen per cup. 10 micromoles of fumarate oxidized for 3 hours, then 10 micro- moles of threonine added. A, 0.03 M phosphate buffer, pH 7; B, 0.003 M phosphate buffer. X, no streptomycin; 0, 10 y per ml. of streptomycin present during fuma- rate oxidation; A, 10 y per ml. of streptomycin added at 3 hours with threonine.

These results lead to a conclusion similar to that reached by Geiger (3) ; i.e., streptomycin prevents the formation or activity of some substance derived from the oxidation of fumarate which is necessary for the oxidation of threonine (or its deamination product, Lu-ketobutyrate). It was, there- fore, desirable to attempt to distinguish between the possibility that strep- tomycin prevented the formation of this substance and the possibility that it prevented its action. If fumarate were oxidized in the absence of strep- tomycin, the substance should be formed. If its action were prevented by streptomycin, then the addition of streptomycin with the threonine should be effective, whereas if its formation were prevented by streptomycin, the latter should be active only when present during fumarate oxidation. The data of Fig. 3 indicate that to be really effective the streptomycin must be

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W. W. UMRREIT 709

present during fumarate oxidation. This agent therefore apparently acts by preventing the formation of the hypothetical intermediate rather than by interfering with its action once it is formed.

Studies with Organic Acids

In efforts to identify the substance which is formed during the oxidation of fumarate and whose formation is inhibited by streptomycin, experiments were done at a low phosphate level, at which effects of streptomycin upon

PI02 PM PER

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FUMARATE ADDED

B

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--t--eL” 3 HOURS

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FIG. 4. Oxidation of fumarate by cells grown under different conditions. All oxidations in 0.003 M phosphate buffer, pH 7, 1 mg. of bacterial nitrogen per cup; streptomycin concentration when present, 10 y per ml. (broken lines). A, cells grown on Medium A, 30°, 16 hours; B, cells on Medium A, 37”, 16 hours; C, cells on Medium A plus 0.1 per cent glucose, 37”, 16 hours; D, cells on Medium B, 30”, 16 hours.

/” A

/

/ I

D

the respiration of fumarate itself may be observed. A comparison of the oxidation of fumarate by cells grown under various conditions is given in Fig. 4. While it is apparent that there is some variation in the rate and extent of fumarate oxidation, depending upon the medium, growth tempera- ture, and doubtless other factors, there is also consistently less complete oxidation of fumarate in the streptomycin-treated cells. It is also apparent from Fig. 4 that the oxygen uptake in all conditions proceeds to at least 1 mole of O2 per mole of fumarate before a marked streptomycin effect is evi-

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710 SITE OF STREPTOMYCIN ACTION

dent. The utilisation of 0.5 mole of 02 per mole of fumarate would bring it to the oxidation state of pyruvate; hence there must be the utilization of 0.5 mole of 02 per mole of pyruvate (at least) before the streptomycin effect is noted. Fig. 5 shows that close to 0.5 mole of 02 per mole of pyruvate added is consumed before any great effect of streptomycin is observable.

The general conclusion that one may draw from these data is that strep- tomycin prevents the “terminal respiration” process in the susceptible

I2 34 I 2 3 4 I 2 3 HOURS HOURS HOURS

FIG. 5. Oxidation of pyruvate. All oxidationa in 0.003 M phosphate buffer, pH 7. Streptomycin concentration when present, 10 y per ml. (broken lines). A, 1 mg. of bacterial nitrogen per cup, cells grown on Medium B, 30”, 18 houre, 10 micro- moles of pyruvate added; B, 0.45 mg. of bacterial nitrogen per cup, cells on Medium B, 30”, 16 hours, 5 micromoles of pyruvate added; C, 1.0 mg. of bacterial nitrogen per cup, celle on Medium A, 30”, 18 houre, 10 micromoles of pyruvate added.

bacteria. This is true of threonine (or the keto acid derived from it), in which a previous oxidation of fumarate was necessary before it could enter the terminal respiration system, as well as for fumarate and pyruvate in which only a partial oxidation is observed when streptomycin is present. For investigation of the terminal respiration process in this strain, two possibilities were considered.

First, the data on pyruvate oxidation shown in Fig. 5 suggest that pyruvate is oxidized to the acetate stage. Thereafter, when streptomycin is present, further oxidation is inhibited. Acetate itself is not readily oxi-

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W. W. UMEZREIT 711

dized by this strain and what oxidation does occur is only slightly affected by streptomycin. In fact, the rate of oxidation observed with pyruvate and streptomycin (Fig. 5) after the 0.5,mole of 02 per mole of pyruvate point corresponds roughly with the ability of the suspensions to oxidize added acetate. Nevertheless, one could postulate that an “active ace- tate” was the result of pyruvate oxidation, and that it was this material which was further oxidized. According to this hypothesis, streptomycin would prevent the formation of “active acetate.”

Second, the data of Fig. 5 could equally well suggest that, in the absence of streptomycin, the pyruvate was oxidized by a process similar to ter- minal respiration in the animal; i.e., condensation with oxalacetate to enter the tricarboxy acid cycle (12). If this process were inhibited by streptomycin, the pyruvate could be oxidized by a different pathway to the acetate stage. The essential difference between these hypotheses is that the first considers that the initial stages of the process of pyruvate oxidation are the same in the presence or absence of streptomycin, whereas the second considers that two different processes are involved.

