The kinetics of potassium exchange in cells of Staphylococcus aureus

54

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 45351 T H E KINETICS OF POTASSIUM E X C H A N G E IN CELLS OF S T A P H Y L O C O C C U S A U R E U S F. GALDIEI~O Institute of Microbiology, The University of Naples, Naples (Italy) (Received November 5th, 1965) (Revised manuscript received January 31st, 1966)

SUMMARY The kinetics of potassium exchange in cells of Staphylococcus aureus with 42K" as a tracer in the extracellular potassium phase, was investigated. In phosphate buffer and KCI, in the absence of glucose, results showed that equilibration of distribution of external specific radioactivity and internal specific radioactivity was not achieved because the cells continuously lose potassium. In the presence of glucose, however, results showed no loss of potassium from the cells, and the potassium phase in the cells remained stationary, so that an equilibrium between intracellular and extracellular specific radioactivity was achieved. Sodium azide and dinitrophenol, added in both the absence and presence of glucose, did not have any effect on exchange as compared with controls. If, however, DL-glyceraldehyde was added in the experiments in the presence of glucose, no utilization of the carbon source, and the same behaviour for the cells as in the absence of glucose, was observed, Incubation temperature has a marked effect" potassium-exchange rate is greatly decreased at a temperature of 4 ° . Increasing concentrations of potassium in the suspension medium did not affect the exchange rate for concentrations higher than 3-4 mequiv potassium per 1.

INTRODUCTION Previous research has established that cells of Staphylococcus aureus, a microorganism having a high potassium content ~-3, assimilate potassium when kept under conditions favouring growth or active metabolism ~,5. Since this action is accompanied by elimination of water, it was postulated that the potassium, or most of it, would have been in a bound state s. In the present study the kinetics of exchange of intracellular potassium with extracellular potassium has been investigated. MATERIAL AND METHODS Staphylococcus aureus strain 22, kindly supplied by Istituto Sieroterapico Itallano, was grown at 37 ° under continuous aeration in io-1 flasks containing 5 1 of Difco Nutrient Broth. Growth was stopped during log-phase and the cells were harvested by centrifugation at 4 ° and washed 3 times with distilled water. A suspension of i g cells (wet weight) in io ml 5° mM sodium phosphate buffer (pH 7.o), 50 mM KC1 and o.I M glucose was incubated at 37 ° for 30 rain. Biochim. Biophys. Acta, 126 (1966) 54-6o

KINETICS OF POTASSIUM EXCHANGE IN STAPHYLOCOCCUS

55

The cells were centrifuged and washed again, and then resuspended in the incubation medium made up of 50 mM sodium phosphate (pH 7.0) and 50 mM KC1 and containing as tracer 4*KC1 (approx. 200 ~C/mmole) (The Radiochemical Centre, Amersham, England). The experiments were carried out in reciprocating shaker-baths at 37 °. Aliquots were removed at various times and the cells were collected on Millipore filters HA-WP-o25. Each sample, rinsed 3 times with deionised water at 4 °, after determination of dry weight s, was ashed at 500 ° for 12 h. The ash was dissolved in 6 M HC1, dried, and finally taken up in a measured quantity of o.i M HC1 in order to check the chemical concentration of the potassium and the radioactivity count. The same determination was carried out on the suspension liquid. The potassium concentration was measured by flame photometry (Beckman Model DU). The radioactivity was measured in a Nuclear Chicago y-counter with a 2 inch by 2 inch well crystal. Counting was carried out until a standard error of 0.02 was achieved. Because of the short half-life of 42K+, all measurements had to be corrected for decay during the course of the experiment. ANALYSIS OF RESULTS

For a closed steady-state two-compartmental system we have, according to SHEPPARD 7 :

aoS1 (l -- e -ps'~/sls2) a2 = ~S--

(i)

whence" a0 $1 = aeq. 0.693 S1S2 1.

'

t~S

(i')

For a closed non-steady-state compartmental system the potassium influx and outflux in ~equiv/h were calculated according to TOSTENSON, CARLSONAND DUNHAMS: dRo. _

_

dS2 _ _

a

2

-

dt pl,2 =

-

dt al - - a2

dS2 ; p2,1 = p l , 2 - - - dt

(2)

dR2/dt and dS~/dt were obtained graphically from the slope (at 30 rain) of the plot of [42K+]2 and [K+]2 against time. R 1 and R~, total radioactivity in medium (Compartment I) and cells (Compartment 2) in counts/rain ; a I and a2, specific radioactivity in Compartments I and 2 at the time of the observation, counts/min per mequiv; a 0, specific radioactivity at zero time in Compartment I ; S, total amount of potassium in the system (mequiv) ; $1 and Sz, amounts of S in Compartments I and 2, respectively; p, exchange rate between compartments; Pl,2 and P~,I, the exchange rate, respectively, from Compartment I to 2 and from Compartment 2 to I. RESULTS

Cells of Staphylococcus aureus suspended in a phosphate buffer solution containing KCl The time dependence of the specific radioactivity of intracellular potassium Biochim. Biophys. dcta, I26 (1966) 54-60

50

F.

