Acids in Micrococcus lysodeikticus

Transcription

1 JOURNAL OF BACTROLOGY, Aug. 1972, p Copyright American Society for Microbiology Vol. 111, No. 2 Printed in U.S.A. ffect of Spermine on the Uptake of Amino Acids in Micrococcus lysodeikticus MIRIAM ARIL' AND NATHAN GROSSOWICZ Department of Bacteriology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 27 April 1972 Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the Vm.x decreased, whereas the apparent Km remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10-1 M to 2.5 x 10-2 M) and dinitrophenol (10-3 M). The intracellular concentration of valine was 100- fold higher than in the medium. Natural polyamines such as spermine [NH2(CH2) 3NH(CH2) NH(CH2)3NH2] and spermidine [NH2(CH2) SNH(CH 2),NH2] are found in animal, plant, and microbial cells. Their biological importance was first recognized by Herbst et al. (4-6) who demonstrated that the growth of Haemophilus parainfluenzae is dependent on the presence of polyamines in the growth medium. The polyamines were also found to be growth factors for Neisseria perflava (11, 15), Pasteurella tularensis (D.. Fleming and L. Foshay, Bacterial. Proc. 21: 106, 1957, and references 11, 14, 21) and Lactobacillus casei (9, 10). The growth-promoting effect apparently stems from the capacity of the polyamines to prevent lysis by protecting the cytoplasmic membrane of sensitive bacteria in media of low tonicity (11-13, 18). Tabor (19, 20) has shown that protoplasts and spheroplasts of Micrococcus lysodeikticus and scherichia coli, respectively, can be protected against lysis by polyamines. We have demonstrated (2) that the protection of M. lysodeikticus protoplasts by spermine was a result of efficient stabilization of the protoplasts against hypotonic conditions. Harold (3) found that This work was submitted to the Senate of the Hebrew University of Jerusalem in partial fulfillment of the requirements for the Ph.D. degree. spermine and other bivalent cations prevented osmotic shock of Streptococcus faecalis protoplasts; approximately 80% of the added spermine, was found to reside in the membrane fraction of the protoplasts. The above findings obviously link the spermine effect with the bacterial membrane. It was therefore of interest to investigate the effect of spermine on the transport of amino acids into the cell, one of the most important functions of the membrane. We also compared the effect of spermine with that of the related polyamines, spermidine and cadaverine. MATRIALS AND MTHODS M. lysodeikticus cells were grown in 250-ml rlenmeyer flasks containing 100 ml of Brain Heart Infusion broth (Baltimore Biological Laboratory). The cells were grown overnight (for 16 to 18 hr) with shaking in a water bath at 37 C. They were then transferred into fresh medium in which they were diluted to a turbidity of 30 to 40 Klett units (measured at 540 nm) and incubated until a turbidity of about 150 Klett units was attained. The exponentially growing cells were collected by centrifugation at 7,500 x g for 5 min, washed twice by centrifugation with M tris(hydroxymethyl)aminomethane (Tris) buffer (ph 7.5), and then suspended in the same buffer. The uptake of amino acids was measured by the 412

2 VOL. 11l, 1972 FFCT OF SPRMIN ON AMINO ACID UPTAK 413 method of Kessel and Lubin (8). The washed cells were preincubated with chloramphenicol (200 jig/ml) in Tris buffer for 15 min at 37 C with shaking; 0.5 ml of the cell suspension (corresponding to 0.88 mg dry weight) was then added to the uptake system which contained Tris buffer (ph 7.5) at a final concentration of M, 0.5% glucose, 200 jg of chloramphenicol per ml, and 0.2 ml of 'IC-amino acid (specific activity 1 jici/jimole, obtained from The Radiochemical Centre, Amersham, Bucks.), at a final concentration of 1 x 10-I M or as otherwise indicated. The final volume of the uptake system was 4 ml. Incubation was carried out with shaking in a water bath at 37 C. At desired times, the uptake was stopped by filtration of the reaction mixture through a membrane filter (pore size 0.45,jm, Millipore Corp., Bedford, Mass.) and washed with 20 ml of buffer. The cells retained on the filters were placed in scintillation vials and dried. A 10-ml amount of toluene scintillation fluid [containing 100 mg of 1,4-bis-2-(5-phenyloxazolyl)-benzene and 3 g of 2,5-diphenyloxazole per liter of toluene] was added to each vial, and the radioactivity was determined in a Tri-Carb liquid scintillation spectrometer model 3310 (Packard Instruments Co., Downers Grove, Ill.). Identification of the incorporated amino acid. Uptake mixtures were prepared in which the amino acid concentration was equivalent to 2 Km values (valine, 3 x 10-5 M; leucine, 7.6 x 10-i M; isoleucine, 5.6 x 10-5 M); the final volume was 10 ml. The other constituents in the uptake mixture were as described above. Duration of uptake was 2 min. The experiment was terminated by centrifuging the cells at 9,000 x g for 3 min at 4 C and then washing them by centrifugation with 5 ml of Tris buffer. The pellet was suspended in 1 ml of distilled water, and the amino acids were extracted by heating in a boiling water bath for 20 min. Cell debris was removed by centrifugation at 9,000 x g for 10 min. The supernatant fraction was dried under vacuum and examined in a Beckman amino acid analyzer model 120B. RSULTS ffect of spermine on the uptake of I C- valine. Two series of the uptake systems were prepared, one without and the other with spermine. From the series without spermine, samples were taken at time zero and after incubation for 2, 5, 10, and 20 min. As seen from Fig. 1, in the absence of spermine there was a considerable uptake of '4C-valine by M. lysodeikticus cells during the first 10 min of incubation. The uptake increased from 7 nmoles/0.88 mg (dry weight) after 2 min to almost 20 nmoles/0.88 mg (dry weight) after 10 min. Spermine (6,imoles/ml final concentration) was added to the uptake system of the other series at the corresponding time intervals. After the addition of spermine, each sample was reincubated for a total period of 20 min. Addition of spermine to the uptake system v C in v min FIG. 1. ffect of spermine on the uptake of 14Cvaline by Micrococcus lysodeikticus cells. Composition of the uptake system was as described in Materials and Methods. '4C-valine concentration, 1 x 10-i M; specific activity, 1,uCi/,imole; cell concentration, 0.88 mg dry weight/4 ml. Spermine (6 Amoles/ml) was added at various intervals as indicated. Symbols: *, amount of valine in the cells in the absence of spermine; 0, amount of valine in the cells after addition of spermine. at zero time resulted in almost no uptake of '4C-valine. Only a slight increase of valine uptake during 20 min of incubation was obtained. On the other hand, when spermine was added after the cells had already taken up "4Cvaline, the radioactivity inside the cells decreased. Thus, while cells took up about 7.0 nmoles of valine per 0.88 mg (dry weight) within 2 min in the absence of spermine, after addition of the polyamine and further incubation for 18 min at 37 C there was almost a 50% reduction in the radioactivity found in the cells. Similarly, addition of spermine to the uptake system after incubation for 10 min caused the cellular radioactivity to decrease by more than half (from 20 to about 9 nmoles/0.88 mg dry weight). ffect of spermine on the uptake of 14C_ leucine and 14C_isoleucine. In. coli, valine, leucine, and isoleucine are transported into the cells by a common system (17). It was therefore of interest to test the effect of spermine on the uptake of leucine and isoleucine. The experiment was carried out in a way similar to the one with valine. As in the case of valine it was found that spermine inhibited the uptake of leucine and isoleucine (see below). Addition of spermine also seemed to enhance the exit of the latter two amino acids from the cells. Uptake of valine, leucine, and isoleucine

