492 BIOCHIMICA ET BIOPHYSICA AC ~

BBA 45404

MEMBRANE ATPase AND ELECTROLYTE LEVELS IN MARSUPIAL

ERYTHROCYTES

E R I C A B A K E R AND W. J. S1MMONDS

Department of Physiology, The University of Western Australia, Nedlands (Western A uslralia)

(Received May 9th, 1966)

SUMMARY

I. Individuals of the Australian marsupial species, the possum (Trichosurus vutpecula), differ in erythrocyte cation concentration. ATPase activity was measured in the fragmented membranes of erythrocytes in which potassium predominates (HK cells) and in which sodium predominates (LK cells). A comparison was made with erythrocyte membrane ATPase activity in the marsupial quokka (Setonyx brachyurus) in which sodium and potassium are present in approximately equal concentration.

2. All erythrocyte preparations showed basal ATPase activity. Significant (Na+-K+)-activated ATPase was present only in HK possum and quokka cells, absolute values and values relative to basal ATPase being higher in HK possum than in quokka cells.

3. These results suggest that the low erythrocyte potassium concentrations in LK possums may be due to the absence of (Na+-K+)-activated ATPase and pump activity in their cell membranes.

INTRODUCTION

It has long been recognized that red blood cells provide a useful tool for study- ing mechanisms by which most cells maintain potassium as the predominant intra- cellular cation while sodium is the predominant extracellular cation. Earlier work explored differences between sodium-rich red cells in certain species (e.g. cat) and the potassium-rich red cells found in most species. Later, individual sheep and goats were found to differ 1, 3. Sheep erythrocytes have been intensively studied ~ to relate such factors as cation permeability, active transport of ions and (Na+-K+)-activated fraction of membrane ATPase to the predominance of sodium or potassium in the erythrocytes of different individuals.

Individual differences in red cell electrolytes comparable to those in sheep have been found in an Australian marsupial species, the possum, Trichosurus vulpe- cula 4. It seemed of interest to see whether differences in membrane functions like those in the sheep could be found between possum erythrocytes containing pre- dominantly potassium (HK cells) and those in which sodium predominates (LK cells). This paper reports comparisons of (Na+-K+)-activated ATPase in fragmented membranes from HK and LK possum erythrocytes and from the marsupial quokka,

Biochim. Biophys. Aeta, 126 (1966) 492-499

ATPase ACTIVITY IN H K AND LK ERYTHROCYTES 493

Setonyx brachyurus, in which erythrocyte sodium and potassium concentrations are approximately equal s .

METHODS

Animals The possum (Trichosurus vulpecula) is common in some scattered Perth envi-

rons, and a total of 54 adults were studied. The quokka (Setonyx brachyurus) is in- digenous to Rottnest Island, Western Australia e. Eight adult quokkas were used in this investigation, after acclimatisation for at least 2 weeks on the mainland. Animals were fed a staple diet of sheep nuts, bread, fruit and vegetables.

Blood sampling Blood was collected by cardiac puncture using heparin as anticoagulant.

Samples were processed within 30 min, since storage of whole blood has a critical effect on the erythrocyte sodium and potassium concentrations ~. All samples and reagents were handled in acid-washed apparatus. The haematocrit was determined by the microtechnique. The standard deviation was i 1.2 % at a haematocrit of 30 % (IO replicates).

Materials Heparin (IOOO units/ml) was obtained from Boots Pure Drug Co. Ltd. and

Blu-tip microtubes and Seal-ease wax from Clay-Adams Inc., New York. The crystal- line disodium salt of ATP was supplied by Sigma Chemical Co. ; crude DNA and l.- histidine (free base, A grade) by Mann Research Laboratories; and ouabain (Ouabaine Arnaud) by Laboratoire Nativelle, Paris. All other chemicals used were of reagent quality.

Electrolyte estimation Sodium and potassium concentrations in diluted whole blood and plasma were

determined using the E.E.L. flame photometer (Mark II). The erythrocyte sodium and potassium levels were then calculated by difference, in conjunction with the haematocrit value, using the relationship described by EVANS 8. This indirect method was preferred because it involves fewer manipulations of the sample than the direct measurement of the electrolyte concentrations in washed packed erythrocytes.

A correction factor was applied for sodium contamination in the heparin, and contamination from glass was avoided by storing standards and samples in polythene vessels. Interference in flame emission between calcium, sodium and potassium at the relative concentrations present in the diluted samples was shown to be negligible.

Estimation of erythrocyte A TPase activity Aliquots of a preparation of isolated fragmented erythrocyte membranes were

incubated in a buffered medium containing ATP. The rate of ATP hydrolysis was followed by measurement of the inorganic phosphate released. Two ATPase compo- nents were distinguished. Considerably activity was present in the absence of added alkali metal ions. This was termed the basal ATPase. Addition of Na + and K + to- gether to the medium produced an increase in activity to a new level termed the total

Biochim. Biophys. Acta, 126 (1966) 492-499

494 E. BAKER, W. J. SIMMONI)S

ATPase. The difference between basal and total activity, i.e. the increment due to the presence of Na + and K +, was termed the (Na~-K ~)-ATPase.

The fragmented erythrocyte membrane suspension was prepared by the method of SEX" AND POST 9. Suspensions were stored at 4 ° and assayed within 2 days of preparation, as preliminary experiments showed an appreciable increase in activity after the second day, and a progressive decline in activity after storage for 5 days.

The incubation procedure was a slight modification of the method of POST el al. 1°. The enzymic reaction was started by adding ATP serially to a series of tubes containing buffered membrane suspension at 37 ° . For the determination of total ATPase activity, the volume of reaction mixture was 2.5 ml and it contained 2 mM ATP, 4 mM Mg 2+, 60 mM Na +, 30 mM K +, 40 mM imidazole-4o mM histidine (ad- justed to pH 7.1 with o.i M HC1) and 0.5 mM H(Tris)a EDTA. Each series of tubes in- cluded appropriate controls for phosphate contamination from the reagents and membrane suspension, and for non-enzymic hydrolysis of ATP. After incubation for 40 rain, the tubes were transferred serially to an ice-water bath, and the reaction was stopped by the addition of 8 % perchloric acid to each tube exactly i min later. The solutions were centrifuged, and aliquots of the supernatant assayed for inorganic phosphate. Preliminary investigations showed that with this method enzymic hydrolysis was linear with time up to ioo rain incubation at 37 °, and the rate was directly proportional to the concentration of membrane suspension.

Inorganic phosphate was estimated by a modification of the method of CHEN, TORIBARA AND WARNER 11. The temperature of incubation with the colour reagent was chosen after preliminary experiments to minimise the non-enzymatic hydrolysis of ATP by the molybdate reagent. Colour reagent, 2.8 ml, at 21 ° was added to 1.2 ml supernatant from the enzymic reaction, or of phosphate standard, the mixture was incubated at 4 ° for at least 20 min and the absorbance then measured within 5 rain at 820 m/~ in a Unicam spectrophotometer. The sensitivity was about 40% of that reported by CHE?L TORIBARA AND WARNER 11 but the correction for non-enzymatic hydrolysis of ATP was not greater than lO-15 O//o of the enzymic phosphate liberation.

RESULTS

Erythrocyte Na + and K + concentrations in individual possums and quokkas Analysis of the concentrations of Na + and K + in the erythrocytes of individual

animals were performed on 6 H K possums, 15 LK possums, and 7 quokkas. The results (Fig. I) indicate the clear division between the cell types. There was no significant difference in plasma electrolyte levels between these groups.

General properties of A TPase preparations Effect of magnesium and A TP concentration. Both (Na+-K+)-activated ATPase

and basal ATPase required magnesium (Fig. 2a). The same pat tern of stimulation was found in all cell types studied. Optimum activity was obtained with a magnesium: ATP ratio of approx. 2:1 (Fig. 2b). In subsequent experiments, 4 mM magnesium and 2 mM ATP were used in preference to higher concentrations to minimise the ATP blank value.

Effect of ouabain. A complete inhibition of the (Na+-K+)-ATPase activity was

Biockim. Biophys. Acla, 126 (1966) 492-499

ATPase ACTIVITY IN HK AND LK ERYTHROCYTES 495

obtained at a ouabain concentration of 5" zo-6 M, No effect was observed on the basal ATPase activity (Table I).

Effect of N a + or K + alone. Sodium ions in concentrations up to IOO mM had no significant effect on the basal activity. Potassium ions in low concentration produced a small but significant increase in basal ATPase activity (Fig. 3)- This increase was however completely inhibited by ouabain. I t was presumably due to the combined

140

12C

100

oo

80 3 E E 6C

o 40

o o

o o

0

x x x x

y,

20 • o o • | ~IIS

% 0 60 80 100 120 No +concn. (mmoles/ I cells)

Fig. I. The concentrat ions of sodium and potass ium in the erythrocytes of 6 H K possums (O), 7 quokkas ( × ) and 15 L K possums (O)-

6 HK possum

, • - 2 6 10

~ 4

2

O I 0

6 r o uokJ~o /

0 v i , i i i

0 2 6 10

M 2+ conch. (raM) 6

2 x

I i i i t 0 1 2 3 4 0 1 2 3 4

ATP concn. (raM)

I LK possum

0 2 6 10

2 S

O0 1 2 3 ~,

(a)

(b)

Fig. 2. The effect on erythrocyte membrane total ATPase (O), basal ATPase (O), and (Na+-K+) - act ivated ATPase ( × ) of (a) increasing magnesium concentrat ions in the presence of 2 mM ATP, and (b) increasing ATP concentrat ions in the presence of 4 mM magnesium. ATPase act ivi ty is expressed as mmoles phospha te released per 1 of erythrocytes per h at 37 °.

Biochim. Biophys. Acta, 126 (1966) 492-499

496 E. BAKER, W. J. SIMMONI)S

effect of K + with the smal l concent ra t ion (4 mM) of Na + from the d i sodium sal t of ATP.

Effect of Na* and K+ added together. To avoid var ia t ion in ionic s t rength between different incubat ion media , a cons tan t to ta l cat ion concent ra t ion of I2o mM was

T A B L E l

THE EFFECT OF OUABAIN ON THE A T P a s e ACTIVITY OF QUOKKA AND POSSUM ERYTHROCYTE MEMBRANES

A T P a s e a c t i v i t y is e x p r e s s e d as m m o l e s p h o s p h a t e re leased/1 of r ed ce l l s pe r h a t 37"- F o r ex- p e r i m e n t a l d e t a i l s see t e x t .

A n i m a l A TPase component Ouabain concentration (M)

H K P o s s u m

Q u o k k a

L K P o s s u m

0 .5"I0-7 5" I0-6 5"I0 o

T o t a l 4.21 2.71 2.05 2.1o B a s a l 2.17 2.15 2.17 2.1o ( N a + - K + ) - a c t i v a t e d 2.o 4 0.56 T o t a l 2.30 1.75 1.2o 1.22 B a s a l i .26 1.38 i .33 1.35 (Na + - K + ) - a c t i v a t e d 1.o 4 o.37 - T o t a l 1.64 1.5o 1.31 1.42 B a s a l 1.4 ° 1.37 i .46 i .5 ° (Na +-K + ) - a c t i v a t e d o.24 o. 13

3 Quokka HK possum

o LK possum

% 4o ;o I~o ,;o °o 4o ;o ,~o ~6o Cation conch. (raM)

Fig. 3. T h e effect on p o s s u m a n d q u o k k a erythrocyte me mb ra n e A T P a s e of s o d i u m ions (O) , o r p o t a s s i u m ions ( O ), a d d e d s e p a r a t e l y . A T P a s e a c t i v i t y is e x p r e s s e d as m m o l e s p h o s p h a t e re leased/1 of erythrocytes per h a t 37 °.

maintained in the first set of experiments (Fig. 4). Sodium and potass ium concen- trations were altered by exchanging one for the other. For both H K possum and quokka erythrocytes , ATPase act ivat ion diminished steeply when the N a : K ratio fell below 1:2 or was greater than 5:1 , while a N a : K ratio near 2:1 (80 mM Na÷: 4 ° mM K +) was opt imal for both. In subsequent experiments the total ion concen- tration was also varied and a Na +: K + ratio of 2 : i again found to be opt imal over the range 60 mM Na+:3 o mM K + to 80 mM N a + : 4 o mM K ÷. Since there was a gradual inhibition of ATPase act iv i ty with increasing total ionic strength, the lower concen- trations (6o:3 o) were preferred. The total osmolari ty of the incubation media after the addition of the buffered membrane preparation was then approx. 300 mosmoles .

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ATPase ACTIVITY IN H K AND L K ERYTHROCYTES 497

As shown in Fig. 4 (dotted lines), ATPase activity in the presence of ouabain (5" Io -e M), was independent of Na + and K + concentrations. Ouabain inhibited only that activity due to the synergistic action of Na + and K +. To avoid any error in (Na+-K+)-ATPase estimation due to differences between the basal and total ATPase incubation media, (Na+-K+)-ATPase was subsequently estimated as the difference between ATPase activity (a) in a medium containing 60 mM Na + and 30 mM K + (total ATPase) and (b) in a medium containing these ions plus 5" lO-6 M ouabain (basal ATPase).

6 Ouokko LK possum

4

tam

No"

HK possum

i i o , o 40 o ' ' 4 0 8 0

0 120 8 0 4 0 0 K ÷ 0 120 80 40 0 0 120 8 0 4 0 0

Cation concn. (raM)

Fig. 4. The effect of varying sodium and po tass ium concentrat ions on the quokka and possum erythrocyte membrane ATPase activity. The cation concentrat ion was mainta ined cons tant (at 12o mM) by exchanging sodium ion for po tass ium ion. Incuba t ion was performed in the presence ( O - - - O ) or absence ( Q - - O ) of 5" IO-e M ouabain. ATPase act ivi ty is expressed as mmoles phos- pha te released per 1 of ery throcytes per h at 37 °.

TABLE I I

A COMPARISON OF BASAL AND (Na+-K+)-ACTIVATED ATPase LEVELS IN QUOKKA AND POSSUM ERYTHROCYTE MEMBRANES

ATPase act ivi ty is expressed as mmoles phospha te released per I of red cells per h at 37 °. Results are given as mean -- S.E. of mean. Parentheses enclose the number of prepara t ions assayed.

Animal Erythrocyte cation concentration (mM)

Na + K +

A TPase activity (Na+-K+)-A TPase

Basal (Na+-K+)-activated Basal A TPa~e

H K possum (25) 37 117 2.44 ± 0.07 2.53 ± o.18 1.o 5 Quokka (17) 78 49 2.13 :J_ o.12 1.21 x~ o.12 0.60 L K possum (20) 12o 13 1.68 4- o.o9 0.06 -4- 0.o6 0.o 3

A TPase levels in possum and quokka erythrocytes A series of determination of membrane basal ATPase and (Na+-K+)-ATPase

was made on HK and LK possum erythrocytes and quokka erythrocytes. The results (Table II) indicate that the LK possum erythrocytes do not contain significant (Na+-K+)-ATPase activity (o.6 < P < 0.7). Stimulation of HK possum and quokka erythrocyte ATPase by Na + and K + amounted to approx. IOO % in the HK possum and 60 % in the quokka. The difference in (Na+-K+)-ATPase activity between the H K possum and quokka erythrocytes is highly significant (P < o.ooi). Basal ATPase

Biochim. Biophys. Acta, 126 (1966) 492-49 9

498 E. BAKER, W. J. SIMMONI)S

activity was highest in the H K possum, intermediate in the quokka, and lowest in the LK possum erythrocytes, these difference being statistically significant (o.or P < 0.05).

DISCUSSION

Erythrocyte membrane preparations from quokkas and H K and LK possums showed basal ATPase activity, but significant (Na+-K+)-stimulated ATPase activity was found only in quokka and H K possum erythrocytes. There was no ATP break- down in the absence of magnesium. Maximum activation of both enzyme components was reached at magnesium : ATP molar ion ratios of approx. 2 : I. Similar magnesium- dependent ATPases have been reported in many membrane preparations including crab nerve 12, human erythrocytes lz and brain microsomes 14. The optimum magnesium : ATP ratio varies, however, between 2 : I and I : I with different preparations. Since the enzyme is membrane bound, and there is a small quanti ty of magnesium associated with the erythrocyte membrane 15, the optimal magnesium:ATP ratio at the site of reaction may not be the same as the ratio in the incubation medium.

Sodium and potassium ions together substantially increased quokka and H K possum ATPase activity. Activation was dependent not only on the presence of both ions, but on their relative concentrations. Very high or low Na+:K + concentration ratios were least activating. An optimal Na+:K + ratio for (Na+-K+)-activated ATPase activity would be consistent with the demonstration of two sites with different affinities for monovalent cations in human erythrocytes 16,17. Departure from the optimal ratio could result in competition of Na + or K + for sites normally occupied by the alternate ion.

The (Na+-K+)-dependent ATPase activity in possum and quokka erythrocyte membranes was completely inhibited by low concentrations of the cardiac glycoside, ouabain. This is consistent with the effect of cardiac glycosides on every mammalian (Na+-K+)-ATPase preparation studied, and on active sodium-potassium transport in intact cells. Ouabain may specifically inhibit a K+-dependent phosphorolysis reaction which normally leads to the release of orthophosphate. In the presence of ouabain, a phosphorylated intermediate (possibly a phosphoprotein) tends to accu- mulate as its turnover is blocked is.

Although other membrane functions have not yet been studied the relationship between (Na+-K+)-stimulated ATPase activity and potassium content of the three types of erythrocytes is significant. Of particular interest was the virtual absence of such activity in preparations from LK possum erythrocyte preparations. This needs to be confirmed using different methods of membrane preparation, storage and assay, since these may influence results. Treatment with deoxycholate 19 and urea 2°, pre- incubation with Tris buffer 21 or freezing and thawing la enhance (Na+-K')-ATPase activity or increase the (Na+-K+)-ATPase:basal ATPase ratio.

The results so far suggest that there may be at least a quanti tat ive difference between the membrane functions of LK possum erythrocytes and those of LK sheep. In sheep, LK erythrocytes show a decrease both in active and passive transfer of potassium and sodium. The (Na+-K+)-stimulated ATPase of LK erythrocyte mem- branes is quite appreciable, about 25 % of that in H K membranes, but the pump: leak ratio in whole erythrocytes is lower in LK cells. In possums results so far suggest a

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ATPase ACTIVITY IN H K AND LK ERYTHROCYTES 499

virtuaiab-~ei~ce of (Na+-K+)-stimulated ATPase activity in K + erythrocyte mem- branes. I t will be interesting to see if, correspondingly, the K permeability for whole erythrocytes is much lower relative to H K cells than in sheep.

ACKNOWLEDGEMENTS

This work was conducted whilst E. B. was a recipient of a Hacket t Scholarship from the University of Western Australia. I t was also supported by a grant from the Medical School Research Grant of the University of Western Australia.

The authors wish to thank Dr. B. M. JOHNSTONE for helpful discussion.

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