Functions of Na+ and K+ in the Active Transport of a ...

THE JOURNAL OF ~~X.OQICAL CHEMISTRY

Vol. 246, No. 12, Issue of June 25, PP. 4066-4074, 1971 Printed in U.S.A.

Functions of Na+ and K+ in the Active Transport of

a=Aminoisobutyric Acid in a Marine Pseudomonad*

(Received for publication, February 1, 1971)

J. THOMPSON AND ROBERT A. MACLEOD

From the Department of Microbiology, Macdonald College of McGill University, and Marine Sciences Center, McGill University, Montreal, Canada

SUMMARY

When cells of the marine pseudomonad B-16 (ATCC 19855) grown in nutrient broth-salts medium containing 0.01 M KCl, were washed in a complete salts solution con- sisting of 0.3 M NaCl, 0.05 M MgS04, and 0.01 M KC1 the intracellular K+ concentration was approximately 0.3 M

and the cells rapidly accumulated cw-aminoisobutyric acid- 14C (AIBJC). Cells washed with 0.05 M MgS04 solution alone lost some 95 to 98% of the total intracellular K+. Transport studies revealed that K+-depleted cells resus- pended in salts solution containing 0.3 M NaCl, 0.05 M

MgS04, and 0.05 M Tris-HCI buffer (pH 7.2) failed to ac- cumulate AIBJ4C. Penetration studies under these con- ditions showed that a-aminoisobutyric acid (AIB) neverthe- less entered the cells, and occupied the total available fluid space in a packed cell preparation of K+-depleted cells. Entry of AlB was Naf dependent, and equivalent concentra- tions of Li+ or I(+ failed to replace this cation in mediating AIB penetration and equilibration. Addition of K+ to the equilibrated system resulted in rapid accumulation of the substrate. The rate of AU3 accumulation was maximal at 0.01 M KC1 and kinetic analysis revealed that K+ increased V max but did not change the K, for transport. Additions of adenine nucleotides further increased the rate of AIB accumulation in the presence but not in the absence of K+. When K+-depleted cells were again loaded with K+ and subsequently transferred to AIB uptake medium lacking K+, there was immediate and rapid accumulation of sub- strate. Active transport of AIB is apparently mediated by two distinct cation-dependent steps. The first stage in- volves the entry and equilibration of AIB into the cells by a Na+-dependent facilitated diffusion mechanism. In the K+-dependent second stage, the cation, acting at the intra- cellular level, brings about the accumulation of AIB against a concentration gradient.

The feature which distinguishes gram-negative marine bac- teria most clearly from their terrestrial counterparts, is the strict

* This work was presented in part at the 70th Annual Meeting of the American Society for Microbiology, Boston, Massachusetts, TJnited States of America, April 26 to May 1, 1970.

dependence of the marine species upon the presence of Na+ in the environment for optimum growth and metabolism (l-5). Drapeau and MacLeod (6) and Drapeau, Matula, and MaoLeod (7) have shown that Naf is required for the transport of (Y- aminoisobutyric acid and D-fUCOSe into cells of a marine pseudo- monad. In a recent publication from this laboratory (8) we were able to show that protoplasts, produced by sequential removal of the four outer layers of the cell envelope, have the same capacity to transport AIB-i4Ci as whole cells. These studies showed that the amino acid transport system was located in, or that it formed an integral part of, the cytoplasmic mem- brane of the pseudomonad. Kinetic analysis of the Na+-de- pendent amino acid transport process (9) suggests that the role for Na+ is to increase the affinity of a carrier or binding protein for the molecule which is to be transported. In this respect, Na+ functions in a manner similar to that proposed for Naf-dependent amino acid and sugar transport mechanisms in mammalian cells (10-13).

It has long been known that while the concentrations of Naf and Kf in the environment can markedly affect both the rate and extent of growth of the marine bacteria examined, only about one-hundredth as much K+ as Na+ is required (1). Tom- linson and MacLeod (14) showed that both Na+ and K+ were essential for the oxidation of exogenous substrates by a marine pseudomonad, and Pratt and Happold (15) showed a specific requirement for Naf and Kf for indole production from trypto- phan by a marine vibrio. Rhodes and Payne (16) concluded that K+ in addition to Na+ was required for the induction of a penetration mechanism in another marine pseudomonad, while Drapeau and MacLeod (6) and Drapeau et al. (7) showed that both K+ and Na+ were required for the transport of AIB into cells of marine pseudomonad B-16.

