NUTRITION AND METABOLISM OF MARINE BACTERIA'

Transcription

1 NUTRITION AND METABOLISM OF MARINE BACTERIA' XII. ION ACTIVATION OF ADENOSINE TRIPHOSPHATASE IN MEMBRANES OF MARINE BACTERIAL CELLS GABRIEL R. DRAPEAU AND ROBERT A. MAcLEOD Department of Bacteriology, Macdonald College of McGill University, Quebec, Canada Received for publication 18 February 1963 ABSTRACT DRAPEAU, GABRIEL R., (Macdonald College of McGill University, Quebec, Canada) AND ROBERT A. MAcLEOD. Nutrition and metabolism of marine bacteria. XII. Ion activation of adenosine triphosphatase in membranes of marine bacterial cells. J. Bacteriol. 85: Isolated membranes of two species of marine bacteria, a Pseudomonas and a Cytophaga, have been shown to possess adenosine triphosphatase activity. The optimal ph for enzyme action of both organisms was 8.8. The enzyme system was found to be capable of splitting inorganic o-phosphate from adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate, and inosine triphosphate but not from inorganic pyrophosphate. Mg++ was required for enzyme activity; with the Pseudomonas species, the optimal Mg++ to ATP ratio was 1:1. Ca++ could not replace Mg++. In the presence of the optimal concentration of Mg+, the enzyme system was further stimulated, nonspecifically, by a number of different salts. Maximal activation was achieved at an ionic strength of 0.3 to 0.4. No evidence of an adenosine triphosphatase specifically activated by a combination of Nat and K+ was obtained with either organism. No effect of ouabain on either the membrane adenosine triphosphatase activity or Nat transport by whole cells could be detected. The results suggest that the mechanism of ion regulation in marine bacterial cells is different from that in animal cells. 1 From a thesis submitted by Gabriel R. Drapeau in partial fulfilment of the requirements for the degree of Master of Science at McGill University. Presented in part at the Annual Meeting of the American Society for Microbiology, Kansas City, Mo., 6-10 May Issued as Macdonald College Journal Series no Skou (1957) reported the presence of an adenosine triphosphatase in the submicroscopic particles of crab nerve which was specifically activated by a combination of Na+ and K+. He concluded that the crab-nerve adenosine triphosphatase seemed to fulfill a number of the conditions that must be imposed on an enzyme thought to be involved in the active extrusion of Na+ from nerve fibers. Post et al. (1960) and Dunham and Glynn (1961) locatedanadenosinetriphosphatase in human erythrocyte membranes which was also specifically activated by Nat and K+. These workers compiled an impressive body of evidence linking Na+ and K+ transport in erythrocytes to the activities of this enzyme. Subsequently, ouabain-sensitive adenosine triphosphatases, specifically activated by a combination of Nat and K+, have been observed in a variety of animal cells and tissues (Bonting, Simon, and Hawkins, 1961). Adenosine triphosphatases have also been detected in the cytoplasmic membranes of bacterial cells. Abrams, McNamara, and Johnson (1960) studied the properties of an adenosine triphosphatase in isolated cell membranes of Streptococcus faecalis but did not report on the effects of monovalent ions on the activity of the enzyme. Weibull, Greenawalt, and Low (1962) found the enzyme in the cytoplasmic membranes of Bacillus megaterium but could not demonstrate its activation by Nat and K+. Since most terrestrial bacteria which have been examined require K+ but not Na+ for growth, one would not expect to find a transport system in the membranes of these cells dependent for activity on the presence of both Na+ and K+. Marine bacterial cells, on the other hand, require both Na+ and K+ for growth and metabolism (1IacLeod and Onofrey, 1957; MacLeod et al., 1958; Payne, 1960; Pratt and Happold, 1960). Since their 1413

