Effects of Nigericin and Monactin on Cation Per- Meability of Streptococcus Faecalis and Metabolic Capacities of Potassium-Depleted Cells

Effects of Nigericin and Monactin on Cation Per- meability of Streptococcus faecalis and Metabolic Capacities of Potassium-depleted Cells

F. M. HAROLD AND J. R. BAARDA Division ofResearch, National Jewish Hospital, Denver, Colorado 80206, and Department of Microbiology, University of Colorado, School of Medicine, Denver, Colorado 80220 Received for publication 15 December 1967

At a concentration of 10-6 M, nigericin and monactin inhibited growth of Strepto- coccusfaecalis, and the inhibition was reversed by addition of excess K+. In the presence of certain antibiotics, the cells exhibited increased permeability to certain cations; internal Rb+ was rapidly lost by exchange with external H+, K+ Rb+, and, more slowly, with Na+ and Li+. No effect was observed on the penetration of other small molecules. Cation exchanges induced by nigericin and monactin were metabolically passive and apparently did not involve the energy-dependent K+ pump. When the cells were washed, the cytoplasmic membrane recovered its original impermeability to cations. By use of monactin, we prepared cells whose K+ content had been completely replaced by other cations, and the metabolic characteristics of K+-depleted cells were studied. Cells containing only Na+ glycolyzed almost as well as did normal ones and, under proper conditions, could accumulate amino acids and orthophosphate. These cells also incorporated '4C-uracil into ribonucleic acid but incorporation of '4C-leucine into protein was strictly dependent upon the addition of K+. When K+ or Rb+ was added to sodium-loaded cells undergoing glycolysis, these ions were accumulated by stoichiometric exchange for Na+. From concurrent measurements of the rate of glycolysis, it was calculated that one mole-pair of cations was exchanged for each mole of adenosine triphosphate produced.

Nigericin is an antibiotic of unknown struc- of nigericin and monactin on Streptococcus ture first described by Harned et al. (15). It faecalis, an organism which relies entirely on inhibits growth of a number of gram-positive glycolysis for the generation of metabolic energy. bacteria and is specifically antagonized by the We show that the mode of action of nigericin and addition of K+ to the medium. Nigericin produces monactin is similar to that previously described complex effects on mammalian mitochondria, for valinomycin and gramicidin (16). Nigericin including inhibition of respiration, release of and monactin markedly increase the permeabil- cations, and unmasking of adenosine triphospha- ity of the cytoplasmic membrane to cations, tase activity (9, 12, 28; S. Estrada-O, S. N. and K+ is lost from the cells, resulting in in- Graven, and H. L. Lardy, Federation Proc., p. hibition of growth which can be reversed by 610, 1967). According to Graven et al. (12), addition of excess K+. However, unlike gramici- the primary site of action of nigericin may be the din or valinomycin, nigericin and monactin are cation-translocating mechanism. readily removed by washing the cells, and washed Monactin and its homologues are also thought cells recover their original impermeability to to act at the level of cation transport in mito- cations. This property of the washed cells af- chondria. The "nactins" are macrocyclic anti- forded an opportunity to prepare cells whose K+ biotics of defined structure (5) which also in- was totally replaced by other cations, and some hibit various gram-positive bacteria (23). The studies on the metabolic characteristics of such "nactins" induce K+ uptake and H+ ejection in K+-depleted cells are described. mitochondria and uncouple oxidative phospho- rylation (13, 14). Their physiological effects are MATERIALS AND METHODS similar to those of the polypeptide antibiotics Since most of our procedures were described in valinomycin and gramicidin (6, 20, 26). earlier papers (16-19), a brief summary will suffice The present report is concerned with the effects here. 816 VOL. 95, 1968 NIGERICIN, MONACTIN, AND CATION PERMEABILITY 817 Growth media. S. faecalis strain 9790 was usually of monactin, their presence was ignored. The antibio- grown on defined media, and both K+ and Rb+ sup- tics were dissolved in ethyl alcohol, and control sus- ported growth (19). Because of the technical con- pensions always received a volume of ethyl alcohol venience of using 8tRb as an isotope of Rb+, cells equal to that of the experimental suspensions (usually grown on Rb+ were used whenever possible. Glucose 1% by volume). served as an energy source, and growth was followed by turbidimetry at 600 m,. In all the experiments RESULTS described here, the cells were grown in medium NaM Inhibition of growth by monactin and nigericin. (19). This medium was buffered with sodium maleate Addition of the antibiotics at concentrations of (200 to 350 mm Na+), and it contained variable 1 to 2 x 10-6 M (1 to 2 jAg/ml) to cells growing amounts of K+ or Rb+. in medium NaM brought about a cessation of Cation content. Loading with 86Rb was accom- plished by growing or incubating the cells for growth. The inhibition could be reversed by several hours in medium containing 10 mM 86Rb of addition of excess K+ (Fig. 1). Inhibition of known specific activity. Under these conditions, the growth was associated with rapid loss of K+ or cells exchanged internal Rb+ for 86Rb (19); at equilib- Rb+ from the cells, suggesting that nigericin rium the radioactivity of the cells was a measure of and monactin interfere with the selective accumu- their Rb+ content. Samples were harvested by filtra- lation of these cations. tion on filters (Millipore Corp., Bedford, Mass.) and Induction of cation exchanges by nigericin and were washed with water or 2 mi MgCla. The radio- monactin. Cells of S. faecalis loaded with K+ or activity of the samples was determined, and, when retain these cations tenaciously when sus- desired, K+, Na+, and Rb+ were extracted and esti- 8tRb mated by flame photometry. pended in solutions of various salts in the ab- Uptake ofradioactive substrates. Cells were prepared sence of a source of energy. However, low con- as described for the individual experiments, and radio- centrations of nigericin or monactin induced active substrates ofknown specific activity were added. rapid loss of t6Rb from the cells by exchange for At intervals, samples were withdrawn, washed with 2 external cations. As shown in Table 1, the ex- mM MgC92, and counted. To measure incorporation of changes were quite selective, and K+, Rb+, and 14C into macromolecules, the cells were extracted first Cs+ exchanged more rapidly than did the other with cold 5% perchloric acid and then with 10% per- cations. The induction of cation exchanges was chloric acid at 95 C. Incorporation of 14C-uracil into dependent upon the temperature. the fraction insoluble in cold acid was taken as a also strongly measure of ribonucleic acid (RNA) synthesis, and No cation exchanges were induced at 0 C under incorporation of 14C-leucine into the final residue was the conditions listed in Table 1. taken as a measure of protein synthesis. Nigericin and monactin also induced a loss of Adenosine triphosphate (ATP) content. Samples K+ and 86Rb by exchange for H+. When anti- containing 5 mg of cells were collected by filtration, biotics were added to a suspension of cells in washed, and extracted with sulfuric acid (10). ATP water, the pH rose, and considerable amounts was assayed by the firefly luminescence method, as modified by Forrest and Walker (10). Preparation of sodium-loaded cells. S. faecalis was I Control grown on medium NaM containing 10 mm 8ORb. The 2 NIG or MAC cells were harvested during exponential growth and 1.08 3 NIG or MAC, 0.2M KCI were washed and resuspended in 0.1 M sodium maleate, .. 0.8 pH 8. Monactin (2 ,gg/ml) was added, and the suspen- 0.6 A. I B. sion was incubated at 37 C for 20 min. The cells were 0)0.4- then collected by filtration, washed twice with sodium 3 3 maleate and three times with water, and resuspended O .2 as desired. Displacement of Rb+ by Na+ was moni- 0- 2 2 tored by loss of radioactivity, which routinely attained 0 0.1 99% or better. Other procedures. Glycolysis was monitored at 0.05 -NIG KC MAC KCI constant pH by automatic titration of the lactic acid 1I I I produced with a radiometer pH-stat. In a few experi- 0 1 2 3 4 5 6 0 1 2 3 4 5 ments, uptake of H+ by the cells was likewise followed Hours Hours by automatic addition of standardized HCl at constant pH. Metabolism of arginine was followed by auto- FIG. 1. Effect ofnigericin and monactin on growth of matic titration of NH3 produced by cells previously Streptococcus faecalis. (A) An exponentially growing grown on arginine (31). culture in medium NaM was divided (arrow) and the Antibiotics. Nigericin was donated by R. L. Harned, following additions were made: (i) control, ethyl alcohol and monactin was a gift from W. Keller-Schierlein. only; (ii) nigericin (NIG), I ug/ml; (iii) nigericin, The latter sample contained minor amounts of non- followed by 200 mM KCI at 2.5 hr. (B) An analogous actin and traces of the higher homologues. Since the experiment with monactin (MAC) at a concentration known effects of these antibiotics are similar to those of 2 g/nml. 818 HAROLD AND BAARDA J. BACTERIOL.

TABLE 1. Release of 86Rb from Streptococcus the induction of cation permeability is the sole faecalis treated with nigericin or monactina effect of the antibiotics at the concentrations employed. Percentage of 16Rb retained Restoration of cation impermeability by wash- Medium ing. Thus far, the effects of nigericin and monac- Nigericin Monactin ho only tin closely resembled those described in detail (16) for gramicidin and valinomycin. However, Water ...... 100 96 100 unlike the latter antibiotics, nigericin and monac- LiCl, 10mM ...... 93 69 94 tin could be readily removed from the cells by NaCl, 10 mm...... 92 42 99 washing with buffers or with water. Washed KCl, 10 mM ...... 29 8 98 cells recovered their original impermeability to RbCl, 10 mm...... 29 8 96 cations (Fig. 2). If the washed cells were placed CsCl,l10 mM ...... 82 32 93 in medium of low K+ content, growth resumed MgCl2, 10 MM ..... 76 70 98 NH4Cl, 10 mM. 21 22 99 after a brief lag. At this point, it became evident that monactin and nigericin could be used to a S. faecalis cells were loaded with 86Rb, as replace K+ completely with other cations, and described in the text. The cells were washed and our interest shifted to the physiology of such K+- resuspended in the various solutions at 0.6 mg/ml depleted cells. (pH 8). Nigericin (1 ,g/ml final concentration), Metabolic capacities of K+-depleted cells: monactin (2 fg/ml final concentration), or ethyl glycolysis. By use of the procedure described in alcohol alone (1% final concentration) were added Materials and Methods, over 99% of the cellular to these suspensions. After 5 min at room temperature, 1-mi samples of the mixture were filtered 86Rb was replaced with K+, Na+, Li+, Cs+, or and washed three times with alkaline water. The NH4+. The cells were washed, resuspended in original cells washed with water contained 4,350 10 mm solutions of the same salt (chloride), counts per min per mg of dry cells. This is taken as and glycolysis was followed on the pH-stat. It 100%, and retention of 86Rb is expressed as the can be seen from Table 2 that, at pH 8, Na+ and percentage of this substance remaining in the NH4+ supported glycolysis almost as well as washed cells after exposure to certain antibiotics. did K+, whereas Li+ and especially Cs+ were less effective. In most cases, glycolysis was stimu- of acid were required to maintain the pH at 6. lated by the addition of K+. The rate of gly- Under these conditions, H+ entered the cells by colysis at apH below 7 was rather variable but was stoichiometric exchange for cellular K+ or 86Rb. usually considerably lower than the rate of Because of the entry of H+ into the cells, the glycolysis at an alkaline pH. This may be due to antibiotics strongly inhibited glycolysis at pH residual traces of monactin, which render the 6 but had little effect at pH 8. Nigericin, a mono- cells somewhat more permeable to H+ than are carboxylic acid (15), was considerably more normal cells. potent at pH 6 than at pH 8. Thus, nigericin and monactin increased the passive cation-permeability of the cytoplasmic membrane but apparently did not affect the E8 0 0 C0o Xla Nigericin Monoctin energy-dependent transport system which medi- u 6000 -*not washed X washed - ates cation exchanges in S. faecalis (17, 19). This E 00-o conclusion is based on the finding that the antibiotics neither inhibited nor stimulated energy- 2000- dependent uptake of K+ from water. These experiments, which were entirely analogous to ° 0 10 20 40 0 10 20 40 0 10 20 40 those described in detail earlier (16), were carried Minutes out at pH 8 on the pH-stat so as to minimize FIG. 2. Reversal of the effects of nigericin and passive loss of K+ from the cells by exchange for monactin by washing. Streptococcus faecalis was grown either H+ or Na+. As far as we know, the anti- on medium NaM supplemented with 86Rb and was har- biotics at the concentrations employed here did vested and resuspended at 0.6 mg/ml in 10 mm NaCl. not affect membrane permeability to other cellu- The suspension was divided into three portions which lar constituents. The antibiotics did not induce received ethyl alcohol only, nigericin (I lAg/ml), or monactin (I ,ug/ml), respectively. After S min, a leakage of amino acids or phosphate from the sample ofeach portion was taken; the cells werefiltered, cells, nor did they cause lysis of protoplasts washed with 10 mm NaCl, and returned to 10 mM NaCI stabilized osmotically by high concentrations of at the original cell density. Incubation was conducted at disaccharides or amino acids. The ready reversal room temperature. Samples (I ml) of all suspensions of growth inhibition by K+ also indicated that werefiltered at intervals, washed, and counted. VOL. 95, 1968 NIGERICIN, MONACTIN, AND CATION PERMEABILITY 819 TABLE 2. Rates of glycolysis by Streptococcus 32P-Pj and '4C-glutamate, required a source of faecalis loaded with various cationsa metabolic energy and external cations. In both cases, 10 mM K+ supported more rapid and ex- of Cells CatIon Glycolysis,pjmolesg of cells(x min) H+/ tensive uptake than did 10 mm Na+, but, at pH 8 or 9, 100 mm Na+ could completely substitute for K+ (Fig. 3). Moreover, it was observed that Normal ...... K+ 90-140 Normal...... Rb+ 70-120 even cells preloaded with K+ required either ex- Monactin-treated ... K+ 100- ternal K+ or high concentrations of Na+ for Monactin-treated ... Na+ 70- maximal uptake of 32P-Pi. The role of K+ in Monactin-treated. . Li+ 40- membrane transport appears to be indirect and Monactin-treated. . Cs+ 4- connected with the maintenance of an electrolyte Monactin-treated. . NH4+ 75- balance. Macromolecular synthesis. Sodium - loaded a Normal cells were harvested during the exponential phase of growth on medium NaM sup- cells were washed and were then incubated on the plemented with Rb+ or K+. These cells contained pH-stat in complete growth medium containing Rb+ or K+ as well as small amounts of Nat Na+ as the sole cation. In agreement with the (17, 20). Monactin-treated cells were loaded with findings of Lubin and Ennis (22) with Escherichia cations, as described in Materials and Methods. coli, sodium-loaded cells readily incorporated The cells were washed and resuspended in 10 mm "4C-uracil into nucleic acids. Incorporation of solutions of the cation present intracellularly "4C-leucine into the protein fraction was, by con- (chloride salt). Glycolysis was followed on the trast, strictly dependent upon addition of K+ pH-stat at room temperature (pH 8). Cell density (Table 3). In the absence of K+, "4C-leucine was 0.6 mg/ml (dry weight). accumulated in the acid-soluble pool, and we were unable to find conditions under which Na+ Membrane transport. We reported previously alone would permit protein synthesis. (16) that gramicidin and valinomycin inhibit Stoichiometry of cation exchanges by Na+- uptake of 32P-Pi (radioactive orthophosphate) loaded cells. When K+ or 86Rb cations were added and "4C-glutamate by cells suspended in sodium to Na+-loaded cells undergoing glycolysis, these maleate buffer, and we attributed this to re- cations were accumulated, whereas stoichio- placement of cellular K+ by Na+. To determine metric amounts of Na+ were extruded from the whether K+ is required for membrane transport, a series of experiments was carried out with cells fully loaded with Na+. The cells were suspended U) in water and the pH was maintained by use of the oNaCI 10 mM pH-stat. This procedure was adopted after we 80 ,*Na CI 100 mM A found that, for reasons which are not under- &.LKCI 10mM /4 stood, tris(hydroxymethyl)aminomethane (Tris) 0) buffer accentuates the K+ requirement of Na+- loaded cells. The results may be summarized as follows: (i) Production of NH3 from the basic amino acid arginine (at pH 6, no glucose) was inde- 41 pendent of K+. (ii) Uptake of the neutral amino acids, "4C- 0~ alanine and 14C-glycine, at pH 8 was stimulated by glucose but did not require K+. In one experi- in 5 ment, sodium-loaded cells were suspended N mM NaCl or 5 mM KC1 with 2.5 x 105 M "4C- CV) 5 10 20 30 alanine. In both suspensions, uptake occurred to the extent of about 3 Amoles of "'C-alanine per g of cells, and the addition of glucose stimulated Minutes rate and extent of uptake fourfold. Somewhat FIG. 3. Uptake of 32P-Pi by sodium-loaded Strepto- the coccus faecalis. Sodium-loaded cells were prepared different results were obtained at pH 7. At this (see Materials and Methods) and suspended in water at pH, glucose was more effective in the presence of 0.6 mg/ml. Suspensions were supplemented with KCI or 5 mM K+ than in the presence of 5 mm Na+, NaCl, as shown, and were allowed to glycolyze at room and this may be due to the fact that glycolysis temperature on the pH-stat at pH 9. After 5 min, nP-P1 was also more rapid in the presence of K+. was added to 0.2 mm, and samples were taken at inter- (iii) Accumulation of the acidic substrates, vals thereafter. 8i20 HAROLD AND BAARDA J. BACTERIO

TABLE 3. Incorporationi of '4C-labeled precursors into macromolecular fractions by sodium-loaded 86 Rb uptake l' No uptake Streptococcus faecalisa .0 -Glycolysi3 o -Clycolyais ro 3 * 86Rb, with glucose Z3 _ 22 Na, with glucose 8 6 22 Count/min per mg of cells Rb, no glucose o- 2o2 No, no glucose in macromolecules 0 2 Cal;)n supplement + _A. +I _B. I 14C-uracil into 14C-leucine into O A RNA protein .1 O0 o U 00 U O 0 f 50 E O _ K+, 10 mm.3,200 2,900 I mM, 0 Na+, 10 mM.6,300 70 IL I I86Rb. _1 / +22Na. 0mM Na+, 100 mM .2,550 42 0 1 2 34 5 0 1 2 3 4 5 Minutes Minutes a 5. faecalis was loaded with sodium by use of monactin, as described in Materials and Methods. FIG. 4. Stoichiometric relationship between glycoly- The cells were suspended at 0.6 mg/ml in a medium sis and the uptake of 86Rb or 22Na by sodium-loaded containing 5 mm sodium phosphate, 2 mM MgC12, Streptococcus faecalis. (A) Sodium-loaded cells were vitamins, and 0.2 mg/ml of the amino acid mixture prepared (see Materials and Methods), suspended in required for growth (29). The suspensions were water (0.6 mg/ml), and allowed to glycolyze at room incubated at 37 C, and a pH of 8 was maintained temperature on the pH-stat at pH 7.5. At 0 min, 86RbCI by use of the pH-stat. 14C-leucine or uracil (2 ,uc) ofknown specific activity was added (final concentration, was added, and, after 20 min, the cells were har- I mM), and samples were taken at 1-min intervals. vested and fractionated. Glycolysis, from the trace ofthe pH-stat, is expressed as ,umoles of H+ produced per 20 ml of suspension; Rb+ uptake is expressed as ,umoles of 88Rb per 20 ml of cells. The exchange of cations was accompanied suspension in the cells. A control was set up in the same by stimulation of glycolysis, but, as cation ex- way with the exception that it received no glucose. (B) change approached completion, the rate of A parallel experiment except that 10 mM 22NaCl was glycolysis declined and returned to almost its added at 0 min. original level. Concurrent measurements of the initial rates of glycolysis and 8fRb uptake are shown in Fig. 4a. The two curves almost coin- 25 o NoCl, I mM one mole- o KCI, ImM cide, suggesting that approximately ciP 2 0 /O pair of cations was exchanged for each mole of lactic acid produced. Similarly, when arginine 1 5 served as the energy source, the ratio of moles of 0 arginine metabolized to moles of cation ex- E 10 0 0i changed approached unity. Sodium-loaded cells also took up 22Na by an 5 exchange for internal Na+, and again the process 1 I required metabolic energy. The stoichiometry 0 5 10 20 30 was a function of the concentration of external 22Na: at 1 mm, less than 0.2 mole of 22Na was Minutes taken up per mole of lactic acid produced, but, FIG. 5. Synthesis and degradation of adeniosine tri- with 10 mM 22Na, the ratio rose to 0.5 (Fig. 4b). phosphate (ATP) by sodium-loaded Streptococcus These findings are consistent with our earlier faecalis. Sodium-loaded cells were resuspended in water at I mg/ml. To 40 ml of this suspension, NaCl or KCl observations that the apparent Km for 86Rb (to I mm final concentration) and 15 ,umoles ofglucose uptake under these conditions is about 0.15 were added. Glycolysis was allowed to proceed at room mm, whereas the Km for Na+ uptake is 15 mM temperature (pH 8) and was monitored by use of the (17). pH-stat (the arrows indicate the points at which glucose In view of these findings, it seemed of impor- was exhausted). Samples were taken at intervals and the tance to determine whether K+ had any obvious A TP content ofthe cells was determined. effect upon ATP turnover. To this end, Na+- loaded cells were allowed to metabolize a limited the stimulation of glycolysis by K+, but it was amount of glucose, and ATP levels were deter- evident that K+ is not essential either for the syn- mined at intervals. As shown in Fig. 5, cells thesis or the degradation of ATP. devoid of K+ rapidly accumulated ATP; when the glucose was exhausted, the ATP was de- DISCUSSION graded. In the presence of K+, the cycle was The mode of action of nigericin and monactin somewhat shorter, as would be expected from on S. faecalis is very similar to the mode of action VoL.95,1968 NIGERICIN, MONACTIN, AND CATION PERMEABILITY 821 described earlier for gramicidin and valinomycin (Table 2). Similar findings were previously re- (16). Nigericin and monactin interact with the ported by Abrams (1) for protoplasts of S. cytoplasmic membrane and markedly increase faecalis 9790 and by Conway and his associates its passive permeability to univalent cations. for yeast (7, 8). The uptake of arginine, glycine, Consequently, in the presence of these anti- alanine, leucine, and uracil was not stimulated biotics, internal 86Rb readily exchanges with by K+. [Mora and Snell (24) reported that K+ cations from the medium. Under the conditions stimulates the uptake of glycine-and alanine by used, the order of exchange preference in the protoplasts of S. faecalis. Their experiments were presence of monactin is K+, Rb+ > Cs+ > Na+ conducted at pH 7.2, some of them in Tris buffer. > Li+. In the presence of nigericin, exchange Under these conditions, we also found that K+ with K+ and Rb+ is also much more rapid than stimulates the uptake of alanine and glycine by is exchange with Cs+, Na+, and Li+ (Table 1). Na+-loaded cells. Swelling and lysis of sodium- The selectivities of the cation exchanges are thus loaded protoplasts suspended in glycylglycine intermediate between those induced by valino- buffer did not require K+, but, even at a high pH, mycin and by gramicidin (16). It may be noted swelling of such protoplasts in sucrose was that monactin and nigericin facilitate entry of strongly stimulated by K+ (1, 2). The role of K+ K+ and Rb+ more than entry of Cs+, even in this phenomenon remains obscure.] Potassium though Cs+ has a smaller hydrated ionic radius. did stimulate the uptake of phosphate and gluta- Mueller and Rudin (25) have considered steric mate, but even here high concentrations of factors which may account for this. sodium could replace K+. Thus, we conclude that The molecular mechanisms by which certain K+ does not play an obligatory role in membrane macrocyclic antibiotics increase the cation transport. Instead, the function of K+ appears permeability of membranes were explored by to be the maintenance of electrolyte balance Mueller and Rudin (25) by use of artificial lipid (27, 30). The characteristics of the cation trans- bilayers. Gramicidin and antibiotics of the port system of S. faecalis are such that K+ is valinomycin, "nactin," and enniatin groups in- transported into the cells in preference to Na+, creased membrane conductance by many orders whereas Na+ and H+ are preferentially extruded of magnitude. On the basis of molecular models, (17, 19). Thus, K+ uptake is favored, but Na+ these investigators proposed that the antibiotics can be accumulated by the cells at a high pH adsorb to the membrane to form artificial pores (17) and it will then replace K+ as a counterion or carriers which mediate cation exchanges. for phosphate and glutamate. The findings reported here and in our previous According to the findings of Lubin and Ennis paper (16) are in full agreement with the model (21, 22), the synthesis of protein uniquely re- proposed by Mueller and Rudin. We would quires K+ rather than Na+. In agreement with stress particularly that monactin, nigericin, their observations, we found that sodium- valinomycin, and gramicidin increase membrane loaded S. faecalis incorporated 14C-uracil into permeability to cations but not to anions. Fur- RNA, but, under all conditions tried, incorpora- ther, the cation exchanges are metabolically tion of 14C-leucine into protein was strictly de- passive, and no evidence could be obtained to pendent upon the addition of K+ (Table 3). indicate any effect on the cation pump of S. When K+ or Rb+ cations were added to so- faecalis. The physiological effects of the anti- dium-loaded cells, the normal ionic composition biotics thus arise from the loss of K+ by passive was rapidly restored by an energy-dependent exchange for Na+ and H+, and these effects are stoichiometric exchange of Rb+ for Na+. To reversed by addition of excess K+ (Fig. 1). estimate the energy cost of cation exchange, we The discovery that monactin and nigericin assumed that ATP is the immediate energy donor are readily removed by washing the cells (Fig. 2) and that glycolysis yields 2 moles of ATP per led us to explore the metabolic capacities of S. mole of glucose, whereas arginine degradation faecalis cells whose K+ was completely (99%) yields only 1 mole of ATP. From concurrent replaced by other cations, especially Na+. Mem- measurements of the rates of 86Rb uptake and brane transport was of particular interest since metabolism of the energy source, it therefore ap- it has long been known that K+ stimulates the peared that up to 1 mole of cations was exchanged uptake of amino acids (11, 24), sugars (1, 2), per mole of ATP produced (Fig. 4). These results and phosphate (18, 27) in streptococci and and extend the conclusion drawn other organisms. Our experiments were conducted confirm by at an alkaline pH on the pH-stat to avoid com- Zarlengo and Schultz (31) in relation to S. plications due to the buffer. Under these con- faecalis cells partly loaded with Na+ and H+. ditions, cells fully loaded with Na+ or NH4+ The finding that not more than 1 mole of cat- glycolyzed nearly as well as did K+-loaded cells ions is exchanged per mole of ATP available to 822 HAROLD AND BAARDA J. BACTERIOL.

the cells is open to several interpretations. It is 6. CHAPPELL, J. B., AND A. R. CRoFrS. 1965. Grami- conceivable that several moles of cations are ex- cidinand ion transport in isolated mitochondria. changed per mole of ATP, but that only a frac- Biochem. J. 95:393-402. tion of the ATP produced by the cells is chan- 7. CONWAY, E. J., AND H. M. GAFFNEY. 1966. The further preparation of inorganic cationic yeasts neled toward cation transport. According to and some of their chief properties. Biochem. J. this view, it is fortuitous that the overall ratio 101:385-391. approaches unity. However, it appears more 8. CONWAY, E. J., AND P. MOOUE. 1954. A sodium- instructive to postulate that 1 mole of ATP is yeast and some of its properties. Biochem. J. required to exchange 1 mole of cations, and that 57:523-528. Na+-loaded cells may expend virtually all the 9. ESTRADA-O, S., S. N. GRAVEN, AND H. L. LARDY. ATP they produce on this process. (The cells 1967. Potassium ion-dependent hydrolysis of studied here were not growing and presumably adenosine triphosphate induced by nigericin in had only minimal energy demands for processes mitochondria. J. Biol. Chem. 242:2925-2931. 10. FORREST, W. W., AND D. J. WALKER. 1965. Syn- other than cation transport.) At the same time, thesis of reserve materials for endogenous let us note that ATP turnover can proceed in the metabolism in Streptococcus faecalis. J. Bac- absence of cation transport (Fig. 5). We can thus teriol. 89:1448-1452. propose that, in the absence of external cations, 11. GALE, E. F. 1953. Assimilation of amino acids by ATP degradation is not coupled to transport. bacteria and the action of some antibiotics When external Rb+ or Na+ cations are added, a thereon. Advan. Protein Chem. 8:285-391. fraction of the ATP turnover is coupled to cation 12. GRAVEN, S. N., S. ESTRADA-O, AND H. L. LARDY. exchange, and this fraction approaches unity if 1966. Alkali metal cation release and respiration the concentration of external cations is suffi- inhibition induced by nigericin in rat liver mitochondria. Proc. Natl. Acad. Sci. U.S. 56: cient to saturate the transport system. The 654-658. degradation of ATP is probably catalyzed by the 13. GRAVEN, S. N., H. L. LARDY, AND S. ESTRADA-O. adenosine triphosphatase which is localized in 1967. Antibiotics as tools for metabolic studies. the cell membrane (3, 4). However, it remains VIII. Effect of nonactin homologs on alkali to be determined whether this adenosine tri- metal cation transport and rate of respiration in phosphatase, like the membrane-bound adeno- mitochondria. Biochemistry 6:365-371. sine triphosphatase found in mammalian cells, 14. GRAVEN, S. N., H. L. LARDY, D. JOHNSON, AND is involved in cation transport. A. RUTrER. 1966. Antibiotics as tools for metabolic studies. V. Effect of nonactin, mon- ACKNOWLEDGMENTS actin, dinactin and trinactin on oxidative phos- phorylation and adenosine triphosphatase in- It is a pleasure to thank R. L. Harned (Commercial duction. Biochemistry 5:1729-1735. Solvents Co.) and W. Keller-Schierlein (Eidgen6s- 15. HARNED, R. L., P. H. HuDY, C. Y. CORUM, AND sische Technische Hochschule, Zurich) for their gifts K. L. JoNS. 1951. Nigericin, a new crystalline of nigericin and monactin, respectively. This investiga- antibiotic from an unidentified Streptomyces. tion was supported by Public Health Service grant Antibiot. Chemotherapy 1:594-596. AI-03568 from the National Institute of Allergy and 16. HAROLD, F. M., AND J. R. BAARDA. 1967. Gram- Infectious Diseases. icidin, valinomycin, and cation permeability of Streptococcus faecalis. J. Bacteriol. 94:53-60. LITERATURE CITED 17. HAROLD, F. M., AND J. R. BAARDA. 1967. Inhibi- 1. ABRAMS, A. 1959. 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