While the function of Na+ in amino acid transport in the marine pseudomonad has been reasonably well documented, participation of K+ in this process has received considerably less attention. In addition to its role in solute transport Na+ was also found to be capable of preventing the release of intracellu- lar solutes from the cell by interaction with, and controlling the porosity of, the cytoplasmic membrane of B-16 (17). Cells of the marine organism, like many of their terrestrial counterparts

(lf9, accumulate K+ against a considerable concentration gradient. When washed in 0.05 M MgS04 solution, the cell membrane becomes permeable to, and the cells are rapidly

1 The abbreviation used is: AIB, a-aminoisobutyric acid.

4066

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depleted of, intracellular K+. At the same time they undergo morphological changes which are indistinguishable from those resulting from classical plasmolysis (19). In the present study we have taken advantage of the fact that we can produce physio- logically active, K+-depleted cells to gain a better understanding of the roles of Na+ and K+ in the transport of AIB into the cells. The functions of Na+ and K+ in amino acid transport in the pseudomonad have been examined and compared with the pro- posed roles of these same ions in transport in mammalian and other bacterial systems.

EXPERIMENTAL PROCEDURE

Orga?zism-Designated B-16, the marine pseudomonad used in these investigations was originally isolated from a marine clam, and has been classified by the Torry Research Group, Aberdeen, Scotland as a Pseudomonas sp. type IV. The or- ganism, which has been deposited in the American Type Culture Collection (ATCC 19855) is also maintained by the Torry Research Station where it is listed as NCMB 19. Studies on the nutrition and metabolism of the marine pseudomonad have been reported in detail in previous communications, including the last paper in this series (19).

Culture Maintenance-The culture was maintained by monthly transfer on slants of a medium containing 0.8% nutrient broth (Difco); 0.5% yeast extract (Difco); and 1.5% agar in a salt solution consisting of 0.22 M NaCl, 0.026 M MgCl2, 0.01 M KCI, and 0.1 mM FeS04(NH&S04.

Growth Conditions-The marine pseudomonad was grown in the same medium as used for culture maintenance, except that the agar was omitted. Cells in. the logarithmic phase of growth were obtained with a serial transfer system which was described previously (8).

Preparation of K+-depleted Cells-Cells were harvested from the growth medium by centrifugation at 16,000 x g at 4” for 10 min, and washed three times by resuspension in and centrifuga- tion from volumes of 0.05 M MgS04 solution equal to the volume of the growth medium. Previous studies (19, 20) have shown that this procedure results in the loss of approximately 98% of the total intracellular Kf from the cells.

Transport Studies with Millipore Filtration Techniques--K+-de- pleted cells were suspended at a final concentration of 100 pg, dry weight, per ml in a basal uptake medium of IO-ml volume, containing (unless otherwise specified) 0.3 M NaCl, 0.05 M MgSOe, 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.2), (Triama HCI, Sigma), and 1.5 X 10m4 M AIB-l-l% (spe- cific activity 0.22 $Zi per pmole). Further additions of K+ or monovalent, cations were added as desired.

In kinetic studies increased K+ concentrations were added to the same system as described above except that the specific activity of AIB-l-14C was increased to 0.88 &i per I.cmole. The initial rate of AIB uptake was determined from the amount of radioactivity taken up during the first lo-min incubation period, since over this interval uptake was linear with time. The velocity of uptake, I’, was expressed as the number of micro- moles of AIB accumulated per min per 100 pg of cells, dry weight.

Measurement of AIB Uptake and Radioactivity Determination- The methods used to measure the amount of radioactivity and hence AIB taken up by the cells have been described in det,ail in a previous communication (9).

Determination of Glycerol, Sucrose, In&n, and A/B Spaces

The percentage of the total available fluid volume in packed cell preparations occupied by glycerol-14C, sucroseJ4C, inulin- 14C, and AIB-14C was determined with procedures described by Buckmire and MacLeod (21), with the exception that the packed cell pellet obtained by high speed centrifugation was resuspended in 0.05 M MgS04 solution to ensure release of any radioactive compounds which had penetrated into the cell. The buffered salts medium used in the penetration studies had the following basal composition: 0.3 M NaCl, 0.05 M MgSOd, 0.05 M Tris-HCI (pH 7.2), and radioactive materials at a final concentration of 3 mM except in the case of inulinJ4C which, because of solubility problems, was added to give a final concen- tration of approximately 0.03%. In the penetration studies Kf-depleted cells were suspended in 10 ml of incubation medium at a concentration of 12 to 15 mg, dry weight, per ml, which after high speed centrifugation yielded a packed cell pellet of 0.5 to 0.7 g, wet weight. Some of the penetration systems contained 0.01 M NaCN or KCN and the reasons for such additions are discussed more fully in the text.

Radioactive Materials-Inulin-carboxyl-14C, sucroseJ4C, glycl erolJ4C, a-aminoisobutyric acid-lJ4C, adenosine-8-14C-5’-tri- phosphate (ATP), and adenosine-8J4C-5’-diphosphate (ADP) were obtained from New England Nuclear.