GALDIERO

follows the course shown in the curve of Fig. I. It m a y be considered as being divided into 2 parts with respect to time. A first part covers a p p r o x i m a t e l y the first 3 h, during which specific r a d i o a c t i v i t y rises rapidly. A second part represents specific r a d i o a c t i v i t y as rising slowly w i t h o u t ever reaching a m a x i n m m . This indicates a loss of potassium b y the cells which prevents the achievement of d i s t r i b u t i o n equilibr i u m between the 2 phases. The loss of potassium b y the cells was d e m o n s t r a t e d by

I

I

1'00 -N"-I.....__ ~

~

al(t)

i

i

I

I

i

r

~

I

1.00

.~

5

o o~c

~ ~ O.lOr I

~_

u

d

c5

© © > >

!

SS 1 o.%

#

~

~

.;,

;

;

~

Time(h)

;

~°°o

~ 1

~

~

4

~

6

~

s

T}me (rain)

Fig. 5. Time dependence of the specific radioactivity of cells ( O O ) relative to the specific radioactivity of the medium at zero time. ~ g of cells (wet weight) was incubated in lo ml of 5° mM sodium phosphate buffer (pH 7.o) and 5° mM of 4zKC1.Temp. 37 ~. The symbols ( O ) indicate the means of io experiments, while each vertical bar indicates the range of the standard deviation of the mean. Q - - O , the time dependence of specific radioactivity in the incubation medium Fig. 2. Size of cellular potassium ( 0 - - 0 ) relative to potassimn content of mediunl (mequiv}. Conditions as in Fig. i. Radioactivity (O - O) of cells (counts/rain) relative to radioactivity of the medium at zero time. Each bar indicates the mean value of io experiments ! S.E.

flame-photometric analysis (Fig. 2). It initially obeys an exponential law of tile first order. Graph i can, therefore, be considered to consist of 2 c o m p o n e n t s : the first (a rapid one) is the exchange; the other (less rapid) is the loss of potassium b y the cells. F r o m Eqn. 2 a n d Table I we m a y o b t a i n the c o n s t a n t s of this system (Table II).

Cells of Staphylococcus aureus in a phosphate buffer solution containing KCl and glucose The presence of glucose in the suspension m e d i u m permits a more extended persistence of potassium in the cells, whereby an equilibrium of d i s t r i b u t i o n of specific r a d i o a c t i v i t y is achieved (Fig. 3). The graph of specific r a d i o a c t i v i t y represents exclusively exchange ; the t o t a l intracellular potassium concentration, in the presence of glucose, r e m a i n e d c o n s t a n t during the course of the i n c u b a t i o n : the cells in fact r e m a i n e d in a steady state. I n this ease the model is provided b y a steady-state system of 2 closed c o m p a r t m e n t s . The c o n s t a n t s of the system shown in Table I I can be calculated from Eqns. I a n d I'.

14iochim. Biophys. Acta, 126 (1966) 54--6o

KINETICS TABLE

OF P O T A S S I U M E X C H A N G E

57

IN STAPHYLOCOCCUS

I

PARAMETERS

NEEDED

FOR

THE

RESOLUTION

OF

EQUATION

2

T h e d a t a r e p r e s e n t t h e m e a n v a l u e s o f t h e r e s u l t s p l o t t e d in F i g s . I a n d 2.

Time (h)

R 1(t) (a o units)

o o.25 o.5 o t.oo 2.00

5oo 488 435 380 364

TABLE

R 2 (t) (a o units) o I2 65 12o 136

a 1(t) (a o units)

a 2(t) (a o mzits)

S 1 (t) (ttequiv)

S 2 (t) (t~equiv)

1 o.961 o.837 o.729 0.666

o o.o37 o.2o8 0.385 0.475

5°o 5o8 52o 52I 546

333 325 313 312 287

II

CONSTANTS

OF THE

SYSTEM

1 g of c e l l s of Staphylococcus aureus ( w e t w e i g h t ) i n c u b a t e d in i o m l of s u s p e n s i o n m e d i u m a s d e s c r i b e d in t h e t e x t . T h e t e m p e r a t u r e w a s 37 °, e x c e p t d u r i n g t h e l a s t e x p e r i m e n t . T h e v a r i o u s s u b s t a n c e s w e r e a d d e d a t t h e f o l l o w i n g c o n c e n t r a t i o n s : s o d i u m p h o s p h a t e b u f f e r , 5 ° m M ; a2KC1, 5 ° raM; glucose, o.i M; 2,4-dinitrophenol, io-aM; sodium azide, IO-2M; DL-glyceraldehyde, lo -2 M.

B u f f e r + 42KC1 B u f f e r + 42KC1 + g l u c o s e B u f f e r + 42KC1 + 2 , 4 - d i n i t r o p h e n o l B u f f e r + 42KC1 + s o d i u m a z i d e B u f f e r + 42KC1 + g l u c o s e + 2,4-dinitrophenol B u f f e r + 42KC1 + g l u c o s e + 2 , 4 - d i n i t r o phenol + glyceraldehyde B u f f e r + 42KCI + g l u c o s e + s o d i u m a z i d e B u f f e r + 42KC1 + g l u c o s e + s o d i u m a z i d e + glyceraldehyde B u f f e r + 42KC1 + g l u c o s e ( t e m p . 4 °)

S1 (t~equiv)

So (zequiv)

p p12(3o') pal(3O') (~equiv/g (~equiv/g (#equiv/g cells cells cells wet wt./h) wet wl./h) wetwt./h)

-5oo ---

-333 ---

-213 ---

293 -288 290

315 -311 312

5o0

33 °

211

--

--

-500

-33 °

-211

291 --

314 --

-500

-306

-69.2

290 --

312 --

Exchange in the presence of enzymatic inhibitors Sodium azide (10-3-1o -2 M) and 2,4-dinitrophenol (I0-5-i0 -4 M) added to the suspension medium do not affect the time dependence of specific radioactivity either in the absence or in the presence of glucose as compared with the controls of Figs. I and 3. Recalling that Staphylococcus aureus is facultatively anaerobic, we investigated the glycolysis-blocking effect of lO-2 M nL-glyceraldehyde. In the presence of DLglyceraldehyde, sodium azide or 2,4-dinitrophenol, the effects of the presence of glucose in the system were inhibited. The system behaved as the controls in the absence of glucose ; it did not achieve equilibrium between internal and external specific radioactivity.

Potassium exchange at low temperature Temperature has a marked effect on the phenomena under investigation. At a temperature of 4 ° the time curve of specific radioactivity (Fig. 4) shows a slow increase with time. The equilibrium between the 2 compartments is attained after Biochim. Biophys. Acta, 126 (1966) 5 4 - 6 0

5~

F. GALDIERO

approx. 0 h. Potassium loss begins later (8-IO h) after the attainment of equilibrium. The curve of Fig. 4 is similar to the theoretical curve described by Eqn. I, though the constants were different (Table II). I

I

[

T--

I

~ q - - T - -

1.00

1.0C ' - - ~ >,

.................

{

Ln 1

. =-~S~

~"

tll 2 =39 min

O

t~ -120rain

~ 0,1C

o.~o

L

X

{- - _{__

._u

u

5

"6

GO' 0

I

Time(h)

I

I

Time (h)

Fig. 3. P o t a s s i u m e x c h a n g e in t h e presence of glucose ( O - - - O ) . C o n d i t i o n s as in Fig. I, e x c e p t t h a t o .i M glucose was added. E a c h b a r i n d i c a t e s t h e m e a n v a l u e of i o e x p e r i m e n t s :~: S.E. Fig. 4. P o t a s s i u m e x c h a n g e a t 4 ° . C o n d i t i o n s as in Fig. 3. E a c h b a r i n d i c a t e s t h e m e a n v a l u e of IO e x p e r i m e n t s ~ S.E.

I

I

I

1

I

I

I

I--

1.0C

O.lC

~ I

~

~ 0 . 1 mM . ~0.SmM £=58,equ,v/h ',~ ~=lP,0~equiv/h "~550m& ~=213Flequiv/h

I

Time (h) Fig. 5. A s e m i l o g a r i t h m i c plot, a c c o r d i n g to Eqn. l ' of t h e d a t a from p o t a s s i u m - e x c h a n g e exp e r i m e n t s . Staphylococcus aureus cells (I g wet weight) were i n c u b a t e d in IO ml of 5o mM s o d i u m p h o s p h a t e buffer (pH 7.o), o.I M glucose a n d o.I mM ( O - - O ) , 0.5 mM ( - ), 5 mM ( 3 ~) or 5 ° mM (A - - A ) 42KC1. P r e i n c u b a t i o n as t h e final m e d i u m . The e q u i l i b r i u m specific a c t i v i t y was 0.60 of t h e i n i t i a l specific a c t i v i t y of t h e m e d i u m for 5 mM a n d 50 nlM t2KC1, o.61 for 0.5 mM a n d 0.62 for o.I mM.

Biochim. Biophys. Acta, ~z6 (~966) 54-6o

KINETICS OF POTASSIUM EXCHANGE IN STAPHYLOCOCCUS

59

Dependence of potassium exchange upon the extracellular K + concentration Experiments carried out in the presence of o.I M glucose showed that an external concentration of potassium above 3-4 mequiv/1 does not affect the time dependence of specific radioactivity. Fig. 5 presents a semilogarithmic plot, according to Eqn. i', of the data obtained with increasing potassium concentrations in the incubation medium. It should be noted that, whereas the exchange rate is constant for concentrations of K + corresponding to 3-4 mequiv/1, for lower concentrations it is proportional to the concentration itself. DISCUSSION

In the system under investigation a potassium concentration gradient exists between cells and incubation medium. Initially the amount of K + is 33 ° t~equiv/g wet wt. cells, a concentration of, say, 44 ° mequiv/1 cell fluid, as compared with a concentration in the medium of 5o mequiv/1. After 6 h incubation in the absence of glucose the cellular potassium content is 225 ~equiv, i.e., a concentration of about 300 t, equiv per 1 as compared with a final concentration in the medium of 61 t~equiv/1. The exchange of potassium in the absence of an energy source and in the presence of enzymatic inhibitors shows that it is not related to active membrane phenomena, but is regulated by laws of a physicochemical nature that involve the relationship between protoplasmic gel potassium and potassium of the suspension medium. The behaviour observed, in fact, is in agreement with the hypothesis of potassium being in a bound state in the polypeptide chains within cells under favourable physiological conditionsa, 9. What is very interesting in this respect is the hypothesis of various workers1°, 11 according to which the counterions form a "mobile monolayer" over the dissociated groups of the polypeptide chains rather than true heteropolar bonds. The effect of temperature on potassium exchange can be attributed to the diffusion of potassium at the change of phases, where there is a high interfacial energy. The effect of the external concentration of potassium on the rate of exchange suggests certain considerations. For extremely low external concentrations of K + the exchange rate is a function of these concentrations, but above a concentration of 3-4 mequiv/1 the rate of exchange becomes independent of the concentration. This behaviour is in agreement with the presence of factors that affect the uptake and exchange kinetics, which may be expressed by asymptotic equations of the Michaelis-Menten type, as has been demonstrated by various workers12, ~3. It does not, however, seem necessary to fall back on the explanation of an active exchange mechanism involving the membrane. Furthermore, the limiting factor can be attributed to the peripheral cellular region, the membrane and cell wall, whose ion-binding capacity could regulate the intracellular and extracellular exchange. This peripheral layer, especially the cell wall and membrane having a more rigid structure of polymers with free carboxylic, phosphate and sulphate groups, could act by adsorbing ions to the dissociated groups and subsequently exchanging them with cellular cytoplasm. ACKNOWLEDGEMENT

This research was carried out with the help of a grant from the Italian Research Council. Biochim. Biophys. Acta, I26 (1966) 54-60

Oo

F. GALDIEI~()

REFE RENCES I 2 3 4 5 0 7 8 9 lo ll 12 13

J. H. t3. CHRISTIAN AND J. A. WALTHO, ./. (;e~Z. Microbiol., 25 (196I) 97. F. GALDIERO AND F. FRAGOMELE, Riv. Isl. Sieroterap. ItaI., 38 (1963) 257. J. H. 13. CHRXSTIAN AND J. A. WAL~HO, J. Appl. lSacteriol., 25 (1962) 309 . t?. GALDIERO AND F. FRAGOMELE, Nit,. Ist. Sieroterap. llal., 4 ° (1965) 33. F. GALDIERO, Nuovo Ann. [g. Microbiol., 14 (1963) 638. F. GALDIERO, Experien/ia, 21 (1965) 4o0. C. \'V. SHEPPARD, Basic Principles of the Tracer Method, W i l e y , N e w Y o r k , 1902. 1). C. TOSTENSON, E. CARLSON AND E. T. DUNHAM, .]. (;en. Physiol., 39 ( i 9 5 6 ) 31. A. S. TROSHIN, in A. I,~t.ElYZELLER AND .\. [(OTVK, Membrane Transport and Metabolism, A c a d e m i c Press, L o n d o n , 1961, p. 45. [.. I'~OT1N AND M. NAGASAWA, J. Am. Chem. Sot., 83 ( 1 9 6 i ) ~o26. S. J. LAPANIE AND S. m. ](ICE, J. A~$i. Che,tl. Soc., 83 (1961) 490. A. ROTHSTEIN ANt) M. BRUC1L J. Cellular Comp. Physiol., 51 (I958) 145. C. W. SLAYMAN AND E. L. TATUM, Biochim. Biophys. Acta, 1o2 (1965) 149.

Bioehim. Biopkys. Acta, i 2 6 (1966) 5 4 - 6 0