Pseudomonad. Received for publication 25 July MgSO4 solution resulted in the loss by the cells. of their intracellular K+, and the cells became

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1975, p Copyright i 1975 American Society for Microbiology Vol. 121, No. 1 Printed in U.S.A. Kinetics of Na+-Dependent K+ Ion Transport in a Marine Pseudomonad H. MOUSTAFA HASSAN'* AND ROBERT A. MAcLEOD Department of Microbiology, Macdonald Campus, and Marine Sciences Center, McGill University, Montreal H9X 3M1, Quebec, Canada Received for publication 25 July 1974 The effect of external Na+ concentration on the transport of K+ was studied using K+-depleted cells of a marine pseudomonad. K+ transport was found to be a saturable process and requires Na+. The initial rates for K+ transport over a range of external K+ concentrations were measured in suspensions containing various fixed concentrations of Na+. Reciprocals of the initial rates for K+ transport were plotted against reciprocals of the external concentration of K+ or Na+ to yield two primary Lineweaver-Burk plots. The experimental data were found to fit bisubstrate enzyme kinetics, with a sequential type mechanism. However, the initial rate data did not allow distinction between ordered or random mechanisms. The results suggest that Na+ and K+ form a ternary complex with a specific K+ carrier molecule on the outer surface of the membrane prior to translocation and the release of K+ inside the cell. Gram-negative marine bacteria generally require Na+ for growth and metabolism (14). The marine pseudomonad B-16 (ATCC 19855) specifically requires Na+ for the active transport of various metabolites into the cell (6, 8, 24). Recently, Na+ was shown to be required for the active transport of organic solutes into Escherichia coli (11), Salmonella typhimurium (19), and Bacillus licheniformis (15). Kinetic ana'sis of the Na+-dependent solute transport into marine pseudomonad B-16 (24) and animal cells (18) demonstrated that Na+ decreases the Km for the transport process, thus suggesting that the role of Na+ is to increase the affinity of a specific carrier or binding protein for the compound to be transported. Na+ has also been shown to prevent the release of intracellular solutes from the cells of the marine pseudomonad B-16 by controlling the porosity of the cytoplasmic membrane (7). On the other hand, potassium is known to be required by all living organisms. In the marine pseudomonad B-16, as in most other types of cells, K+ is accumulated inside the cells against a concentration gradient (21). K+ was also found to be required for the transport of amino acids into the cells (6, 8). The role of K+ in amino acid transport was reported to be at the intracellular level (21). Thompson et al. (20) demonstrated that washing and resuspending the marine pseudomonad B-16 in 0.05 M ' Present address: Department of Microbiology, University of Maine at Orono, Orono, Me MgSO4 solution resulted in the loss by the cells of their intracellular K+, and the cells became plasmolyzed. Deplasmolysis was observed when K+ was added back to the complete reaction mixture and the cells regained their intracellular K+ concentration. However, deplasmolysis did not take place when Na+ was omitted from the complete reaction mixture, and the intracellular K+ concentration remained low. Thus, deplasmolysis required the presence of both Na+ and K+ in the suspending medium. This dependence of K+ accumulation upon the presence of Na+ was attributed to the ability of Na+ to cause the membrane to retain the K+ accumulated by the cells (20). The present communication is an attempt to elucidate the role of Na+ in K+ transport into K+-depleted cells of the marine pseudomonad B-16. (Part of this work was presented at the 24th Annual Meeting of the Canadian Society of Microbiologists, 18 to 21 June, 1974, Macdonald Campus, McGill University, Montreal, Quebec, Canada.) MATERIALS AND METHODS Organism. The organism used was the marine pseudomonad designated as B-16, variant 3 (12), and was recently reclassified as Alteromonas haloplanktis (17). The organism has been deposited in the National Collection of Marine Bacteria (NCMB 19) and in the American Type Culture Collection (ATCC 19855). Medium. The medium used for growth and maintenance of the culture was reported previously (21). Growth of cells. The cells were grown at 25 C on a 160

2 VOL. 121, 1975 KINETICS OF NA+-DEPENDENT K+ TRANSPORT rotary shaker to late log phase in liquid medium according to the protocol reported previously (5). Preparation of K+-depleted cells. The cells were harvested from the growth medium (200 ml) by centrifugation at 16,000 x g at 4 C for 10 min. The cell pellet was resuspended once in an equal volume of 0.05 M MgSO4 solution to deplete them of K+ (20) and was centrifuged imntediately. The K+-depleted cell pellet was washed once in Mg++-Tris(hydroxymethyl)aminomethane(Tris)-HCl-PO4 buffer (0.05 M MgSO4, 0.05 M Tris-hydrochloride, M H,PO4, ph 7.2) and resuspended in 50 ml of the same buffer. In some experiments, this buffer contained 200 mm NaCl or 200 mm LiCl (to avoid damage to the cell membrane). Bacterial cell mass was determined from a standard curve relating absorbance at 660 nm (A,0o) (Gilford-N300 spectrophotometer with 1-cm cuvettes) to dry weight as determined by the ashing method reported before (10). One milligram (dry weight) of the K+-depleted cells per milliliter corresponded to A,,0 of This relationship was linear up to an A,,60 value of 0.9. Transport of 42K+ by K+-depleted cells. The reaction mixture for 42K+ uptake contained: various final concentration of NaCl (10 to 160 mm) and 42KCI (1 to 20 mm), 25 mm ethanol which serves as an oxidizable substrate (23), and the K+-depleted cells (100 gg [dry weight] per ml) in the Mg++-Tris-HCl- P04 (ph 7.2) mentioned above. For the time course experiments, the final volume of the reaction mixture was 5 ml contained in a 50-ml Erlenmeyer flask. For initial velocity determinations, the final volume of the reaction mixture was 1 ml placed in a test tube (85 by 15 mm). The uptake assays were run in duplicate. The reaction mixtures were aerated by shaking on a rotary water bath shaker (Gyrotory Shaker, model G-76, New Brunswick Scientific Co.) operated at 250 rpm and 25 C. At the specified time intervals, 0.5-ml samples were removed by a Gilson micropipette and filtered through 0.45-Mm membrane filters type HA (Millipore Corp., Bedford, Mass.). Cells retained on the filter pads were immediately washed by drawing through the filter 6 ml of buffered solution having the same composition as the one used in the reaction mixture, except that K+ was omitted. Measurement of radioactivity. The filter pads with the adhering washed cells were placed in 20-ml screw cap scintillation vials and dried slowly under an infrared lamp. To each vial, 10 ml of water was added and Cerenkov light emission (16) was measured in a Nuclear Chicago Isocap 300 liquid scintillation spectrometer using the tritum 1B program. This system measures 42K+ with an efficiency of 50%. The concentration of K+ taken up by the cells at any time was determined from radioactivity counts per unit (dry weight) of cells and the specific activity of 42K+ at the time of counting. The intracellular K+ concentration (mm) was calculated by using the average value for the intracellular fluid volume (the volume inside the cytoplasmic membrane) reported previously, i.e., 1.6 psliters per mg (dry weight) of cells (22). Radioactive materials. '2K+ was obtained from New England Nuclear Corp., Boston, Mass., as a KCl solution in water. The initial specific activity varied from batch to batch, but it was in the range of 0.9 to 1.28 ACi/mg. RESULTS Time course of 42K+ uptake by K+-depleted cells. To determine accurately the initial rates for K+ transport, it was necessary to establish the period during which the uptake was time dependent. The results in Fig. 1 show that 42K+ uptake began immediately after the addition of the cells to the reaction mixture. Accumulation of 42K+ was linear for at least 1 min. Therefore, 1 min was used for initial rate determinations in all subsequent experiments. The results in Fig. 1 also show that the concentrations of both Na+ and K+ in the reaction mixture affect the rate and the extent of K+ uptake. The intracellular concentration for K+ was calculated (Table 1). The highest level obtained was 421 mm ob- w 12 10, o8-4 a. 6- (A) INCUBATION PERIOD (min) FIG. 1. Time course of 42K+ uptake by K+-depleted cells. A, 1 mm K+; B, 5 mm K+ and either 10 mm Na+ (open circles) or 80 mm Na+ (closed circles) in the suspending medium. (B) TABLE 1. Effect of extracellular concentrations of Na+ and K+ on the intracellular steady-state level of K+ Extracellular Intracellular concn" steady-state 42K+ Na+ (mm) (mm) weight] of cells) Intracellular level of K+ concn of K+ (MM) (MM) (jsmol/g [dry (MM) a LiCl was added to maintain the final concentration of Na+ plus Li+ at 200 mm.