2 1414 DRAPEAU AND MAcLEOD J. BACTERIOL. quantitative requirements for these ions are similar to those of animal cells in tissue culture, marine bacteria might be expected to transport the ions by a similar mechanism. Accordingly, efforts were made to determine whether an adenosine triphosphatase activated specifically by a combination of Na+ and K+ is present in the membranes of marine bacterial cells. MATERIALS AND METHODS Cultures. Two organisms were employed in this study. One, designated B-9, is a gram-negative rod which exhibits gliding motility and is capable of digesting agar. It is probably a member of the genus Cytophaga (MacLeod and Matula, 1962). The other, B-16, is a gram-negative, polarly flagellated rod best classified as a Pseudomonas. Both organisms require Na+ and K+ for growth (MacLeod and Onofrey, 1957). Media. The cultures were maintained by monthly transfer on slants of a medium containing 0.8% nutrient broth, 0.5% yeast extract, and 1.5%o agar in a salt solution consisting of 0.22 M NaCl, M MgCl2, 0.01 M KCI, M CaCl2, and 0.1 mm FeSO4(NH4)2SO4. The basal liquid medium employed contained the same constituents with the agar omitted and 0.5% potassium citrate added to reduce metal ion precipitation. Isolation of cell membranes. The organisms were grown in 250 ml of medium contained in 2-liter Erlenmeyer flasks incubated on a rotary shaker at 25 C for 16 to 18 hr in the case of B-16, 36 to 40 hr for B-9. The cells were harvested by centrifugation and washed three times by resuspension in and sedimentation from 200-ml volumes of 1.0 M NaCl. After the final sedimentation, the cells were resuspended in 250 ml of 0.5 M NaCl. The suspension was made 0.05 M with respect to tris(hydroxymethyl)aminomethane (tris) buffer (ph 8) and 4.3 mm with respect to ethylenediaminetetraacetic acid (EDTA). Lysozyme was added at a concentration of 100,g per ml, and the mixture was stirred slowly. After 1 hr, lysozyme was again added at a level of 100 ug per ml. Usually, by the end of the second hour all the cells could be observed by phase-contrast microscopy to have changed from rod forms to spheres. When the conversion was complete, the spheres were centrifuged from the suspending salt solution and resuspended in 200 ml of cold 0.1 mf tris buffer (ph 8.0). Lysis was instantaneous and complete. The disrupted spheres were separated from the soluble cytoplasmic fraction by centrifugation at 50,000 X g for 20 min at 4 C. The particulate material collected was washed five times by repeated centrifugation from and resuspension in 100-ml portions of cold solutions of either 0.05 M MgSO4 or 0.2 M NaCl. The choice of washing solution depended on whether monovalent or divalent cation requirements were to be investigated. Only trace amounts of protein could be detected after the fifth washing. The membrane fraction was finally suspended in 15 to 20 ml of either 0.05 M MgSO4 or 0.2 M NaCl. This suspension was divided into a series of 2-ml volumes which were frozen and stored at -20 C. To obtain a homogeneous suspension for enzyme studies, the samples were treated for 2 min in a sonic oscillator after thawing. Measurement of adenosine triphosphatase activity. Unless otherwise specified, the reaction mixture contained (in a final volume of 1.0 ml) 0.1 M tris (chloride) buffer (ph 9.0), 5 X 10-3 M adenosine triphosphate (ATP), 5 X 10-3 M MgSO4, 0.3 M KCl, and 0.1 ml of the enzyme preparation (containing about 0.50 mg of protein). After 30 min of incubation at 25 C, the reaction was stopped by adding an equal volume of 5% perchloric acid. The precipitated protein was removed by centrifugation. The inorganic phosphate (Pi) released was measured by the method of Fiske and SubbaRow (1925). Suitable corrections were made for any nonenzymatically released Pi present. Transport by whole cells. To determine the effect of ouabain on the transport of Na+, cells of B-16 were washed five times with 0.05 M MgSO4. Samples consisting of 2.5 g (wet weight) of cells in 7.5 ml of suspension were added to tubes with and without ouabain and incubated for 10 min. A solution of salts was then added to the suspensions to give the following concentrations in a final volume of 10 ml: NaCl, 200 mm containing about 0.1,uc of Na22; KCI, 10 mm; and MgSO4, 50 mm. After incubating for 30 min at 25 C, the cells were centrifuged from the suspending medium at 50,000 X g, and the supernatant liquid was discarded. The cells were resuspended in water, and the volume was adjusted to 5 ml. Radioactivity was measured in a scintillation well counter. More than 10,000 counts were recorded for each sample to reduce the probable error due to counting to less than 1 %.