Electron Microscopy---Cells were harvested in the logarithmic pha,se of growth. The solution used for the fixation procedure had the following composition: 5% glutaraldehyde, 0.2 M so-

dium potassium phosphate buffer (pH 6.2), and complete salts (0.3 M NaCl, 0.05 M MgSO+ 0.01 M KCl). One volume of the fixative was added to 9 volumes of the cell suspension and preliminary fixation was permitted for 30 to 60 min at room temperature. The previously fixed cells were centrifuged, enrobed in agar, and were then transferred to the fixative solu- tion for a further a-hour period. The cores were washed five times in phosphate buffer containing the salts shown above, and then were postfixed in the same solution containing 2Y, osmium tetroxide for 1 hour. The preparations were washed five times in Verona]-acetate buffer (pH 6.2) and then were stained for 1 hour in a 1% aqueous solut,ion of uranyl acetate. The cores were washed five times in Verona]-acetate buffer, dehydrated through an acetone series, and finally embedded in Vestopal. Sections were cut with a Porter-Blum MT 2 ultra- microtome and were stained with uranyl acetate and lead cit- rate. Thin sections were examined and photographed with an AEI EM-6B electron microscope.

RESULTS

Preparation of K+-depleted Cells

Fig. 1 shows an electron micrograph of the marine pseudo- monad. The organism has the characteristic envelope of gram- negative bacteria in having two outer double track structures, there being an outer double track or wall layer (O.L.), and an inner double track which is the cytoplasmic membrane (C.X.). Also illustrated in the figure are the distribution and concentra- tions of the major physiologically active cations. While Na+ freely equilibrates across the cytoplasmic membrane, there is ,a considerable accumulation of K+ against .a concentration gra- dient (19, 22). It has been found (19) that the intracellular K+ concentration approximates to 0.3 M when the cells are grown in growth medium which contains only 0.01 M KCI. Exposure of the cells to a solution of 0.05 M MgSO4 alone, results in im-

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Amino Acid Transport in a Marine Pseudomonad Vol. 246, No. 12

. ,.

‘.” ,

3.

,:

+/ L1 2, I I I !

0 60 SO 100 120

INCUBATION PERIOD hrn.,

FIG. 2. AIB-1% uptake by K+-depleted cells in the presence of: 1, buffered complete salts solution; 0.3 M NaCl, 0.01 M KCl, 0.05 M MgS04, and 0.05 M Tris-KC1 buffer (pH 7.2); R, buffered salts minus NaCl; and 5, buffered salts minus KC1 until 60-min incuba- tion period when KC1 was added to a final concentration of 0.01 M.

mediate porosity changes within the cytoplasmic membrane and concomitant loss of about 98% of the total intracellular K+ as well as other low molecular weight solutes (9, 17, 20). These Kf-depleted cells were used throughout the current investigation.

Na+ and K+ Requirements for AIB Transport in K+-depleted Cells

The results in Fig. 2 (Plot I) show that K+-depleted cells, resuspended in a buffered complete salts solution containing 0.05 M Tris-HCl (pH 7,2), 0.3 M Na+, 0.01 M K+, and 0.05 M

WP, rapidly accumulate AIB. In the absence of either Na+ or K+ (Plots d and 3, respectively) no accumulation of substrate was observed. The subsequent addition of K+ to medium already containing Naf, was accompanied by rapid uptake of the substrate. These observations confirm previous findings by Drapeau et al., (7), which showed that Na+ and K+ must be present together in the medium for active transport of substrate to occur in Mg++-washed cells.

Inability of Other Monovalent Cations to Replace K+

Other monovalent cations were added to buffered complete salts uptake medium lacking K+, and uptake of AIB was fol- lowed. The results presented in Table I show that of the monovalent cations studied, namely, Li+, NHd+, Rb+, Cs+, and Kf, only the latter was effective in the restoration of the active transport process.

Effect of Increased Extracellular K+ Cancentration on Rate of AIB-14C Uptake

The effect of varying the external K+ concentration on the initial rate of AIB uptake was investigated. Kinetic analysis <

FIG. 1. Electron micrograph of a thin section of a whole cell of the marine pseudomonad B-16. Note the cell envelope which is composed of two double track structures: outer-double track layer (O.L.); and the inner-double track, the cytoplasmic membrane (C.M.) X 91,500. Also shown are the distributions of Na+ and K+ ions across the cytoplasmic membrane, and the relative pene- trability of inulin, sucrose, and glycerol into the cell. The bar on this electron micrograph equals 0.1 nm.

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Issue of June 25, 1971 J. Thompson and R. A. MoxLeod 4069

TABLE I

Effect of monovalent cations on AIB-W uptake by K+-depleted cells of marine pseudomonad B-16

Cations were added to a basal medium containing 0.3 M NaCl, 0.05 M MgSO,, and 0.05 M Tris buffer (pH 7.2). Final concentra- tion of added cations = 0.01 M.

Cation added

None............................ Rbf cs+.............................. K+ Li+... _................._....... NH,+. .

-

.-

-

Incubation period

20 miu 1 40 mill 1 60min

352 342 352

5,004 325 296

CM 522 490 522

9,247 419 448

652 704 630

11,359 519 496

I 1’. ” I ’ I I ! I 0.4 0.2 0.2 04 0.6 0.6 I.0

-- +- I 7 md

FIG. 3. Lineweaver-Burk plot of the effect of increased external K+ concentrations on the initial rate of uptake of AIB.

of the results showed that at the optimal Na+ concentration of 0.3 M, the rate of AIB uptake as a function of the K+ concen- tration in the medium exhibited Michaelis-Menten kinetics. The Lineweaver-Burk plot of the results (Fig. 3) indicated that the K,,,, the external concentration of K+ which gave half- maximal rate of AIB uptake, was approximately 5.5 X 1OW M.

Kinetic Analysis of K+-stimulated Uptake

Kinetic analysis of the K+-stimulated active transport of AIB was investigated in experiments in which both Kf and AIB concentrations were varied simultaneously in systems con- taining 0.3 M NaCl, 0.05 M Tris-HCI buffer (pH 7.2), and 0.05 M MgS04. A double reciprocal plot of the data (Fig. 4) shows that increasing Kf concentrations while increasing V,,, does not change the K, of the transport process. The latter re- mained constant at 2.2 X 10ms M.

Effect of Preliminary Incubation in Presence of Kf upon Initial Rate of AIB Uptake

The rate of AIB uptake by cells of the marine pseudomonad was proportional to the K+ concentration in the suspending medium if the AIB and K+ were added to the cell suspension

FIG. 4. Lineweaver-Burk plot of the results of a kinetic experi- ment showing the effect of simultaneous variation of external K? and AIB concentrations upon the initial rate of AIB uptake by K+-dedeted cells. Experimental conditions are described under “Mat&ials and Metho&.”

b 4

k CONCENTRATION p&

FIG. 5. Effect of increased K+ concentration upon AIB uptake by K+-depleted cells. The cross-hatched bars illustrate uptake during a 15-min incubation period, and the solid black bars show AIB accumulation during 15-min incubation by cells which had been previously incubated in the medium for 30-min prior to addi- tion of substrate (AIB). The basal salts medium contained 0.3 M NaCl, 0.05 M MgSO,, and 0.05 M Tris-HCl buffer, pH 7.2.

together. If, however, the cells were initially incubated for 30 min in the presence of varying K+ concentrations before addition of substrate, the rate of AIB uptake was no longer proportional to the external K+ concentration. In fact, it was found (Fig. 5) that after preliminary incubstion the rate of AIB uptake was essentially maximal at the lowest level of Kf tested.

Localization of Site of K+ Action

The kinetic studies concerning the Kf stimulation of active transport did not indicate the site of action of the ion, i.e. whether internal or external to the cytoplasmic membrane. In a previous publication (19), it has been shown that Kf-

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Amino Acid Transport in a Marine Pseudomonad Vol. 246, No. 12

0 , ~-~-~---~-------- ,---_2----o---

0 I I 1 20 40 60

INCUBATION PERIOD (min.) -

FIQ. 6. Capacity of Kc-depleted or K+-preloaded cells to take up AIB in the absence of external Kf. The experimental pro- cedure is described in the text, and AIB uptake was followed when either K+-loaded (1) or K+-depleted (2) cells were resuspended in buffered salts solution containing 0.3 M NaCl, 0.05 M MgSO,, and 0.05 M Tris-HCl, pH 7.2.

depleted cells have a residual internal K+ concentration approximately 0.004 M. After 30-min preliminary incubation in presence of 0.01 M K+, the intracellular K+ level is restored to the level found in normal cells (0.3 M). This marked ac- cumulation of K+, together with the observation that after preliminary incubation, AIB transport is no longer dependent on the external K+ concentration (Fig. 5) suggested that the role of K+ in active transport of AIB might be at the intracellular level. Further evidence for this postulate was obtained in a subsequent experiment. A suspension of Ktdepleted cells was divided into two parts, the cells in each were centrifuged, and the supernatant fluids were removed. The cells in Frac- tion 1 were resuspended in a complete salts buffer system, while those in Fraction 2 were suspended in an identical medium except for the omission of K+. Both systems were incubated for 30 min and the cells were then centrifuged from the sus- pending medium. The supernatant fluids were removed and the cells were washed by resuspension in a buffered solution containing 0.3 M NaCl and 0.05 M MgS04. The cells were resuspended and centrifuged from fresh washing solution twice more. Cell samples from Fractions 1 and 2 were then assayed for their capacity to transport AIB-i4C in the standard uptake medium lacking K+. The results of the experiment (Fig. 6) showed that only cells from Fraction 1, that is, those cells initially incubated with Kf and containing a high internal Kf concentration, had the capacity to accumulate the substrate in the absence of extracellular K+.

Penetration Studies

As previously stated, when cells of the marine pseudomonad are suspended in a solution of 0.05 M MgS04, there is an imme- diate loss of accumulated intracellular solutes such as AIB-14C and K+ from the cells. It seemed logical to believe that under these conditions AIB would equally readily diffuse into the cells since the semipermeability properties of the cytoplasmic membrane had been lost. This possibility was investigated by comparing the penetrability of AIB into cells of the marine pseudomonad with that of inulin, sucrose, and glycerol. Previ- ous studies (21) in this laboratory have led us to conclude

= Complete 100 ---p ____ --- __ _ __ ---Penetration

s

SUCROSE &

FIQ. 7. Relative capacities of AIB-r4C, inulin-*4C, and sucrose- 1°C and alvcerolJ4C to OCCUDV the available fluid snace in nacked

-1 * I

cell preparations of K+-depleted cells, suspended in a solution containing 0.05 M MgSO, and 0.05 M Tris-HCl buffer (pH 7.2). All compounds were present at 3 mM, except inulin which was ap- proximately 0.030/0. T.A.F.V., total available fluid volume.

that inulin is unable to penetrate the cell wall of this organism, sucrose can penetrate to the level of the cytoplasmic mem- brane, and glycerol can cross the cell membrane and occupy the total available fluid space in a packed cell preparation (see Fig. 1). With these compounds in their radioactive forms it was found (Fig. 7) that inulin-14C, sucrose-14C, and glycerol-i4C occupied 58 f 1.7, 71 f 0.8, and 107 f 7.2% of the total available fluid volume in cell pellets obtained by centrifugation of thick suspensions of K+-depleted cells. Surprisingly how- ever, AIB occupied a volume of only 71 f 0.8’%, similar to that occupied by sucrose and thus is apparently unable to penetrate the cytoplasmic membrane of cells suspended in Tris-buffered 0.05 M MgS04 solution.

Naf-dependent Penetration of AI&It was found however that when 0.3 M Na+ was added, AIB occupied 166 f 10.8% of the total available fluid volume suggesting that in the pres- ence of Naf, AIB can. penetrate the cytoplasmic membrane (Fig. 8 (I)). The value of 166% is rather more than the 100% figure expected if entry and equilibration had taken place. Since we know that there is 0.004 M residual K+ in Kf-depleted cells (lQ), it was thought that! this K+ might lead to some accumulation of AIB and hence could account for the penetra- tion value exceeding 100%. When 0.01 M NaCN was added to identical systems to prevent energy generation, the accumu- lation component was reduced and a penetration value of approximately 118% was obtained (Fig. 8 (2)). The results of penetration studies with cells of the marine pseudomonad which had been washed in complete salts are shown in Fig. 8 (3). In these cells, which contain their normal high K+ concen- tration, the presence of Naf results in an AIB penetration figure many times greater than the theoretical 100% value, indicating that accumulation of the substrate had occurred.

Specij’icity of Na+ Requirement for AIB Penetration

Several experiments were conducted in which K+-depleted cells were resuspended in Tris-buffered salts-cyanide solution containing 0.3 M concentrations of either Ii+, K+, or Na+. The penetrability and volumes occupied by inulin, sucrose, glycerol, and AIB were determined. The results (Fig. 9) show that only in the presence of Na+ was AIB able to enter

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Issue of June 25, 1971 J. Thompson and R. A. MacLeod 4071

the cell. In the presence of equivalent concentrations of Li+ or K+ the substrate could only occupy a volume similar to that occupied by sucrose indicating penetration only to the level of the cytoplasmic membrane.

Nature of Na+ Requirement for AIB Entry

Competition Xtudies-Competition studies were performed to determine whether the Naf-mediated penetration of AIB into the cells was stereospecific and hence possibly involved a

1220 -4

carrier mechanism. Various amino acids, structurally related to AIB have been shown to inhibit uptake of AIB by whole cells of this organism (7). Such amino acids were examined for their capacity to prevent Na+-dependent penetration of AIB into the total available fluid space in a packed cell prepara- tion and the results are shown in Fig. 10. In the presence of high concentrations of threonine, glycine, alanine, serine, and AIB-12C, the radioactive AIBJ4C could only occupy about 80% of the total available fluid volume. The branched chain com- pounds, valine and isoleucine, did not interfere with AIB-‘*C penetration.

1200 1.

m

f

, , 160 m

100 -----_-- --- - - -_ -- Complete

-_ ----penatrot1on

Cont. &SER. ALA. GLY THR. VAL ILEU --- - -. - +---I* l----22----1 +3---+

FIG. 8. Relative capacities of the different radioactive com- pounds to occupy the total available fluid space in packed cell pellets of: f, K+-depleted cells; 2, K+-depleted cells plus cyanide; 3, complete salts washed cells. All systems contained: 0.3 M Na+, 0.05 M Mg++, 0.05 M Tris-HCl buffer (pH 7.2), except 2 which also contained 0.01 M NaCN. C.P., complete penetration; C.M., cell membrane; C.W., cell wall; T.A.F.V., total available fluid volume.

6 I ffj 60 F 2 s 40

8

3 20

FIG. 10. Effect of the presence of various amino acids on the capacity of AIB to penetrate and occupy the available fluid in the pellet obtained from a thick suspension of K+-depleted cells. The incubation medium contained 0.3 M NaCl; 0.05 M MgSO,; 0.05 M Tris-HCl buffer (pH 7.2); and AIB-“C, 3 mM. The amino acids were added at a concentration of 50 mM. Incubation period was 10 min prior to ultracentrifugation of the thick cell suspension. Control system contained only AIBJ4C. T.A.F.V., total available fluid volume.

Complete --_----- --------- Penetration

;E q 5

z E = q - -- -E- -- -- --=--- -To Cell

i Membrane =

--- - ----

AIB GLY SUC IN -* L -*L 0

Fro. 9. Relative capacities of AIBJ4C, inulinJ4C, and sucrose- HCl buffer (pH 7.2) together with the penetrants in radioactive 14C and glycerol-‘% to occupy the available fluid in packed cell form. Where indicated, Na+, Li+, or K+ as the chloride salt was pellets of K+-depleted cells. The cells were suspended in a basal included in the basal medium at 0.3 M final concentration. medium containing 0.05 M MgSOa, 0.01 M NaCN, and 0.05 M Tris- T.A.F.V., total available fluid volume.

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4072 Amino Acid Transport in a Marine Pseudomonad Vol. 246, No. 12

E$ect of Low Temperature on Na+-dependent AIB Penetration- A further argument against a Na+-dependent passive diffusion theory of AIB penetration into the cell was indicated when lowering of the temperature of the incubation medium to 0”, resulted in the AIB occupying only 71% of the total available fluid volume even in the presence of optimum Na+ concentra- tion.

Stimulation of AIB Uptake by Adenine Nuxleotides

Scarborough, Rumley, and Kennedy (23), observed that the rate of p-gala&se transport was greatly reduced in cells of Escherichia coli ML-308 treated so as to deplete them of intra- cellular K+ and nucleotides. Addition of ATP to such cells in the absence of K+ increases the rate of transport. When K+ and ATP were added together the rate of O-nitrophenyl- fl-n-galactoside transport was depressed while the level of ac- cumulation was increased. Since we were also dealing with K+-depleted cells, it was felt that nucleotides might also play a

,103 I I I I

IO-

0 20 40 60 80

INCUBATION PERIOD (min.1

FIG. 11. Uptake of AIB by K+-depleted cells in the presence or absence of K+ and adenine nucleotides. The basal incubation medium contained: 0.3 M NaCl, 0.05 M MgSOr and 0.05 M Tris- HCl buffer (pH 7.2). The adenine nucleotides (0, AMP; a, ADP; and 0, ATP) were present at 1 X 1O-3 M concentrations, and the pH of these systems was adjusted to pH 7.2 by addition of solid Tris.

TABLE II Penetration of adenine nucleotides into a packed cell pellet

of K’-depleted cellsa

Radioactive pen&ant Percentage of occupation of T.A.F.V. in packed cell pelletb

Inulin . Sucrose............. Glycerol................ ADP (lm~)........... ATP (1 mM).

63 i 2.0 77 f 1.0

110 i 2.0 95 * 0.5 94 I 1.0

0 The K+-depleted cells were resuspended at a final coneentra- tion of 12 to 15 mg, dry weight, per ml in 10 ml of incubation me- dium containing the radioactive compounds, 0.3 M NaCl, 0.05 M

MgSOb, 0.01 M NaCN, 0.01 M KCl, and 0.05 M Tris-HCl buffer (pH 7.2). The nucleotide solutions were adjusted to pH 7.2 with NaOH solution before use.

b The figures represent the average and average deviations of the results of at least three separate experiments. T.A.F.V. = total available fluid volume.

role in the AIB transport process. Fig. 11 shows the results of an experiment in which adenine nucleotides were added to incubation media in the presence and absence of K+. Addition of any of the three nucleotides AMP, ADP, and ATP to the system resulted in more rapid uptake of AIB than in the control system containing only K+. In the absence of K+ the cells did not take up AIB whether nucleotides were present or not.

Penetration Studies with ADPJ4C and ATP-“C

The data in Fig. 11 suggest that the maximal rate of uptake of AIB is the result of a synergistic action of K+ and adenosine phosphate but gives no information as to the site of action of the nucleotides. Table II shows the results of penetration experi- ments with ADP-‘4C and ATP-‘4C, and it appears that both compounds can penetrate the cytoplasmic membrane.

DISCUSSION

The marine pseudomonad is grown on a synthetic sea water- nutrient broth medium in which the Kf concentration is approxi- mately 0.01 M. When cells are washed with complete salts solution (0.3 M NaCI, 0.05 M MgS04, and 0.01 M KCI), the intracellular Kf concentration approaches 0.3 M. The marine bacterium, in common with most cell forms (18), has therefore the capacity to accumulate K+ against a considerable concen- tration gradient. Quantitative analysis has also shown (22) that Na+ freely equilibrates across the cytoplasmic membrane, and the distributions of both Na+ and K+ under normal physio- logical conditions are illustrated in Fig. 1.

Previous studies with cells of the marine pseudomonad in which the intracellular K+ concentration had been maintained at 0.3 M, were primarily concerned with the role of Na+ in the active transport of AIB into the cell (6, 7, 9). These investiga- tions have revealed two major functions of Na+ in cellular metabolism. One is to mediate the uptake of AIB by the or- ganism, and the second function is to prevent leakage of the substrate once inside the cell.

When cells are washed with a solution of 0.05 M MgSO4, that is in the absence of Na+, the cytoplasmic membrane becomes instantaneously permeable and although the cells remain intact, some 95 to 98% of the total intracellular Kf is lost (19, 20). A preliminary study (7) revealed that Kf, in addition to Naf, was now required for AIB uptake, and in the present report we show that this requirement for K+ for AIB accumulation can only be detected in K+-depleted cells.

Control (Ktcontaining) cells, resuspended in buffered solu- tion containing 0.3 M Na+ and 0.05 M Mg*, rapidly accumulate AIB from the medium. K+-depleted cells under identical conditions did not take up the amino acid. If however, 0.01 M

Kf was added to the system, there was rapid accumulation of the analogue. This restoration of active transport was specifi- cally caused by Kf ion, and other monovalent cations such as Rb+ and Cs+, which in some metabolic reactions can partially spare or replace K+, were without effect upon the transport process. A previous communication (19) showed that when Kf-depleted cells were resuspended in buffered complete salts containing K+, the latter ion could be reaccumulated from the medium to normal intracellular concentrations. We have been able to show in the present report that these cells can now con- centrate AIB in the absence of extracellular K+, and this ob- servation clearly shows an intracellular role for K+ in AIB up- take by the marine pseudomonad.

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Issue of June 25, 1971 J. Thompson and R. A. MacLeod 4073

Transport studies with thin cell suspensions of K+-depleted cells suspended in the presence of Na+, showed that no ac- cumulation of AIB had occurred. The experiments gave no indication as to whether or not AIB had actually entered the cell. The thick cell suspension technique was used to answer this important question, and comparison of the penetrabilities of AIB, inulin, sucrose, and glycerol into Kf-depleted cells showed that AIB could penetrate the cytoplasmic membrane under these conditions. Thus penetration and equilibration of AIB required the presence of Na+. Equivalent concentrations of KCI, or LiCl failed to substitute for Na+ for entry. In the absence of cyanide, and in the presence of Na+, little accumula- tion of AIB occurred in the K+-depleted cells as evidenced by the fact that AIB occupied only slightly more than 100% of the total available fluid volume in the packed cell pellet. That this slight accumulation was caused by the presence of residual K+ in the cells, was indicated by the enormously greater per- centage of penetration into cells which had been washed in complete salts and which contained the normally high intra- cellular K+ level.

A characteristic feature of mammalian cells is their ability to effect an asymmetric distribution of Na+ and K+ ions across the cell membrane. It is believed that the distribution of the ions against their electrochemical gradients is achieved by con- tinued operation of the (Na+ + K+)-activated ATPase complex often called the ‘Na+ pump’ (24). Furthermore a close correla- tion between Na+ and solute transport in these tissues has long been known (25). In the model for Na+-dependent solute transport proposed by Crane (11) and Crane, Forstner, and Eichholz (12), the potential energy of the inwardly directed, downhill Naf gradient produced by the ATPase, indirectly provides the energy necessary for active transport of substrates. If the Crane hypothesis is functionally correct then a reversal of the normal Naf gradient in intestinal tissue should be fol- lowed by the extrusion of solute from previously loaded cells into the medium. Crane obtained evidence (26) in support of this hypothesis when he found that when Na+ was replaced by Tris (so that the external Na+ concentration was less than the intracellular level), 6-deoxyglucose moved out of the cells into the medium against its own concentration gradient. Kimmich however, with epithelial cells of chick intestine, found that transport of sugar into the cells continued at the same rate and to the same extent even when the Na+ gradient was re- versed (27). Since it seemed therefore, that the energy required for active transport of sugar need not necessarily be derived from the downhill Na+ gradient, Kimmich (27) proposed an alternative to the Crane model. In this scheme it was sug- gested that Na+-dependent metabolite transport might be de- pendent upon the Na+-dependent formation of a high energy intermediate produced by the (Naf + K+)-activated ATPase complex, and that subsequent K+-dependent hydrolysis; of the intermediate could provide the necessary energy for either ion transport or for active transport of sugars.

In the marine pseudomonad no Naf gradient is maintained, and under normal growth conditions internal and external concentrations of the cation are the same (22). Obviously, in this instance energy for AIB uptake cannot be derived from a previously existing Na+ gradient. Furthermore, in the two marine bacteria examined, an ATPase requiring a combination of Naf + K+ for activation is apparently lacking in the cyto- plasmic membrane (28, 29). However, in the marine pseudo-

monad B-16, the presence of Na+ is essential for entry of AIB. Kinetic analysis of the Natdependent entry process indicates that an increase in the Na+ concentration is followed by a decrease in the K, of the carrier system (9). In this respect Na+-dependent transport in the pseudomonad is similar to that found by Crane with mammalian preparations (12). While the model proposed by Kimmich elaborates upon the energetics of sugar transport in mammalian systems it, does not explain why Na+ decreases the K, for transport either in mammalian cells or in the marine pseudomonad. The present report shows that in the marine bacterium, even after K+ depletion and poi- soning with CN-, there is still a requirement for Na+ for the entry of AIB. The Na+-dependent entry of AIB into K+- depleted cells was competitively inhibited by structurally similar amino acids such as glycine, serine, and alanine. Branched chain compounds, e.g. valine and isoleucine which do not apparently share the same carrier as the other amino acids (7) did not inter- fere with AIB penetration into the cells. We believe therefore, that entry of AIB occurs via a Na+-dependent, carrier-mediated process rather than being simply caused by a physicochemical effect of Na+ upon the cell membrane which would permit AIB to enter the cell by passive diffusion. These results strongly suggest that in the marine pseudomonad Na+ has a direct effect on a carrier putting it, as Crane suggests for animal cells (11, 12), into a configuration having greatest affinity for the substrate. Thus in the marine pseudomonad the specific role of Na+ in transport would seem to be to effect the facilitated diffusion of the substrate into the cell. Whether Na+ has a role to play in the accumulation process is less clear. That K+ has such a role is evident from the data presented. If Na+ does have a role in active transport. in the marine pseudomonad it is certainly not through the maintenance of a Na+ gradient or the operation of a (Naf + K+) ATPase complex.

A tentative model to account for Na+- and K+-dependent active transport of AIB in the marine pseudomonad is pre- sented in Fig. 12. This model combines certain features of

INTERNAL

FIG. 12. Proposed model for the roles of Na+ and K+ ions in the active transport of AIB by the marine pseudomonad B-16. In this hypothetical scheme, Cr. represents carrier or permease, which is represented white when loaded and black in the unloaded state. Stage 1, binding or recognition; Stuge 2, translocation; Stage 8, release of AIB(Sub) ; Stage 4, reactivation.

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4074 Amino Acid Transport in a Marine Pseudomonad Vol. 246, No. 12

the model proposed by Crane (11) to account for Na+-dependent solute transport in animal cells, with mechanisms suggested to account for active transport in bacteria (30, 31). In Stage 1, in the scheme illustrated, Na+ combines with the permease or carrier (32-34), thereby putting it into a conformation having affinity for the substrate. The Na+-carrier-substrate complex then crosses the cytoplasmic membrane, Stage W. In K+- depleted cells equilibration of the substrate would occur via this facilitated diffusion system. In the presence of energy and intra- cellular Kf, it is suggested that the affinity of the Na+ carrier complex for the substrate is in some way reduced (31, 35) and the substrate is released and accumulates within the cell, Stage 3. Finally, the carrier, after release of the substrate would return to the outside surface of the membrane. At some stage in the translocation, by an as yet unknown process it would be converted to a high affinity form once more, Stage 4.

The exact nature of the K+-dependent stage in active trans- port in the marine pseudomonad is still obscure, but in view of the stimulation of AIB transport by nucleotides in the presence of K+, it is tempting to speculate that, K+ is a cofactor in the coupling of phosphate bond energy or a high energy intermedi- ate to the transport process. Nucleotides are known to be lost from cells of this organism under the same conditions which lead to the loss of intracellular K+. The penetration studies with adenine nucleotides show that these compounds can cross the cytoplasmic membrane, and if utilized in energy-yielding reactions could account for the stimulation of AIB transport produced by these compounds in the presence of K+.

Scarborough et al. (23) with E. coli treated with cold Tris- HCl buffer under conditions causing the cells to become de- pleted of nucleotides and K+, observed stimulation of the trans- port of galactosides by subsequent addition of ATP and K+ to the incubation medium. From these observations it was con- cluded that K+ acted internally but ATP functioned on the outside surface of the membrane during transport of galacto- sides. In these experiments the capacity of ATP to penetrate the cytoplasmic membrane of E. co& was not examined, and, because of the prior treatment to which the cells had been exposed they might well have been leaky. It is thus not clear from their experiments whether ATP was functioning on the inside or the outside of the cell membrane during sugar transport.

Acknowledgments-We should like to express appreciation of the help given by Dr. J. W. Costerton and Mrs. J. M. Weilandt during the electron microscopic stages of this work. The re- search program was supported by a grant from the National Research Council of Canada.

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J. Thompson and Robert A. MacLeodMarine Pseudomonad

-Aminoisobutyric Acid in aα in the Active Transport of + and K+Functions of Na

1971, 246:4066-4074.J. Biol. Chem. 

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