Search for the formation of “active acetate” was not experimentally suc- cessful. In our hands, the hydroxylamine method of Lipmann and Tuttle (9) has not revealed “active acetate” (not necessarily acetyl phosphate) at any stage of the process. Recourse was therefore had to vacuumdried cell preparations in the hope that conditions could be obtained in which some of the side reactions were eliminated. In the case of the reactions studied here, the dried preparations were relatively unstable and retained the activities described for about 24 hours, thereafter becoming completely in- active. As might be expected, occasional preparations were entirely in- active. Data from active preparations were typical of those shown in Fig. 6. In this case, as for other preparations, pyruvate could be replaced by threonine with essentially the same results except that the reactions were somewhat slower.

The first portion of Fig. 6 shows that when fumarate is added there is a lag period of approximately 90 minutes before the maximum rate of oxida- tion is observed. During this period there is a gradually increasing rate of oxygen uptake and by the time the maximum rate has been reached (90 minutes) 0.5 mole of 02 per mole of fumarate has been used. This would be sufficient to oxidize the fumarate to the oxalacetate stage. One may presume that the rate of oxidation thereafter is controlled by the oxalacetate and pyruvate (its decarboxylation product) condensations and indeed, since the addition of pyruvate at 3 hours has no effect, the rate-controlling factor is probably oxalacetate. When streptomycin is present, there is a slight initial oxidation which soon stops, and addition of pyruvate at 3 hours has no effect.

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712 SITE OF STREPTOMYCIN ACTION

In the second portion of Fig. 6, the addition of pyruvate causes 6rst an oxygen uptake essentially uninfluenced by streptomycin except in the later stages. However, the oxygen taken up falls far short of that necessary for oxidation even to the acetate stage. The addition of fumarate now results in a rapid oxidation with a lag period of only 30 minutes and the uptake of only 26 microliters of OS before the maximum rate is reached. When streptomycin is present, fumarate does not initiate any oxidation.

. FUMARATE

500

400

300

200

100

PY RUVATE

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FUMARATE

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HOURS FIG. 0. Oxidation of fumarate and pyruvate by dried preparations of Escherichia

wli. Each flask contained 20 mg. of vacuum-dried preparation of cells of E. coli grown on Medium A, 30”, for 16 hours, 0.003 M phosphate buffer, pH 7; 10 micromoles of fumarate or pyruvate added ae indicated. Streptomycin when present at 20 y per ml. (broken Knee).

These results point definitely to the second hypothesis; that is, that pyruvate can be oxidized by way of condensation with oxalacetate, and that in dried cell preparations, in which the oxidation to acetate is weak, the condensation reaction is the principal pathway. Streptomycin prevents the condensation reaction. This mode of action of streptomycin appears to be relatively clear. However, the mechanism by which it prevents this condensation is still obscure. The data of Fig. 3 demonstrate that

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W. W. UMBREIT 713

streptomycin prevents the formation and not the activity of the sub- stance which is formed from fumarate oxidation. The data of Fig. 6 indicate this substance to be oxalacetate. In addition the data of Fig. 1 show that at higher phosphate levels the oxidation of fumarate proceeds at the same rate with or without streptomycin; yet the ability of the cell to oxidize threonine is impaired when streptomycin is present. Until these reaction systems are untangled experimentally, it is not safe to ascribe the inhibition exerted by streptomycin to any stage in this evidently com- plex process. Thus from these experiments it is permissible to conclude only that the net result of streptomycin inhibition is to prevent pyruvate (and evidently other keto acids) from entering the terminal respiration system by way of oxalacetate condensation. It, is further evident that, since the observable reaction inhibited is also an important one in animal metabolism, it is as yet difficult to explain why the streptomycin is able to kill the bacteria without comparable harm to the host. At least two possibilities, that streptomycin does not penetrate to the active centers of this reaction in the host, or that the condensation in the bacterium differs in some subtle manner from that occurring in the host, are matters for further study.

SUMMARY

1. In the Gratia strain of Escherichia coli the oxidation of threonine is stimulated by the previous oxidation of fumarate.

2. This stimulating effect is prevented by streptomycin. 3. Streptomycin can be shown to inhibit the oxidation of fumarate and

pyruvate under suitable conditions. 4. The mode of action of streptomycin is to inhibit the terminal respira-

tion process. 5. The terminal respiration process apparently involves a pyruvate-

oxalacetate condensation and it is close to this reaction that streptomycin exerts its activity.

Thanks are due Mr. James G. Waddell for technical assistance.

BIBLIOGRAPHY

1. Bernheim, F., and Fitzgerald, R. J., Science, 106,417 (1947). 2. Fitzgerald, R. J., and Bernheim, F., J. Bact., 64,071 (1947). 3. Geiger, W. B., Arch. Biochem., 16,227 (1947). 4. Henry, J., Henry, R. J., Berkman, S., and Housewright, R. D., Conference on

antibiotic research, National Institute of Health, Washington, Feb., 1947. 5. Wight, K., and Burk, D., Federation Proc., 7, 199 (1948). 6. Friedemann, T. E., and Haugen, G. E., J. Biol. Chem., 147,415 (1943). 7. Carter, H. E., Handler, B., and Melville, D. B., J. Biol. Chem., 120, 359 (1939).

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714 SITE OF STREPTOMYCIN ACTION

8. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric technic related methods for the study of tissue metabolism, Minneapolis (1941

9. Lipmann, F., and Tuttle, L. C., J. Biol. &em., 169, 21 (1946). 10. Chargaff, E., and Sprinson, D. B., J. Biol. Chem., 161, 273 (1943). 11. Long, C., and Peters, R. A., Biochem. J., 37, 249 (1939). 12. Krebs, H. A., in N&d, F. F., and Werkman, C. H., Advances in enzyme

related subjects, New York, 3,191 (1943).

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W. W. UmbreitSTREPTOMYCIN

A SITE OF ACTION OF

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