3 162 HASSAN AND MAcLEOD J. BACTERIOL. tained in the presence of 10 mm K+ and 80 mm Na+. This value is close to the value of 0.44 M reported before and determined by a different method (22). Effect of external Na+ concentration on 42K+ uptake. The relationship between the external concentrations of NaCl and the initial rate of K+ uptake by the K+-depleted cells is shown in Fig. 2. In this experiment, the external concentration of 42K+ was maintained constant at 5 mm, whereas the external NaX concentration was varied between 10 and 160 mm. Meanwhile, the molarity of the salt solution was kept constant by using LiCl. The data clearly show that increasing the concentration of Na+ in the reaction mixture greatly stimulated the initial rate of K+ uptake. The possibility that Na+ acts only to prevent the leakage of K+ from the cells was eliminated by the fact that we are dealing with initial rates, and therefore, the problem of leakage is not encountered. Moreover, the salt concentration was kept constant by using Li+, an ion that was shown to be effective in preventing release of metabolites from cells of this organism (24). Kinetic analysis of Na+-stimulated K+ uptake. The kinetics of the effect of Na+ on the uptake of K+ by the K+-depleted cells were examined in experiments in which K+ and Na+ concentrations were varied. Reciprocals of the initial rates for K+ transport were plotted against reciprocals of the external concentration of K+ or Na+ to yield two primary Lineweaver- Burk plots (Fig. 3). In each case, straight lines A 260 [K+J. mm. [Na. mm. FIG. 3. Primary plots of the kinetics of '2K+ transport into K+-depleted cells of marine pseudomonad, B-16. A, Effect of increasing external K+ concentrations on the initial rate of K+ uptake at different constant external Na+ concentrations; B, effect of increasing NaCI concentrations on initial rate of 42K+ uptake at different constant external K+ concentrations. C E E a- +y O No ClI Li Cl CONCENTRATION (mm). FIG. 2. Effect of external Na+ concentration on initial rates of 42K+ uptake by cells of the marine pseudomonad, B-16. The total salt concentration was maintained constant at 200 mm by using LiCI. Potassium concentration was maintained constant at 5 mm. were obtained with a common point of intersection above the abscissa. The results show that Na+ increases the affinity (11Km) and the capacity (Vmax) of the K+-transport system. The fact that variation of the initial rates of K+ uptake as a function of Na+ and K+ concentrations followed Michaelis-Menten kinetics suggested that we were dealing with a bisubstrate type mechanism. Therefore, the intercepts and the slopes, obtained from the two primary plots (Fig. 3), were replotted against the reciprocals of the opposite substrate (4, 9) to yield secondary intercept and slope plots (Fig. 4 and 5, respectively). The kinetic parameters for the initial rate equation of a bisubstrate reaction were calculated (Table 2), according to the method of Florini and Vestling (9). The initial rate equation for a bisubstrate reaction is = V/v, 1 + (KmA/A) + (KmB/B) +