3 VOL. 85, 1963 ADENOSINE TRIPHOSPHATASE IN BACTERIAL CELL MEMBRANES 1415 t. i f' A 76 A FIG. 1. lvhole cells (left), spherical forms (center), and cell membranes (right) of marine bacterium B-16 as seen with a phase-contrast microscope. The optical conditions were the same in all three photographs. Protein determination. Protein was estimated by the biuret method (Gornall, Bardawill, and David, 1949) with crystalline pepsin (Worthington Biochemical Corp.) as a standard. Chemicals. ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP), and inosine triphosphate (ITP) were obtained from the Sigma Chemical Co. as Na;+ salts. ATP was rendered Na±-free by passage through a Dowex 50W-X8 cation-exchange column in the tris buffer form. This treatment removed 99.9% of the Na+ as established by flame photometry. All inorganic salts used were of reagent-grade quality. Na" as NaCl was obtained from Atomic Energy of Canada Ltd. RESULTS Cell membrane preparations. Figure 1 is a photomicrograph showing whole cells, spherical forms, and membranes of B-16 as seen with a phase-contrast microscope. Membranes showed little contrast as compared with the spherical forms or the rod-shaped cells. Localization of adenosine triphosphatase. When the membrane and cytoplasmic fractions of cells of B-I16 were tested separately, both were found to possess adenosine triphosphatase activity (Table 1). Repeated washing of the membranes did not lower the activity of the membrane preparations, suggesting that the adenosine triphosphatase present was an intrinsic part of the membrane structure. Although the rate of release of Pi increased with increasing amounts of membrane preparation, the response was not quite linear (Fig. 2). Membranes prepared from organism B-9 also contained adenosine triphosphatase activity which could not be removed by repeated washing. TABLE 1. Relative capacity of membrane preparations and cytoplasm of marine bacterium B-16 to release Pi from adenosine triphosphate Preparation Pi liberated* Cell membranes Soluble cytoplasmic fraction * Expressed as j.amoles per mg of protein.

4 b~~~~~~~~~~~ 1416 DRAPEAU AND MAcLEOD J. BACTER1 OL 0.8 nearly 3 moles of Pi per mole of ATP present, indicating that ATP, ADP, and AMP must all 0.7 have been hydrolyzed. This w was confirmed by -J showing that each of the compounds could serve [ as a substrate in the system (Table 2). In addition, ITP was hydrolyzed but not inorganic 0.5 pyrophosphate. Membrane preparations of B-9 w had an identical substrate specificity..4 ir Mg++ activation. Figure 4 shows that when 5 w 0.3,umoles of ATP were present in the reaction system, dj maximal enzyme activity was obtained 0.2 with approximately 5,umoles of Mg++. When the ATP concentration was raised to 10,umoles, ,umoles of Mg+ gave an optimal response. Thus, the optimal Mg+ -ATP ratio for the membrane adenosine triphosphatase of marine bacterium PROTEIN ADDED (MG) B-1 6 is 1:1. Ca++ could not be substituted for FIG. 2. Effect of increasing concentrations of the Mg++ in this system. membrane preparation of marine bacterium B-16 on Monovalent cation effects. When the effect of the rate of release of inorganic phosphate (Pi) from monovalent cations on the membrane adenosine adenosine triphosphate. triphosphatase of B-16 was examined, any one of a number of salts was found capable of stimulating enzyme activity. Figure 5 shows the re-.1 TABLE 2. Substrate specificity of membrane J -i preparations of marine bacterium B Substrate tested Pi liberated am jsmoles J I -c X \tt ATP ADP AMP ITP '7 8.0 Pyrophosphate ph FIG Relation between ph and the rate of release of inorganic phosphate (Ps) from adenosine triphosphate by membrane preparations of marine -j bacterium B-16. Optimal ph of membrane adenosine triphosphatase. This was found to be about ph 8.8, as shown for B-16 (Fig. 3). The enzyme from B-9 had an almost identical optimum. Ammediol (2-amino-2-methyl-1,3-propanediol) buffer was chosen to obtain the ph response shown because of its high ph range (7.8 to 10). Since this buffer was found to be somewhat inhibitory for the enzyme, tris buffer was routinely used in the assay system. Substrate specificity. On extended incubation, the membrane preparations of B-16 released a: ax -j.: Mg** ADDED (UMOLES) FIG. 4. Relation between Mg++ concentration and the rate of release of inorganic phosphate (Pi) from adenosine triphosphate (A TP) by membrane preparations of marine bacterium B-16 at two levels of substrate.

5 VOL. 85, 1963 ADENOSINE TRIPHOSPHATASE IN BACTERIAL CELL MEMBRANES 1417 (0 I7 w 0.5A 04 I~~~~~. KCI 3. NoCI w 2. N H4CI 4. LiCI IONIC STRENGTH FIG. 5. Stinmulation of membrane adenosine triphosphatase of marine bacterium B-16 by various salts. sponse to the chloride salts of four monovalent cations. All four salts had a capacity to stimulate enzyme action. The sulfate salts of the same cations produced a similar response. This nonspecific capacity of a number of different salts to stimulate enzyme activity indicated an ionic strength effect. As Fig. 5 shows, optimal activity was obtained when the ionic strength due to the added salts was 0.2 to 0.3. Ionic strength contributed by the substrate, buffer, and Mg++ salt present was about Thus, the total ionic strength for optimal activity of the enzyme was 0.3 to 0.4. Effect of Nat plus K+. The effect of adding NaCl to a system already containing KCl was examined. Adding sufficient KCl to increase the ionic strength by 0.2 produced the expected increase in enzyme activity (Table 3). Adding more KCl had little further effect. The addition of NaCl to the system already activated by KCl produced no more effect than would be expected from the increase in ionic strength. Flame photometric analysis of the enzyme system without added NaCl showed only 2.9 X 10-i M Na+ present as contaminant. The enzyme system from B-9 responded in the same way as B-16 to combinations of Na+ and K+. Since no evidence for the presence in the isolated membranes of an adenosine triphosphatase specifically activated by a combination of Na+ and K+ was obtained, the possibility was considered that such an enzyme might be labile and hence destroyed during isolation of the membranes. Accordingly, washed suspensions of both B-9 and B-16 were disrupted by brief sonic treatment (5 min), and the complete mixture of membranes and cytoplasm was tested for adenosine triphosphatase activity. No evidence for an enzyme specifically activated by a combination of Na+ and K+ was found. Effect of ouabain. Post et al. (1960) showed that the ouabain concentration required for halfmaximal inhibition of the Na+- and K+-activated adenosine triphosphatase in human erythrocyte membranes was 10-7 M. Ouabain produced no inhibition of the membrane adenosine triphosphatase of either B-9 or B-16, even when tested at concentrations considerably in excess of those found to inhibit the enzyme in animal cells as shown in Table 4 with a membrane preparation from B-16. In human red cells, ouabain inhibits transport by preventing the efflux of Na+. For this reason, red cells suspended in NaCl containing labeled Na+ accumulate radioactivity if 10-6 to 10-8 M ouabain is present (Gill and Solomon, 1959). Cells of marine bacterium B-16, when suspended in solutions containing NaCl labeled with Na22, did not accumulate radioactivity in the presence of ouabain (Table 5). TABLE 3. Comparison of the effect of K+ and of combinations of Na+ and K+ on adenosine triphosphatase activity of membrane preparations of marine bacterium B-16 KCI Ionic strength NaCl P1 liberated itmoles TABLE 4. Effect of ouabain on the adenosine triphosphatase activity of membrane preparations of marine bacterium B-16 Ouabain added M Pi liberated 1jmoles X X X

6 1418 DRAPEAU AND MAcLEOD J. BACTERIOL. TABLE 5. Effect of ouabain on Na22 accumulation by whole cells of marine bacterium B-16 Ouabain added Radioactivity* M 0 5, X ,709 4: 43 3 X , X ,637 i 18 * Couints per min per g of wet cells. DISCUSSION The evidence indicates that one or more enzymes capable of releasing inorganic phosphate from ATP are present in the cell membranes of the two marine bacteria examined. No indication was found that this adenosine triphosphatase activity was due, even in part, to an enzyme specifically activated by a combination of Na+ and K+. Furthermore, no effect of ouabain either on membrane adenosine triphosphatase activity or on Na+ transport by the whole cells could be detected. Thus, no evidence of a link between the transport of Na+ and K+ and membrane adenosine triphosphatase activity was found in these marine bacterial cells. If the Na+-K+activated ouabain-sensitive