3 414 ARIL AND GROSSOWICZ J. BACTRIOL. as a function of concentration. Since uptake systems display enzymatic kinetics, the nature of the inhibition by spermine was studied. For this purpose the uptake of labeled valine, leucine0 and isoleucine as a function of their concentration was determined upon incubation in / the presence and absence of spermine. The H range of concentrations used was between 5 x 10-7 and 1 x 10-1 M. The incubation period - was limited to 2 min, a time interval within the linear phase of uptake. The results were plotted according to Lineweaver-Burk, i.e., 1/v as a function of 1/s. From Fig. 2, 3, and 4 the 4A values of the apparent Km and Vmax were de- 4 * 20. termined. FIG. 3. Lineweaver-Burk plot of the 14C-leucine It can be seen (Table 1) that spermine did uptake in the absence and presence of spermine. not affect the apparent Km values of the above xperimental design as in Fig. 2. Specific activity of amino acids whereas the Vmax was measurably 14C-leucine, uci/mmole. Symbols: 0, without sperdecreased in all three cases. mine; A, in the presence of spermine. Is the uptake of valine, leucine, and isoleucine mediated by the same transport system in M. lysodeikticus? When two or more amino acids share the same uptake system, a competition between them is expected _5 to occur. The uptake of "IC-valine (10-5 M) was determined in the absence and in the presence / of increasing concentrations (from 5 x 10-I to : 5 x 10-3 M) of unlabeled valine, leucine, or iso- O leucine. It can be clearly seen (Table 2) that the three amino acids compete for the same / uptake system. Addition of any of the unla- ' beled amino acids reduced the uptake of 11C- _.-/ valine. There was no difference among the three amino acids in this respect. The dilution of the radioactivity of valine by leucine or iso- _ leucine was similar to the dilution by unla beled valine. Thus, it seems that the three I/ lo,. FIG. 4. Lineweaver-Burk plot of 14C-isoleucine uptake in the absence and presence of spermine. xperimental design as in Fig. 2. Specific activity of.4c-isoleucine, 1 MCi/Mmole. Symbols: 0, without spermine; A, in the presence of spermine. J/sXOS FIG. 2. Lineweaver-Burk plot of the 14C-valine uptake in the absence and presence of spermine. Composition of the uptake system was as described in Materials and Methods. Duration of uptake, 2 min; spermine, 6 umoles/ml; specific activity of 14Cvaline, 1 MCi/,smole. Symbols: 0, without spermine; A, in the presence of spermine. TABL 1. ffect of spermine on Km and Vmax of the uptake of neutral aliphatic amino acids Spermine Vmax Amino acid added (6 (K10-5 M) (nmoles/mg /Lmoles/ml) x dry wt/min) Valine Leucine _ Isoleucine _ Alanine _ Glycine _

4 VOL. 111, 1972 FFCT OF SPRMIN ON AMINO ACID UPTAK 415 amino acids are transported into the cells of M. lysodeikticus by the same system. Nature of the uptake system. xperiments were carried out to determine whether the uptake system for valine, leucine, and isoleucine is an active one. (i) ffect of temperature on the uptake system. Figure 5 shows that the uptake of "ICvaline by M. lysodeikticus cells was temperature-dependent. Maximal uptake occurred at 25 C; at 37 C the uptake was slightly lower. On the other hand, at 10 C the uptake was considerably reduced. Moreover, whereas at 25 and 37 C maximal uptake was achieved within 10 min, at 10 C uptake reached the maximal value only after 20 min. At 0 C there was almost no uptake; the very low counts obtained were probably due to nonspecific adsorption of valine to the cells. (ii) ffect of NaN8 and 2,4 dinitrophenol (DNP) on uptake system. Since sodium azide is a potent inhibitor of the respiratory system, its effect on the uptake of "IC-valine by M. lysodeikticus was tested. From Table 3 it can be seen that azide (at concentrations between 2.5 x 10-2 M and 1 x 10-' M) inhibited the uptake of "IC-valine; an 80% inhibition of valine uptake was obtained in the presence of 10-1 M NaN,. DNP proved more effective than TABL 2. Uptake of "4C-valine in the presence of unlabeled valine, leucine, or isoleucinea "4C-valine Unlabeled amino acid in the cells "4C-valine added (M) (nmoles/mg uptake (%) dry wt/min) No addition Valine, 5.0 x 10-' x 10-' x x x 10-' Leucine, 5.0 x 10-' x 10-' x x x Isoleucine, 5.0 x x 10' x 10-' x x 10-I a Composition of the uptai4e system was as described in Materials and Methods. Duration of uptake 2 min. Concentration of "4C-valine, 1.0 x 10-I M; specific activity, 1 uci/amole. c -. u In GD 0 c min FIG. 5. Kinetics of "4C-valine uptake at various temperatures. Composition of the uptake system was as described in Materials and Methods. Total volume, 4 ml; amount of cells equivalent to 0.88 mg dry weight; '4C-valine concentration, 1 x 10-5 M; specific activity, 1 gci/mmole. Symbols:+, uptake of 14C-valine at 0 C; A, uptake of 14C-valine at 10 C; 0, uptake of 14C-valine at 25 C; U, uptake of 14C-valine at 37 C. sodium azide; a 40% inhibition was obtained at a DNP concentration of 10-3 M. (Mii) Glucose dependence of the uptake system. Omission of glucose from the incubation mixture caused almost no change in the uptake of valine by M. lysodeikticus cells. (iv) Concentration of "IC-valine in the cells. Addition of chloramphenicol to the uptake system prevents utilization of the amino acids of the cellular pool for protein synthesis (8). Indeed, in presence of chloramphenicol in the reaction mixture, precipitation with 5% trichloracetic acid removed only 2% of the label, showing that essentially all of the valine remained in the free amino acid pool of the cell. On the assumption that the cellular water of

5 416 ARIL AND GROSSOWICZ J. BACTRIOL. M. lysodeikticus cell is 4.0 gliters/mg (dry weight) (7), it was calculated that the intracellular concentration of "IC-valine was 100-fold higher than in the medium. (v) Identification of amino acids taken up. xamination of the accumulated radioactivity has shown that about 85% of valine and 60% of leucine and isoleucine remained unaltered in the cells under the experimental conditions employed. ffect of spermine on the uptake of various amino acids. From the above it may be seen that spermine inhibited the uptake of the three aliphatic amino acids: valine, leucine, and isoleucine. It has also been shown that the transport of these amino acids is mediated by the same uptake system. Therefore, it was of interest to establish whether the inhibitory effect of spermine is specific to this particular uptake system, or whether it would affect the uptake of other amino acids as well. For this purpose, the effect of spermine on the uptake of neutral, acidic, basic, and aromatic amino acids was determined. It was found that spermine inhibited the uptake of two additional neutral amino acids: alanine and glycine. The inhibition seems to be similar to that of valine, leucine, and isoleucine (Table 1); the inhibition was of the noncompetitive type. On the other hand, spermine did not inhibit the uptake of acidic amino acids (glutamic acid), basic amino acids (arginine and histidine), or aromatic amino acids (tyrosine and phenylalanine). Table 4 presents the results obtained with glutamic acid. ffect of valine on the uptake of I C- spermine. Spermine is very rapidly absorbed by the cells; 90% of the total count was obtained at zero time. The uptake of spermine (10-2,umole/ml; 0.1,uCi/umole) was not affected by valine (10- lgmole/ml). It would thus seem that spermine is not a substrate for the valine transport system. TABL 3. ffect of NaNs on valine uptakea NaN, added Valine taken Inhibition (M) up (nmoles/mg dry wt/min) (%) No addition x x x 10' x a Valine concentration was 1.0 x 10- I M; duration of uptake was 2 min at 37 C. Composition of the uptake system as described in Materials and Methods. TABL 4. Uptake of "4C-glutamic acid in the absence and presence of sperminea '4C-L-glutamic acid in the "C-L-glutamic acid cells (nmoles/mg dry wt/min) added (M) Without With spermine spermine (6 Mmoles/ml) 1.0 x 10' x x x x 10' x 10-' a Composition of the uptake system as described in Materials and Methods. Duration of uptake, 2 min at 37 C. "C-glutamate specific activity, 1 ACi/;Lmole. Comparison between spermine and other polyamines. Spermidine (a triamine) and cadaverine (a diamine) were compared to spermine (a tetramine) in their ability to inhibit the uptake of amino acids into M. lysodeikticus cells. Radioactive valine was used in these experiments. Figure 6 shows that all three polyamines inhibited the uptake of valine into M. lysodeikticus cells, though the extent of inhibition was different. Spermine was the most effective, spermidine less so, and cadaverine the least active. In Table 5 the kinetic parameters obtained for the uptake of 14C-valine in the presence of the various polyamines are given. DISCUSSION The stabilizing effect of bacterial protoplasts by spermine (2, 3, 11-13, 18-21) prompted us to investigate the effect of the polyamine on the transport of amino acids across the bacterial membrane. It was found that spermine inhibits the uptake of the neutral aliphatic amino acids: valine, leucine, isoleucine, alanine, and glycine. On the other hand, the uptake of acidic amino acids (glutamic acid), basic amino acids (arginine, histidine), and aromatic amino acids (tryosine and phenylalanine) was not affected by spermine. Friedman and Bachrach (1) have shown that in Staphylococcus aureus spermine inhibits the incorporation of valine, leucine, phenylalanine, arginine, and lysine into protein, whereas the incorporation of alanine, glycine, and threonine is not affected. The above authors raised the possibility that the effect observed by them may have been due to inhibition at the uptake level although incorporation into protein was the only parameter meas-

6 VOL. 111, 1972 FFCT OF SPRMIN ON AMINO ACID UPTAK 417 TAML 5. ffect ofpolyamines on "4C-valine uptake by M. lysodeikticus cells Inhibitor added K. V.m/ (6 umoles/ml) (x 10-I M) (nmole/mg 20. n 20 0 H> i/s X 105 FIG. 6. ffect of spermidine and cadaverine on '4C-valine uptake (a Lineweaver-Burk plot). xperimental design as in Fig. 2. Concentrations of spermine, spermidine, and cadaverine were 6 gmoles/ml each. Specific activity of 14C-valine, 1 4uCi/Mmole. Symbols: *, without any addition;+, in the presence of cadaverine; *, in the presence of spermidine; A, in the presence of spermine. ured. No strict comparison can be drawn between the above work and ours, because the bacterial strains as well as the systems examined were different. The present work deals solely with the transport of amino acids across the membrane. It was found by Oxender et al. (17) that valine, leucine, and isoleucine are transported into. coli cells via a common uptake system. Indeed, competition experiments carried out by us showed that in M. lysodeikticus these three amino acids share a common carrier. (The possibility of the presence of additional specific carriers was not tested.) The temperature dependence of the transport of valine, leucine, and isoleucine into M. lysodeikticus cells, inhibition of the uptake by azide and DNP, and accumulation of the No addition Spermine Spermidine Cadaverine amino acids in the cell against a concentration gradient strongly suggest an active transport system. Lack of dependence of the system on an external glucose supply could be due to adequate intracellular energy sources. Mills and Dubin (16) found that spermine affected the potassium flux as well as early stages in the synthesis of protein and ribonucleic acid. The authors proposed that the effect of the potassium flux is a specific effect of spermine on the potassium carrier. Our data are in accordance with the explanation that spermine binds specifically to the carrier of the above amino acids, thereby inhibiting their uptake. The Lineweaver-Burk plots show that the inhibition is of a noncompetitive nature. Noncompetitive inhibitors do not tend to bind to the same site on the enzyme or carrier to which the substrate binds. However, if the binding site were close enough to the active site, the inhibitor might affect the reaction by steric hindrance or electrostatic change. The interesting feature of the spermine effect is its specificity. Spermine does not affect uptake of amino acids in general, but rather the uptake of certain amino acids. We assume therefore that spermine binds to exposed reactive groups located on the carriers of the amino acids affected. Possibly these carriers are characterized by certain amino acids which project outward from the steric structure of the protein. This is to say that the determining factors for attachment of spermine are the presence of specific groups and their availability due to the steric structure of the protein. From the experiments (Fig. 1) it would seem that spermine not only inhibited the uptake of valine by the cells but also increased the amino acid exit from the cells; however, one cannot exclude the possibility that by inhibiting the uptake there was only an apparent increase in the efflux of the amino acid. It seems plausible to assume that the amino groups of the polyamines bind to a reactive group of the carrier. The lower the amine, the weaker the effect; thus, spermidine showed a weaker effect than spermine, and cadaverine

7 418 ARIL AND G,ROSSOWICZ J. BACTRIOL. was the least active of all three. It may be that the chain length of the inhibitor is also of importance. LITRATUR CITD 1. Friedman, M.., and U. Bachrach Inhibition of protein synthesis by spermine in growing cells of Staphylococcus aureus. J. Bacteriol. 92: Grossowicz, N., and M. Ariel Mechanism of protection of cells by spermine against lysozyme-induced lysis. J. Bacteriol. 85: Harold, F. M Stabilization of Streptococcus faecalis protoplasts by spermine. J. Bacteriol. 88: Herbst,. J.,. B. Glinos, and L. H. Amundsen An analysis of the putrescine requirement of Hemophilus parainfluenzae. J. Biol. Chem. 214: Herbst,. J., and.. Snell Putrescine as a growth factor for Hemophilus parainfluenzae. J. Biol. Chem. 176: Herbst,. J., and.. Snell Putrescine and related compounds as growth factors for Hemophilus parainfluenzae J. Biol. Chem. 181: Kepes, A., and G. N. Cohen Permeation, p In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol IV. Physiology and growth. Academic Press Inc., New York. 8. Kessel, D., and M. Lubin Stability of a-hydrogen of amino acids during active transport. Biochemistry 4: Kihara, H., and.. Snell Spermine and related polyamines as growth stimulants for Lactobacillus casei. Proc. Nat. Acad. Sci. U.S.A. 43: Kihara, H., and.. Snell Peptides and bac- terial growth. VIH. The nature of streptogenin. J. Biol. Chem. 235: Mager, J Influence of osmotic pressure on the polyamine requirement of Neisseria perflava and Pasteurella tularensis for growth in defined media. Nature (London) 176: Mager, J The stabilizing effect of spermine and related polyamines on bacterial protoplasts. Biochim. Biophys. Acta 36: Mager, J Spermine as a protective agent against osmotic lysis. Nature (London) 183: Mager, J., A. Traub, and N. Grossowicz Cultivation of Pasteurella tularensis in chemically defined media: effect of buffer and spermine. Nature (London) 174: Martin, W. H., Jr., M. J. Pelczar, Jr., and P. A. Hansen Putrescine as a growth requirement for Neisseria. Science 116: Mills, J., and D. T. Dubin Some effects of spermine on scherichia coli. Mol. Pharmacol. 2: Piperno, J. R., and D. L. Oxender Amino acid transport system in. coli K-12. J. Biol. Chem. 243: Razin, S., and R. Rozansky Mechanism of the antibacterial action of spermine. Arch. Biochem. Biophys. 81: Tabor, C. W The stabilizing effect of spermine and related amines on mitochondria and protoplasts. Biochem. Biophys. Res. Commun. 2: Tabor, C. W Stabilization of protoplasts and spheroplasts by spermine and other polyamines. J. Bacteriol. 83: Traub, A., J. Mager, and N. Grossowicz Studies on the nutrition of Pasteurella tularensis. J. Bacteriol. 70: Downloaded from on October 4, 2018 by guest