4 VOL. 121, 1975 KINETICS OF NA+-DEPENDENT K+ TRANSPORT 163 1/I[KI mm.*- - I I/ pa1 mm --- FIG. 4. Secondary intercept plot. The intercepts of the Lineweaver-Burk plot shown in Fig. 3A were plotted against the reciprocals of NaCI concentration (line 1); the intercepts from Fig. 3B were plotted against the reciprocals of KCI concentration (line 2). 02 Q- 0 J 0-1 C,) 0 I 0 *2 *4 *6 *8 1.0 I/Kt -mm - 0 *02-04 *06 *08 *I I/Nd? mm *-- FIG. 5. Secondary slope plot. The slopes of the Lineweaver-Burk plot shown in Fig. 3A were plotted against the reciprocals of NaCI concentration (line 1); and the slopes from Fig. 3B were plotted against the reciprocals of KCI concentration (line 2). TABLE 2. Parameter K.A KmB K8A V Kinetic parameters for the transport of 42K+ Value mm 1.13 mm 50.8 mm Mmol/min per g ([K8A KmB]IAB) (2), taking Na+ as substrate A and K+ as substrate B where: KmA is the limiting Michaelis constant for Na+; KmB is the limiting Michaelis constant for K+; K8A is the dissociation constant for Na+; V is the limiting maximal velocity; and v1 is the initial velocity. From Fig. 4 line 1, the slope is equal to KmA/V and the intercepts on the Y and X axes are equal to 1/V and - 1/KMA, respectively. From line 2 of the same figure, the slope is equal to KmB/V and the Y- and X-axis intercepts are equal to 1/V and - 11Km, respectively. In Fig. 5, the slopes of line 1 and 2 were the same and equal (K.A Km )/V. Thus, from the data in Fig. 4 and 5, we were able to determine KmA, Kmi, V, and K8A (Table 2). DISCUSSION In this study, we determined the effect of various conditions on the initial rates of K+ uptake by K+-depleted cells of the marine pseudomonad. Also, by using the average value for the intracellular volume (22), we were able to show that the cells used in this study had the normal capacity to concentrate K+ intracellularly against a concentration gradient (Table 1). Since a source of energy is required for K+ accumulation (23), K+ uptake is an active i process (13). The results clearly show that K+ transport into this organism is a saturable system and requires Na+. Increasing concentrations of Na+ were found to increase the Vmax and decrease the Km for the K+ transport process. These results suggest that K+-transport is a carriermediated process and Na+ interacts with the carrier in some way to increase its maximal velocity and affinity toward the substrate (i.e., K+). If this was the case, one would expect bisubstrate kinetics. Generally, bisubstrate enzyme mechanisms are divided into two classes, sequential and ping pong (1, 2). The data in Fig. 3A show a common point of intersection above the abscissa. This is indicative of a sequential rather than a ping pong type mechanism (2, 9, 25). The present results do not allow us to conclude whether the mechanism is random or ordered. By using the graphical method of Florini and Vestling (9), we were able to determine the kinetic parameters for the Na+-K+ carrier complex, supporting the conclusion that Na+ and K+ form a ternary complex with a specific K+ carrier molecule on the outer surface of the membrane prior to translocation and the release of K+ inside the cell. This mechanism is similar

5 164 HASSAN AND MAcLEOD J. BACTERIOL. to that proposed for Na+-dependent transport in animal cells (3). The kinetic data do not permit us to conclude that Na+ and K+ are co-transported, although this is a possibility. Previous studies have shown that the Na+-dependent transport of the amino amino analogue, a-aminoisobutyric acid, into cells of this organism is not affected when the gradients of Na+ and K+ are abolished (22). The Na+-dependent uptake of amino acids in this marine pseudomonad has the same (bisubstrate) kinetic characteristics as the uptake of K+ reported here (G. D. Sprott and R. A. MacLeod, manuscript in preparation). ACKNOWLEDGMENTS This research was supported by a grant from the National Research Council of Canada. LITERATURE CITED 1. Alberty, R. A The relationship between Michaelis constants, maximum velocities and the equilibrim constant for an enzyme-catalyzed reaction. J. Amer. Chem. Soc. 75: Cleland, W. W The kinietics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim. Biophys. Acta 67: Crane, R. K Na+-dependent transport in the intestine and other animal tissues. Fed. Proc. 24: Dalziel, K Initial steady state velocities in the evaluation of enzyme-co-enzyme-substrate reaction mechanisms. Acta Chem. Scand. 11: DeVoe, I. W., J. Thompson, J. W. Costerton, and R. A. MacLeod Stability and comparative transport capacity of cells, mureinoplasts, and true protoplasts of a gram-negative bacterium. J. Bacteriol. 101: Drapeau, G. R., and R. A. MacLeod Na+-dependent active transport of a-aminoisobutyric acid into cells of a marine pseudomonad. Biochem. Biophys. Res. Commun. 12: Drapeau, G. R., and R. A. MacLeod A role for inorganic ions in the maintenance of intracellular solute concentrations in a marine pseudomonad. Nature (London) 206: Drapeau, G. R., T. I. Matula, and R. A. MacLeod Nutrition and metabolism cf marine bacteria. XV. Relation of Na+-activated transport to the Na+requirement of a marine pseudomonad for growth. J. Bacteriol. 92: Florini, J. R., and C. S. Vestling Graphical determination of the dissociation constants for twosubstrate enzyme systems. Biochim. Biophys. Acta 25: Forsberg, C. W., J. W. Costerton, and R. A. MacLeod Separation and localization of cell wall layers of a gram-negative bacterium. J. Bacteriol. 104: Frank, L., and I. Hopkins Sodium-stimulated transport of glutamate in Escherichia coli. J. Bacteriol. 100: Gow, J. A., I. W. DeVoe, and R. A. MacLeod Dissociation in a marine pseudomonad. Can. J. Microbiol. 19: Kaback, H. R Transport across isolated bacterial cytoplasmic membranes. Biochim. Biophys. Acta 265: MacLeod, R. A The question of the existence of specific marine bacteria. Bacteriol. Rev. 29: MacLeod, R. A., P. Thurman, and H. J. Rogers Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis. J. Bacteriol. 113: Parker, R. P., and R. H. Elrick Cerenkov counting as a means of assaying a-emitting radionuclides, p In E. D. Bransom (ed.), The current status of liquid scintillation counting. Grune and Stratton, New York. 17. Reichelt, J. L., and P. Baumann Change of the name Alteromonas marinopraesens (Zobell and Upham) Baumann et al to Alteromonas haloplanktis (Zobell and Upham) Comb. nov. and assignment of strain ATCC 2382 (Pseudomonas enalia) and strain c-al of DeVoe and Oginsky to this species. Int. J. Syst. Bacteriol. 23: Schultz, S. G., and P. F. Curran Coupled transport of sodium and organic solutes. Phys. Rev. 50: Stock, J., and S. Roseman Sodium-dependent cotransport system in bacteria. Biochem. Biophys. Res. Commun. 44: Thompson, J., J. W. Costerton, and R. A. MacLeod K+-dependent deplasmolysis of a marine pseudomonad plasmolyzed in a hypotonic solution. J. Bacteriol. 102: Thompson, J., and R. A. MacLeod Functions of Na+ and K+ in the active transport of a- aminoisobutyric acid in a marine pseudomonad. J. Biol. Chem. 246: Thompson, J., and R. A. MacLeod Na+ and K+ gradients and a-aminoisobutyric acid transport in a marine pseudomonad. J. Biol. Chem. 248: Thompson, J., and R. A. MacLeod Specific electron donor energized transport of a-aminoisobutyric acid and K+ into intact cells of a marine pseudomonad. J. Bacteriol. 117: Wong, P. T. S., J. Thompson, and R. A. MacLeod Nutrition and metabolism of marine bacteria. XVII. Ion-dependent retention of a-aminoisobutyric acid and its relation to Na+-dependent transport in a marine pseudomonad. J. Biol. Chem. 244: Wong, J. T.-F., and C. S. Hanes Kinetic formulation for enzymic reactions involving two substrates. Can. J. Biochem. Physiol. 40: