ION ANTAGONISMS AFFECTING GLYCOLYSIS BY BACTERIAL SUSPENSIONS*

Transcription

1 ION ANTAGONISMS AFFECTING GLYCOLYSIS BY BACTERIAL SUSPENSIONS* BY HIROSHI TSUYUKIt AND ROBERT A. MAcLEOD (From the Department of Biochemistry, Queen s University, Kingston, Ontario, Canada) (Received for publication, December 12, 1959) The phenomenon termed ion antagonism, whereby the inhibitory action of one inorganic ion can be overcome by adding sufficient of another, has been observed in many biological systems (1). Until recently, no exact explanation for the mechanism of ion antagonism has been offered. In a study of the mineral requirements of lactic acid bacteria, MacLeod and Snell observed examples of ion antagonism which could be visualized best as a competition between the antagonists for an enzyme surface (24). Further support for the hypothesis presented would be forthcoming if the same relationships between inorganic ions which have been demonstrated in growth experiments were found to apply to enzymes isoiated from the organism. The ultimate objective of the present studies is to determine whether or not this is so. Inorganic ions have been shown to be required in several of the enzymatic reactions involved in the physiological breakdown of glucose to lactic acid (5). The possibility exists that some of the ion antagonisms influencing growth of the lactic acid bacteria might be doing so by acting upon enzymes involved in glycolysis. The results of studies with resting cell suspensions of Lactobacillus arabinosus, presented below, indicate that this may indeed be the case. Methods Preparation of Cell Suspensions--Cells of L. arabinosus ATCC 8014 were grown in an amino acid medium prepared as previously described (3), except that the required amounts of acetate and phosphate were added as salts of triethanolamine. The ph of the medium was adjusted to 5.0, since growth was more rapid at this ph in the presence of triethanolamine than at higher values. To obtain K+-deficient cells sufficient Mn++ (100 y per 10 ml.) but insufficient K+ (20 y per 10 ml.) for maximum growth * Supported by a grant from the Division of Medical Research of the National Research Council of Canada. Presented at the annual meeting of the Canadian Physiological Society at Ottawa, Canada, October 17, t From a thesis submitted by Hiroshi Tsuyuki in partial fulfilment of the requirements for the degree of Master of Arts, Queen s University. 711

2 712 ION ANTAGONISivl AND GLYCOLYSIS was added to the medium. Mn++-deficient cells were harvested from a medium supplemented with 100 y of K+ and 1 y of Mn+f per 10 ml. After 36 to 40 hours of incubation at 37 the cells from 100 ml. of appropriately supplemented basal medium were harvested by centrifugation and washed with three 10 ml. portions of distilled water. The washed cells were suspended in water and diluted to a turbidity equivalent to 1 mg. of dry weight of cells per ml. Turbidity, measured in an Evelyn calorimeter with a 660 rnp filter, was related to dry weight by a previously calibrated curve. Manometric Methods-The rate of glycolysis was determined manometrically in a Warburg respirometer by measuring the CO2 released from a bicarbonate buffer by acid produced from glucose. Unless otherwise stated, each flask contained the following components: 0.3 ml. of 0.1 M glucose in the side arm; 0.2 ml. of 0.3 M NaHC03, 0.1 ml. of 0.01 M NaH2P04*HzO, 0.5 ml. of cell suspension, metal ion solutions, and water to 2.7 ml. in the main compartment. For experiments with K+ and related ions, 1 pm of Mn++, and for Mn++ studies, 500 PM of K+ were added per flask. The suspending medium was adjusted to ph 6.2 with an atmosphere of CO2 and maintained at a temperature of 37. After a period of equilibration, glucose was introduced into the main compartment from the side arm and manometric measurements were made at intervals over a 3 hour period. The same kinds of salts as those employed in previous investigations were used to prepare metal ion solutions (2, 3, 6). Distilled water, redistilled in a glass-distilling apparatus, was used in the preparation of all media and solutions. Glassware was cleaned with a hot mixture of concentrated nitric and sulfuric acids and rinsed frrst with tap water and finally with distilled water. Results Effect of K+ and Related Ions on Glycolysis-A previous investigation has shown that NH,+, Naf, and Cs+ inhibit the growth of L. arabinosus if insufficient K+ is present and that this inhibition of growth can be overcome by adding enough K+ (2). The data presented indicate that a similar relationship affects glycolysis. Fig. 1 shows that K+ stimulates glycolysis by resting cell suspensions of L. arabinosus. The addition of NH 4+, Na+, or Cs+ inhibits glycolysis (Figs. 1 and 2). The inhibition produced by each of these ions can be overcome by the addition of sufficient K+. As the concentration of each of the inhibitory ions is increased, the amount of K+ required to overcome the resulting inhibition is increased, indicating qualitatively that the inhibitions are competitive in nature.

3 H. TSUYUKI AND R. A. MACLEOD 713 Further evidence for the competitive nature of the inhibitions would be obtained if the molar ratios of inhibitor to metabolite at one-half maximum reversal of the inhibitions were constant at a number of different 4 tl CURVE 3 200,M N CURVE I OrM N: 100 CURVE2 moum N; CURVE JIM Ni Of-----A IO 20 MICROMOLES K+ PER FLASK FIG. 1. Glycolysis by resting cell suspensions of L. arabinosus. The effect of K+ on the inhibition of glycolysis by NHd+ and Na+. The amounts of NH4+ and Na+ indicated refer to amounts added per flask. &cot = microliters of COz released per mg. of cells per 3 hours. IO ;M K PER FLASK FIG. 2. Glycolysis by resting cell suspensions of L. arabinosus. The effect of KC on the inhibition of glycolysis by Csf. The amounts of Cs+ indicated refer to amounts added per flask. &coz as in Fig. 1.

4 714 ION ANTAGONISM AND GLYCOLYSIS concentrations of the inhibitor. This has been found to be the case for NH,+ and Cs+. The NH4+:K+ ratio calculated from these and other data is 350. The Cs+:K+ ratio is 30. The Na+:K+ ratio, on the other hand, decreases progressively from a high of 4800 at a concentration of 250 PM of Na+ to a low of 300 at 1000 PM of Na+ per flask. Rb+ is the nutritional equivalent of K+ for the growth of Streptococcus faecalis R and Lactobacillus casei but only partially replaces K+ for the 2 R;+soo~M N; 3 R i+soowm NH: J,M R: PER FLASK 3 K++ IOOJJM C; 4 R+B +~oopm C: FIG. 3. A, glycolysis by resting cell suspensions of L. arabinosus. The effect of Rb+ on the inhibition of glycolysis by Na+ and NH*+. Curve 1, response to Rb+; Curves 2 and 3, response to Rb+ in the presence of the indicated amounts of Naf and NHa, respectively, per flask. B, the comparative ability of Kc and Rb+ to stimulate glycolysis by resting cell suspensions of L. arabinosus and to overcome the inhibition of glycolysis by Cs+. Curves 1 and 2, response to K+ and Rb+ respectively; Curve 3, response to K+; Curve 4, response to Rb+ in the presence of 100 pat of Cs+ per flask. &co2 as in Fig. 1. growth of L. arabinosks (2). For glycolyzing cell suspensions of L. arabimwus, Rbf carries out all of the functions of added K+ (Fig. 3, A and B). Rb+, like K+, stimulates glycolysis and overcomes the inhibitions of glycolysis produced by adding Na +, NHk+ (Fig. 3, A), and Cs+ (Fig. 3, B). The comparative ability of Rbf and K+ to stimulate glycolysis and to overcome Csf inhibition is shown in Fig. 3, B. Rb+ is slightly less effective than K+ in both capacities. It has been observed consistently that, although Na+ and Csf can inhibit glycolysis completely, NH,+ is not able to do so in the range of concentrations used to demonstrate the antagonisms. The comparative effect of adding increasing concentrations of Na+ and NHd+ on the rate of

5 H. TSUYUKI AND R. A. MACLEOD 715 glycolysis is shown in Fig. 4. Although NHd+ is more inhibitory than Na+ at low concentrations, increasing concentrations of NHJ+ do not significantly decrease the rate of glycolysis beyond approximately 50 per cent of the initial rate. An explanation for the nature of the inhibitory effect of NH,+ on glycolysis would be forthcoming if this ion inhibited the enzyme triose phosphate isomerase. If this were the case, dihydroxyacetone phosphate would not give rise to lactic acid and only 1 mole of lactic acid per mole of glucose would be produced instead of 2 (7), thus adequately accounting for the-effect of NH4+ on glycolysis. This possibility was tested by de- 350,,,,, 1,,,, 1,,,,,,,, xi 100 so-,l xl CURVE CURVE I NH; 2 Ni II I I I I I I I I h MICROMOLES NH: OR N: PER FLASK Fig. 4. The comparative effect of adding increasing concentrations of NH*+ and Na+ on the rate of glycolysis by resting cell suspensions of L. arabinosus. &co2 as in Fig. 1. termining the ratio of lactic acid produced to glucose utilized in the presence and absence of NHd+. Warburg flask contents after 3 hours incubation were analyzed for lactic acid and glucose by the methods of Barker and Summerson (8) and Park and Johnson (9), respectively. The results, corrected for endogenous activity, are presented in Table I. A ratio of 2 : 1 of lactic acid produced to glucose utilized was obtained both in the presence and in the absence of NH4+. The effect of this ion upon glycolysis, therefore, is not due to its effect upon the enzyme triose phosphate isomerase. The results in Table I also serve to demonstrate that within the limits of experimental error the amount of CO2 released from the bicarbonate buffer is an adequate measure of lactic acid production under the conditions used in this investigation.

6 716 ION ANTAGONISM AND GLYCOLYSIS E$ect of Mn++ and Related Ions on Glycolysis-A previous study has shown that Zn* inhibits the growth of L. arabinosus and that the inhibition can be overcome by the addition of Mn++, Mg++, Ca++, or Sr++ (3). TABLE I E$ect of NHJ+ on Glucose Utilization and Lactic Acid Production by Resting Cell Suspensions of L. arabinosus Additions* per flask CO% released Lactate produced Glucose utilized * The flask contents are the same as those described for use in studies with ions related to K+ (see the text). t Micromoles of lactic acid produced against micromoles of glucose utilized I.0 MM Mfi PER FLASK NM Mr PER FLASK FIG. 5. Glycolysis by resting cell suspensions of L. arabinosus. The effect of Mn++ and Mg++ on the inhibition of glycolysis by Zn++. The amounts of Zn indicated refer to amounts added per flask. &co2 as in Fig. 1. Zn++ also inhibits glycolysis by resting cell suspensions of L. arabinosus and the inhibition can be overcome by the addition of Mn++ (Fig. 5). As the Zn+f concentration is increased, the concentration of Mn++ required to overcome the toxicity is increased. The molar ratio of Zn++: Mn++ at one-half maximum reversal of the inhibition is about 0.8 and remains quite constant over the range of Zn++ concentrations tested. The

7 H. TSUYUKI AND R. A. MACLEOD 717 range of concentrations which can be tested, however, is limited by the tendency of Zn* and Mn++ to precipitate as phosphates at higher concentrations. In contrast to its effect in growth (3), Mg++ IS unable to overcome the Zn++ inhibition of glycolysis (Fig. 5). As can be seen, however, Mg++ stimulates glycolysis to about the same extent as Mn++. In growth, Ca++ is the most effective ion in reversing Zn++ toxicity (3). For glycolyzing cell suspensions, Catt- neither overcomes Zn++ toxicity nor stimulates glycolysis. DISCUSSION Although the importance of inorganic ions in glycolysis has been demonstrated by many investigators (5), few examples of ion antagonism in glycolysis have been observed. Utter has shown that Naf inhibition of glycolysis in homogenates of nervous tissue of the cotton rat can be overcome only partially by K+ (10). Boyer et al., using rat muscle homogenates, have demonstrated that Na+ antagonizes the function of K+ in the enzymatic transfer of phosphate from phosphopyruvate to the adenylic system (11). The results obtained in this investigation reveal that many of the ion antagonisms affecting growth of L. arabinosus also affect glycolysis by the intact resting cell. When the molar ratios of antagonist to metabolite giving one-half maximum reversal of the inhibition of glycolysis are compared with the ratios permitting one-half maximum growth of the organism, some striking similarities are observed. The Cs+:K+ ratio for growing cells, calculated from data previously published (2), is about 25 and for resting cells 30. The Zn++:Mn* ratios calculated from growth data (3) and for glycolysis are both approximately 1.0. The corresponding values for the NHJ+:K+ ratios are 140 (2) and 350, respectively. No satisfactory comparison of the Na+:K+ ratios can be made, since the values obtained with resting cells are not constant. The inhibition ratios in growing and resting cells are sufficiently similar in the first three cases to suggest that the corresponding ion antagonisms could be influencing growth by affecting glycolysis. Early workers believed ion antagonisms to be due to the opposite effect of ions on membrane permeability (cf. Falk (1)). Results obtained by MacLeod and Snell with growing cells of lactic acid bacteria suggested the possibility that the ion antagonisms encountered were due to competition between the antagonists for an enzyme surface (4). Since intact resting cells were used in this investigation, no conclusions regarding either of these hypotheses can yet be drawn. Studies with cell-free extracts of L. arabinosus are now proceeding.

8 718 ION ANTAGONISM AND GLYCOLYSIS Although there are many similarities between the effects of inorganic ions on growth of and on glycolysis by intact cells of L. arabinosus, there are also some significant differences. For instance, NH4+ can prevent growth completely but can inhibit glycolysis only partially. This finding would suggest that in growing cells either NH,+ can inhibit some other system essential for the growth of the organism or it can affect the glycolytic system in such a way that the partial inhibition the ion produces prevents completely the growth of the organism. Another difference is observed in the response of growing and resting cells to Rb+. This ion cannot replace K+ completely in growing cells of L. urubinosus (2); yet in resting cells, Rbf and Kf appear to be interchangeable. If Rb+ and K+ are equivalent in glycolysis, the failure of Rbf to replace Kf completely in growth would indicate that there is an essential role of K+ in the latter process, apart from its function in glycolysis, in which Rbf cannot act. In growing cells, Mg++, Ca*, and Mn++ overcome inhibition by Zn++, but in glycolysis by resting cells only Mn++ is active in this respect. No adequate explanation for this difference in response can be offered at the present time. SUMMARY Glycolysis by resting cell suspensions of Lactobacillus arabinosus 8014 is inhibited by Na+, NHI+, and Cs+ if insufficient Kf is present. The extent of the inhibitions produced by each of these ions is dependent upon the ratio of their concentrations to that of K+ and not upon the absolute amounts present. Except for those involving Na+, the ratios at a given level of glycolysis are relatively constant over the range of concentrations tested. Rb+ can overcome the inhibition of glycolysis produced by Na+, NH4+, and Cs+. When the ability of K+ and Rbf to stimulate glycolgsis and to overcome Cs+ inhibition is compared, Rb+ is found to be slightly less effective than K+. Whereas Na+ and Cs+ inhibit glycolysis completely, NHd+ can do so only partially. NH*+ has been shown to reduce by approximately onehalf the total amount of glucose converted to lactic acid by the resting cells. Zn++ has been found to inhibit glycolysis and the extent of the inhibition has been shown to be dependent upon the concentration of Mn* present. Mg++ and Ca*, which are known to overcome the inhibition of growth by Zn*, have no detectable effects upon the inhibition of gly- colysis by this ion. The results, which indicate that some of the ion antagonisms influenc-

9 H. TSUYUKI AND R. A. MACLEOD 719 ing the growth of L. arabinosus could be doing so by affecting glycolysis, have been discussed. The authors wish to express their appreciation to Dr. Patricia MacLeod and to Dr. J. M. R. Beveridge for their constructive criticism of the manuscript. BIBLIOGRAPHY 1. Falk, I. S., Abstr. Bact., 7, 33, 87, 133 (1923). 2. MacLeod, R. A., and Snell, E. E., J. Biol. Chem., 176,39 (1948). 3. MacLeod, R. A., and Snell, E. E., J. Bact., 69,783 (1950). 4. MacLeod, R. A., and Snell, E. E., Ann. New York Acad. SC., 62, 1249 (1950). 5. Lardy, H. A., in Respiratory enzymes, Minneapolis, revised edition (1949). 6. MacLeod, R. A., and Snell, E. E., J. Biol. Chem., 170,351 (1947). 7. Meyerhof, O., and Beck, L. V., J. Biol. Chem., 166, 109 (1944). 8. Barker, S. B., and Summerson, W. H., J. Biol. &em., 138, 535 (1941). 9. Park, J.,T., and Johnson, M. J., J. Biol. Chem., 181, 149 (1949). 10. Utter, M. F., J. Biol. Chem., 186, 499 (1950). 11. Boyer, P. D., Lardy, H. A., and Phillips, P. H., J. Biol. Chem., 149,529 (1943).

10 ION ANTAGONISMS AFFECTING GLYCOLYSIS BY BACTERIAL SUSPENSIONS Hiroshi Tsuyuki and Robert A. MacLeod J. Biol. Chem. 1951, 190: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1