adenosine triphosphatases in the membranes of animal cells are involved in ion transport, then the results reported here indicate that significant differences in the mechanism of ion regulation exist between marine bacterial cells and animal cells. The properties of the membrane adenosine triphosphatase of the two marine bacterial species were surprisingly similar and differed in a number of respects from those of membranes of human red blood cells (Post et al., 1960), B. megaterium (Weibull et al., 1962), S. faecalis (Abrams et al., 1960), and the Na+-K+-activated adenosine triphosphatase of crab nerve (Skou, 1957, 1960). The ph optimum of 8.8 for the adenosine triphosphatase activity of membranes of marine bacterial cells was appreciably higher than the ph 7.5 optimum reported for the adenosine triphosphatase of red blood cells. The activity of the enzyme from B. megaterium, however, increased as the ph increased, the highest level tested being ph 8.5. The adenosine triphosphatase of marine bacterium B-16 was activated by Mg+-+ but not by Ca+, whereas the enzyme from B. megaterium was activated by both these ions. The crab-nerve adenosine triphosphatase was activated by Mg"+ and inhibited by Ca++. The Mg-ATP ratio for maximal activity of the adenosine triphosphatase of marine bacterium B-16 was 1:1, the same as reported for S. faecalis but different from the ratio of 1:2 found for B. megaterium and of 2:1 observed for the crabnerve enzyme. The crab-nerve adenosine triphosphatase hydrolyzed ATP and ITP but not ADP. The enzymes of both B. megaterium and S. faecalis hydrolyzed ATP but not ADP or AMP. In contrast, the membrane preparations of the marine bacterial cells split Pi from all the nucleotides tested. Whether this was due to the action of one or of more than one enzyme was not established. ACKNOWLEDGMENT This work was supported by grant T 1112 from the National Research Council of Canada. LITERATURE CITED ABRAMS, A., P. MCNAMARA, AND F. B. JOHNSON Adenosine triphosphatase in isolated bacterial cell membranes. J. Biol. Chem. 235: BONTING, S. L., K. A. SIMON, AND N. M. HAWKINS Studies on sodium-potassium activated adenosinetriphosphatase. Arch. Biochem. Biophys. 95: DUNHAM, E. T., AND J. M. GLYNN Adenosine triphosphatase activity and the active movements of alkali metal ions. J. Physiol. 156: FISKE, C. H., AND Y. SUBBAROW The colorimetric determination of phosphorus. J. Biol. Chem. 66: GILL, T. J., AND A. K. SOLOMON Effect of ouabain on sodium flux in human red cells. Nature 183: GORNALL, A. G., C. J. BARDAWILL, AND M. M. DAVID Determination of serum protein by means of the biuret reaction. J. Biol. Chem. 177: MAcLEOD, R. A., C. A. CLARIDGE, A. HORI, AND J. F. MURRAY Observations on the function of sodium in the metabolism of a marine bacterium. J. Biol. Chem. 232: MAcLEOD, R. A., AND T. MATULA Nutrition and metabolism of marine bacteria. XI. Some characteristics of the lytic phenomenon. Can. J. Microbiol. 8:

7 VOL. 85, 1963 ADENOSINE TRIPHOSPHATASE IN BACTERIAL CELL MEMBRANES 1419 MACLEOD, R. A., AND E. ONOFREY Nutrition and metabolism of marine bacteria. III. The relation of sodium and potassium to growth. J. Cell. Comp. Physiol. 50: PAYNE, W. J Effects of sodium and potassium ions on growth and substrate penetration of a marine pseudomonad. J. Bacteriol. 80: POST, R. L., C. R. MERRITT, C. R. KINSOLVING, AND C. D. ALBRIGHT Membrane adenosine triphosphatase as a participant in the active transport of sodium and potassium in the human erythrocyte. J. Biol. Chem. 235: PRATT, D., AND F. C. HAPPOLD Requirements for indole production by cells and extracts of a marine bacterium. J. Bacteriol. 80: SKOU, J. C The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim. Biophys. Acta 23: SKOU, J. C Further investigations on a Mg++ + Na+-activated adenosintriphosphatase, possibly related to the active linked transport of Na+ and K+ across the nerve membrane. Biochim. Biophys. Acta 42:6-23. WEIBULL, C., J. W. GREENAWALT, AND H. Low The hydrolysis of adenosine triphosphate by cell fractions of Bacillus megaterium. I. Localization and general characteristics of the enzymic activities. J. Biol. Chem. 237: