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Red cell ouabain binding sites, Na+K+-ATPase, and intracellular Na+ as individual characteristics
Life Sciences, Vol. 29, pp. 371-381 Printed in the U.S.A.
Pergamon Press
RED CELL OUABAIN BINDING SITES, Na+K+-ATPase, AND INTRACELLULAR Na+
AS INDIVIDUAL CHARACTERISTICS
GGnther Sc~ualzing, Erich Pfaff, and Ursula Breyer-Pfaff Department of Toxicology, University of T~bingen, W-Germany (Received in final form May 26, 1981)
Summary Red cells were used as model cells to assess the relationship between the number of ouabain binding sites and the Na+K+-ATPase activity in humans. (3H)Ouabain binding to intact red cells was found to be a reversible reaction and rectilinear Scatchard plots were obtained. Interindividual comparison revealed identical slopes, whereas the abscissa intercepts differed, indicating variations in the number of ouabain binding sites. The ntm~ber of sites ranged from 330 to 890 (~ : 453 ± I04 SD) per red cell in 73 individuals and was intraindividually stable during the course of 3-12 months. The number of sites was significantly lower in the middle-age females cor~oared to younger ones. A linear relationship (r = 0.96) was found between the total U~K~r of ouabain binding sites of intact red cells and the --ATPase activity of red cell ghosts. The catalytic activity varied little among individuals; a turnover number of 5500 + 250 (SD) molecules ATP bydrolyzed per site and per rain was calculated. A highly significant correlation was established between [Na+li of red cells in vivo and the number of ouabain binding sites. Red cell meznbranes frequently serve as models for studying the properties of membrane-bound enzymes. Among these, the Na+K+-activated ATPase has attracted considerable attention due to its 9~oortance for the maintenance of the interior milieu. Its assessment in human red cells was carried out by measurLug either the number of (3H)ouabain binding sites (for a synopsis, see I) or the enzyme activity in red cell ghosts (2-5). Both procedures resulted in large deviations among the values reported by different groups. In contrast, the interindividual differences were small (I, 6). Joiner and Lauf (I) assumed that the deviations in the number of (3H)ouabain binding sites were mainly due to incorrect estimates of the specific activity of (3H)ouabain. Variations of the Na+K+-ATPase activities of red cell ghosts m y reflect differences in membrane preparation procedures, but such variations were also observed with red cell ghosts prepared from single subjects on successive occasions (5). Differences and alterations in the surrounding lipid organization would provide an explanation of variations+of the catalytic activity of an integral ma~rane protein (7, 8) like the Na+K -ATPase. The present study examines this hypothesis by analysing the relationshi~ between the number of ouabain binding sites per intact red cell and the Na~K~-ATPase activity of red cell ghosts. The number of enzyme molecules and the Na+K+-ATPase activity proved to be intraindividually constant and closely correlated, but they differed among individuals.
0024-3205/81/040371-I1502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Number and Activity of Red Cell Na+ Pumps
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Materials and Methods Chamic~s. (3H)Ouabain (32 Ci/mol) was obtained from Amersham Buchler GmbH (Braunschweig, GFR). The radiochemical purity was 98 %, as determined by thin-layer chromatography and radioscanning. (3H)Ouabain was stored in portions of about 9 uCi in ethanol/benzene (9:1) at -20° C. Under these conditions the substance was stable for at least one year. Just before use the solvent was evaporated under a stream of nitrogen a~d the substance dissolved in the incubation medium. N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes) buffer substance was purchased from Serva (Heidelberg, GFR), ethylphenylpolyethyleneglycol (Nonidet ®) from Fluka (Neu-Ulm, GFR) and silicone oil AP 100 from Wacker Chemie (FCtnchen, GFR). Unisolve I (Zinsser, FrsrAcfurt, GFR) was used as a scintillator. Test reagents from Boehringer (Mannheim, GFR) served to perform hematological analyses. All other substances were analytical grade and obtained through Merck (Darmstadt, GFR). Preparation of blood samples. Blood was drawn from hematologically healthy humans and heparinized. Red cells were washed by repetitive centrifugation and resuspension in a standard medium without K + (concentrations are given in raM: 144 NaC1, 2 MgCI2, 1.2 CaCI2, 3 phosphate buffer of pH 7.4) at room temperature. The buffy coat was carefully removed with attention to minimize the loss of the lightest red cells. Part of the washed erythrocytes were resuspended in an equal volume of the standard medium with I0 mM glucose for ouabain binding experiments. The remaining bulk of red cells were lysed immediately to prepare red cell ghosts. 6-i0 ml of blood per person were necessary to carry out the experiments described below. (3H)Ouabain binding. In order to establish Scatchard plots, 200 ul aliquots of red cell suspensions in standard medium with glucose were mixed with 50 ul of (3H)ouabain solutions to achieve final concentrations between 2 and 100 riM. Since the radioactive compound was used without dilution, the added radioactivity ranged between 0.01 and 0.5 ~Ci per sample. An incubation time of 6 h was found to be necessary to reach equilibrium at low ouabain concentrations. Each measurement was done in duplicate. In parallel samples to each assay, the 'nonspecific binding' was determined by measuring the radioactivity in the sediment in the presence of a lOOOfold higher concentration of unlabelled ouabain, using the same amount of radioactivity as in the assay for the specific binding. For the majority of blood samples, the number of binding sites was determined only at an ouabain concentration of about 100 nM by adding 1 uCi of (3H)ouabain dissolved in 100 ~I standard medium to 400 ul of red cell suspension. This volume allowed quadruplicate determination of binding sites. 'Nonspecific binding' was measured in parallel to each assay. The reaction mixtures were analyzed for total hemoglobin content using the cyanohemoglobin method and the number of cells was computed by means of the mean corpuscular hemoglobin content (31 pg per cell). In order to minimize interference of (3H)ouabain in the medium, 100 ~laliquots of the reaction mixture were quickly diluted at the predetermined time with 1.3 ml of standard medium layered over 100 ~I of silicone oil in Eppendorf polypropylene vessels. After centrifugation for 30 s in a Beckman microfuge, the red cells were pelleted below the layer of silicone oil. One-half ml of the supernatant was mixed with 4 ml of scintillator (Unisolve I). The residual supernatant over the layer of silicone oil was discarded. The sides of the vessel were washed twice with methanol without disturbing the red cells below the layer of silicone oil. After washing, the silicone was partially removed, and the cells were hemolyzed with 0.1 ml of water. Decolourization was achieved by the method of Neame (9). The radioactivity was estimated by liquid scintillation spectrometry in a Packard Tricarb® 2660. Counting efficiency, determined using both an internal standard and the channel ratio, was 35 % in 10
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Number and Activity of Red Cell Na+ Pumps
373
ml of Unisolve I and did not change by more than 1 % within one year. The dilution prior to separation of the cells by silicone oil filtration greatly reduced unbound (3H)ouabain between the cells. Nevertheless, the 'nonspecific binding' resulted mainly from entrapped radioactivity. At 100 nM ouabain, it was below 5 % of the specific binding and ranged from 350 to 450 cpm, depending on the hematocrit of the reaction mixture. At a concentration of 2 nM ouabain, 'nonspecific binding' was only slightly above the background. Preparation of red cell ~hosts. Membranes were prepared from washed red cells in analogy to the procedure of Dodge et al. (10), using 10 mM Hepes/NaOH buffer of pH 7.6 as a standard procedure. The ,.~potonically lysed red cells were pelleted in a refrigerated centrifuge at 4 C with a fixed angle rotor at 34 000 g for 20 rain. The membranes were washed 3 times with Hepes buffer containing 1 mM ethylene diamine tetraacetic acid (EDTA) and 2 times with Hepes buffer and 0.1 mM ethyleneglycol-bis(S-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Due to the inclusion of EGTA, the ATPase ~tivities were better preserved during storage (without EGTA traces of Ca~ may activate membrane-bound proteases ). After the final wash the ghost suspensions prepared at pH 7.6 seemed virtually hemoglobin-free. The pellet was resuspended in a small volume of buffer. An aliquot of this ghost suspension was diluted with NaOH (final concentration 0.5 raM) and incubated at 37° C for 30 rain. Bovine serum albumin was handled in the same way. Protein was measured using the Coomassie method (11). According to this protein determination, the ghost suspension was adjusted to 1 mg protein per ml with buffer and dithioerythritol (final concentration 2 raM). A second protein determination served as a control of the adjustment. ATPase ass~v. ATPase activities were assayed in terms of the liberation of inorganic phosphate b~ conventional procedures giving maximal activities in pilot tests. Mg ~+, Na+K -ATPase activity was determined in a total volume of 1 ml containing (n/N): 2 MgC12, 2 Na2-ATP , 10Q NaC1, 20 KC1, 0.1 EGTA and 100 mosM Tris/HCl of pH 7.4 at 37° C; for the M~+-ATPase activity KC1 was omitted and 0.1 mM ouabain was added. The reaction was started by the addition of 0.5 ml of red cell ghosts (0.5 mg protein) to the prewarmed incubation medium. After 5 and 20 m/n, 0.2 ml aliquots were transferred into 0.4 ml Beckman polypropylene vessels containing 0.15 ml of CHC13. The two phases were quickly intermixed to stop the reaction (the whole procedure requires about 10 s). After short centrifugation in a Beckman microfuge, the precipitated protein was found at the water/CHC13 interface. The samples were stored on ice until phosphate was measured The Na+KV-A~Pase activity was calculated as ouabain inhibitable activity by subtracting Ng ~ -ATPase activity from Mg 2+, Na+K+-ATPase activity. Inorganic phosphate assa~. Phosphate was measured spectrophotometrically in duplicate by a modification of the Penney method (12). The reaction was carried out in a semi-microcuvet. To 650 ~l of the acid-molybdate reagent (13.5 g ammonium molybdate/1 1.25 N HC1), 20 -50 ul of the san~ple or the phosphate standard and subsequently 50 ~i of malachite green were added (600 mg malachite green/1 aqua bidest, with 0 . 1 % Nonidet®, v/v). The dye solution was thoroughly mixed by means of the pipette. At a wavelength of 624 r~n the absorbance was observed to increase rapidly, reaching a plateau within 30 s, followed by a slow decline. Analysing samples with residual ATP, this colour loss occurred markedly slower. The cause presumably was an acid hydrolysis of ATP. The hydrolysis proceeds still after reaching maximal absorbance and new colour will be formed delaying the decrease. This behaviour did not impair the accuracy of the determination, when the absorbance was read within 1-2 rain after adding the dye solution. During the increase of the absorbance, the acid hydrolysis was negligible, since the period of colour development was very short. The addition of Nonidet® (13) was necessary for increasing the accuracy of the assay. Moreover, the standard curve was linear up to an absorbance >1. The various quantities
374
Number and Activity of Red Cell Na+ Pumps
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of phosphate liberated by the ATPases at the different times could be measured with sufficient sensitivity and accuracy, when the volume of the sample was varied between 20 and 50 ul. P~spholipid-P of intact red cells and red cell ~hosts. Intact red cells were washed 3 times with I00 mM MgC12. Lipids were extracted according to the method of Rose and 0klander (14). After evaporation of the solvent, the residue was digested with 0.5 ml of perchloric acid (70 %, w/v) at 180° C. In the case of red cell ghosts, an aliquot of the suspension corresponding to 0.1 mg of protein was mixed directly with the perchloric acid. Phosphate standards were handled in the same way. The colourless and cooled digest was diluted with 2 ml of aqua bidest. Phosphate was measured using malachite green as described above Na + and K + concentrations of red cells. One-tenth ml of whole blood was mixed'with iO ml of 100 mM MgC12, layered over 0.3 ml of silicone oil in conical polycarbonate tubes. Red cells were pelleted below the silicone oil layer by centrifugation at 3000 g for 8 rain. After discarding the supernatant, the sides of the tubes were washed twice with aqua bidest, without affecting the red cells below the oil layer. A Beckman atomic absorption spectrophotometer was used to estimate the Na + and K + content of the red cells after lysis with 5 ml of 0.I % Nonidet® in aqua bidest. The volume of the cells was computed from the hemoglobin concentration of whole blood.
Result s (3H) 0uabain binding. The time course of (3H)ouabain binding was measured at different (3H) ouabain concentrations in order to define equilibrium conditions (Fig. 1). For promoting ouabain binding, external K + was omitted (15-17).
o 600C free
~. 500"tD
e99 nMI e34 nM
-~ 400-
---e 13 nM
.~ 200" 100L
L
L
I
L
L
1
2
3
4
5
6
L
7
8 t~ne (h)
FIG. I Time and concentration d e p ~ e n c e of (3H)ouabain.binding. H ~ a n red cells were incubated in a K free medzum at various concentrations of (3H)ouabain and the amount of bound (3H)ouabain was measured at the times indicated. According to the Scatchard plots constructed from the equilibrium data presented here and shown in Fig. 2 with the same symbols (e), the total number amounted to 525 ouabain binding sites per red cell. Cfree means: final concentration of unbound ouabain.
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Number and Activity of Red Cell Na+ Pumps
375
Because of the concentration dependence of the binding rate, incubation times as long as 6 h were required at the lowest ouabain concentration of about 2 nM; at I00 nM, 2.5 h were sufficient. In a series of experiments, (3H)ouabain binding was terminated by the addition of excess non-radioactive ouabain (0.i mM), and the subsequent release of bound (3H)ouabain was monitored. Sen/logarithmic plots of these data vs. time showed a linear decline. A half-life time of dissociation of 9 h at 37° C was calculated. Straight lines were obtained when the equilibrium data ware plotted according to Scatchard (18), as shown in Fig. 2. The extrapolated number of ouabain binding sites on the abscissa intercept differed among individuals. Irrespective of the total number of sites, the slopes of these plots, calculated from least squares regression lines, yielded dissociation constants between 2.6 and 3.2 oM (E = 2.8 + 0.2 SD) for red cells from 8 subjects. Therefore, the majority of ouabain binding experiments was carried out at a nearly saturating ouabain concentration (100 nM). According to the law of mass action, this concentration labels 97 % of the total number of binding sites at a Kd of 2.8 nM. The corrected values agreed well with those obtained by Scatchard plots. In quadruplicate determinations of binding sites, the confident interval (p = 0.05) was found to be + 1.4 % of the mean.
JD les
100
200
300
400
500
600
700
oust~n modec~es bound per red ca~
FIG. 2 Scatchard plots for (SH)o~bain binding to intact red cells. Human red cells were incubated at 37° C with a range of (SH)ouabain concentrations between 2 and 100 nM for 6 h. The ratio bound/ free is the number of ouabain molecules bound per red cell divided by the f~ee ouabain concentration in nM. Accordirg to the intersections on the abscissa, the following total numbers of ouabain binding sites were calculated: (o) 355; (e) 525; (m) 612. Open symbols represent data fram a female, filled symbols data from 2 male volunteers.
376
Number and Activity of Red Cell Na + Pumps
Vol. 29, No. 4, 1981
The total number of ouabain binding sites per red cell from 6 individuals did not change during the course of one year (Fig. 3). Moreover, such a constancy was observed with red cells from additional subjects, in whom binding sites were determined two or more times during the course of several months. An exception was observed in the case of a 26 a old woman: during an interval of 5 months the number of ouabain binding sites declined from 550 (5 baseline determinations within one year) to 380 (3 determinations within half a year). Sin~ltaneously, the Na+K+-ATPase activity of red cell membranes decreased and the Na + content of erythrocytes increased. I I
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March April
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l~e. 3 Ouabain binding sites per red cell during the course of one year in different subjects between 20 and 45 years of age ( o m A males, oa females). Confident interval (p = 0.05) + 1.6 % in 2 subjects (oo). Three of the subjects were identical with those in Fig. 2, and their data were depicted with the same symbols.
The number of ouabain binding sites ranged from 330 to 890 in red cells from 73 subjects. In all cases, serum K + was within the normal range. The mean value of ouabain binding sites was significantly (p < 0.01) lower in the middleage (>40 a, ~ = 403, N = 16) than in young females (<40 a, E = 508, N = 20). The number of males above 40 years was too small to permit statistical comparison. In red cells from men below 40 a, a mean value of 443 ouabain binding sites was obtained (N = 29). Rh null cells of 4 subjects in our series were found to have 350, 386, 482, and 504 ouabain binding sites. ATPase activity. In order to check whether the nLm~0er of ouabain binding sites per intact red cell correlates with the Na+K+-ATPase activity, leaky red cell ghosts were prepared. However, hemolysis in a solution of low ionic strength may result in significant alterations of lipid-protein interactions. Therefore, different procedures for the preparation of red cell ghosts were reinvestigated. An increase of the pH value of the hypotonic lysis and washing buffer led to a decrease of protein content (including hemoglobin) relative to phospholipid-P of red cell ghosts. In parallel, ATPase activities increased. Red cell ghosts prepared with 10 mM buffer of pH 7.6 were y'i~ually hemoglobinfree and exhibited maximal ATPase activities. The lower Na'K -ATPase activities of membranes prepared at a pH below 7.2 were probably caused by a restricted
Vol. 29, No. 4, 1981
Number and Activity of Red Cell Na+ Pumps
377
accessibility of the hydrolysis sites for ATP (2); this could be demonstrated by increasing the activity by procedures which are thought to increase permeability, viz freeze-thawing, sonication, and addition of deterrents. In contrast, these procedures did not influence or even decreased the Na+KW-ATPase activity of untreated pH 7.6 membranes. From an individual's red cells, ghosts were prepared alternatively by initial isosmotic (310mosM imidazole buffer of pH 7.6) an~+hypotonic lysis. Both procedures yielded nearly identical Na+K+-ATPaseand Mg~ -ATPase activities provided that the washing was carried out with a l O m M buffer of pH 7.6; the nature of the buffer substance (Hepes, Tris, or imidazole) did not influence the results. A highly significant correlation (p < 0.001) was established between the number of ouabain binding sites of intact red cells and the Na+K+-ATPase activity of erythrocyte membranes in 50 subjects (Fig. 4). In contrast, the Mg 2+ATPase activity of red cell ghosts did~not depend on the Na+K+-ATPase activity and ranged between 590 and 480 r~ol PO~- liberated per ~mol phospholipid-P per hour. The protein content rarg~d from 0.84 to 0.92mgproteinper ~nol phospholipid-P in differe~ series of me~rane preparations within one year. Repeated measurements of Mg ~ -ATPase and Na K~-ATPase activities in a number of individuals revealed intraindividual stability of ATPase activities in relation to phospholipid-P. The extraction of phospholipids from intact red cells in several cases allowed to relate the amount of bound ouabain to the quantity of phospholipid-P. Since the Na+K+-ATPase activitywas usually referredto phospholipid-P, the number of ATP molecules hydrolyzed per m in and per site could be calculated (Table I). In II subjects, a mean value of 5500 z 250 (SD) molecules per min and per site was obtained.
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o females ( n , 2 5 ) • maJes ( n . 2 5 ) oeo •
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o
f
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y-0.00148 x - 0.008 r-0.96
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s~es ~
~
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FIG. 4 Relationship between the ntm~0er of ouabain binding sites and Na+K+-ATPase activity in 25 females (o) and 25 males (oI age 20-57 years. ATPase activities a r e expressed as ~nol PO~liberated per ~Bol phospholipid-P per hour.
378
Number and Activity of Red Cell Na+ Pumps
Vol. 29, No. 4, 1981
TABLE I The Hydrolysis of ATP per Ouabain Binding Site in il Individuals Subject
Males
Binding sites Amount of per red cell bound ouabain
Na+K+-ATPase activity
Turnover _i number (min )
26 26 29 31 31
a a a a a
401 490 510 540 612
1.77 1.99 2.20 2.40 2.66
0.57 0.70 0.72 0.75 0.90
5300 5900 5400 5200 5700
Females 45 33 27 20 32 33
a a a a a a
355 425 480 490 495 5O0
1.47 1.82 2.07 2.33 2.23 2.00
0.48 0.57 0.66 O. 81 0.79 0.68
5500 5300 5300 5800 5500 55OO
The amount of bound ouabain (in ~mol) is referred to 1 ~mol phospholipid-P of ~ntact red cells. Na+K÷-ATPase activity is expressed as ~mol PO~- liberated per ~ o l phospholipid-P of red cell ghosts per hour. Turnover number means: ATP hydrolyzed per min and per ouabain binding site.
Na + and K + concentrations. The Na + content per liter of red cells varied by a factor of 2 and was negatively correlated (p <0.001) with the number of ouabain binding sites (Fig. 5) and the Na+K+-ATPase activity. •
I% °
°° ~
°o oo eo
+~ z E
~ females,
n-24
•
n-27
males,
o
o
y- - 0.0086 X + 10.8 r- 0.94
ouabain b~qdlng sites per red cell
FIG. 5 Relationship between the intracellular Na + concentration and the number of ouabain binding sites in red cells from 24 females (o) and 27 males (e).
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Number and Activity of Red Cell Na+ Pumps
379
Red cells' Na + concentration was significantly (p <0.05) higher in middle-age females (>40 a, ~ = 7.2 meq/l of cells, N = 16) than~in young ones (<40 a, = 6.3 meq/l of cells, N = 20). The intracellular K- concentration ranged from 96 to 104 meq/l of red cells and did not correlate with the number of ouabain binding sites. During the course of several months, constant levels of both cations were measured in red cells From the 6 individuals, in whom the number of ouabain binding sites remained constant (Fig. 3).
Discussion As with the Na+K+-ATPase of other sources (15, 16, 19), ouabain binding to intact human red cells was found to be a reversible reaction, although dissociation proceeds very slowly with a half-life time of 9 h. This is in agreement with the ouabain binding to human red cell ghosts reported by Erdmann and Hasse (6), but in contrast with Joiner and Lauf (1), who termed ouabain binding to human red cells as "irreversible for practical purpose", because they did not observe subnsximal steady state levels upon decreasing the ouabain concentration. The discrepancy is due to the facts that they incubated for 3 h only and that their lowest ouabain concentration was still nearly sufficient for saturation of the binding sites; this results frcm a comparison with the dissociation constant found here (Fig. 2). On the other hand, the small variation in the number of binding sites during the course of years and the mean value they reported (E = 470 ± 30 SE, N = 6) agree well with data presented here (Fig. 3; = 453 ± 104 SD, N = 73). Presumably because they investigated red cells from 6 subjects only, Joiner and Lauf (1) did not report interindividual differences. Hypokalaemia as a possible reason for an increased number of binding sites (20) could be excluded in our series. In contrast to a report of Lauf and Joiner (21), the number of ouabain binding sites was found to be not increased in Rh null cells. •
.
.
.
.
.
.
.
+
Concermng the questlon of nnterlndiwdual varlatlon of Na -pump molecules, one can refer to previous studies on the variation of intracellular Na + concentration, since both values were closely correlated in the present series. Considerable interindividual differences were reported: The subject-to-subject variation of [Na+]i was significantly greater than the day-to-day variation for an individual (22) and amounted to a factor of 2.5, whereas the intracellular K + levels were similar (22-24). Accordingly, the interindividual differences (Fig. 3) are not likely to be a special feature of the population investigated here. As in previous studies (22, 25), the red cells of the older fsnales contained more Na" (p <0.05); this agrees well with the observation of HokinNeaverson et al. (25), who found a higher active sodium efflux from erythrocytes of 18-39 a old females compared with the cells of 40-70 a old females. The significantly lower nt~nber of ouabain binding sites in the older group supports these findings. In our study the cause was not an over-all decrease in the number of sites, but a lack of females with a very high number of sites, as they were found in the group of the younger ones (i.e. 4 females <30 a with 650 to 890 binding sites). The true value of Na+-pumps per single cell renmins unknown, since the measured number of ouabain binding sites per erythrocyte represents a mean of scme 10 y cells. The cell population may consist of two types of cells with either a high or a low number of sites. This hypothesis would explain interindividual differences as being due to varying fractions of the two erythrocyte types. These may mature from two different types of stem cells in the bone marrow. The switchoff of stem cells which produce erythrocytes with a high number of Na+-pu~ps may explain the lower average number of sites in the group of the older females and the decrease observed in the case of the 26 a old female. Differences in
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Number and Activity of Red Cell Na+ Pumps
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the number of ouabain binding sites between light and heavy red cells (26) were correlated with the age of the cells with a decline of binding sites in heavier (older) red cells. However, these findings may also be brought about by a parallel partial separation of subpopulations of red cells that differ in their specific weights and number of binding sites, but not in their age. The results on properties of membranes prepared by different procedures confirm and extend findings of other working groups. Dodge et al. (I0) showed that ghosts became hemoglobin-free when prepared in 10-20 mosM phosphate buffer at pH 7.4-8.0. At this pH, non-hemoglobin nitrogen was minimal. Bramely et al. (2) recognized that ghosts prepared at low ionic strength at pH 7.4 with accordingly low hemoglobin content exhibited high ATPase activities, which were not increased by sonication or detergents. They proved that low ATPase activities are principally due to the impermeability of the preparation to ATP. Such findings were confirmed by Hanahan and Ekholm (3), but later these authors proposed initial lysis in an isotonic buffer with saponin (4) as the most suitable method. However, we found that ATPase activities of leaky ghosts prepared in 10 mosM buffers at pH 7.6 remained unchanged or were reduced when saponin or other detergents were added. Saponin may be stimulatory when not all ghosts are leaky The correlation between Na+K+-ATPase activity and the total number of ouabain binding sites could only be obtained indirectly, since the first parameter was measured on red cell ghosts and the second on intact red cells. However, [Na+]i in vivo and the number of ouabain binding sites were also closely correlated. Therefore, the highly significant correlation between enzyme activity and the number of ouabain binding sites seems to indicate that the activity reported here is representative of a physiological parameter and not a fortuitous value produced as an artifact. Even if bound ouabain is referred to the phospholipid-P content of intact red cells, individual differences in the catalytic activity were small (Table I). This additionally demonstrates that the relation of binding sites to hemoglobin in healthy subjects is correct as well. A turnover number of 5500 ATP molecules hydrolyzed per ~i~e and per min agrees well with previously reported hydrolysis numbers of Na-K--ATPase from different sources (6, 19; for a synopsis, see 1). The physiological or pathophysiological significance of the interindividual differences in the intracellular sodium concentration depends on the question whether red cells are representative as model cells and whether interindividual differences can be expected in excitable cells.
Acknowled~nt We thank Ms. Petra Kutschera for excellent technical assistance and Prof. Dr. Winne for a valuable discussion. Mr. Ulrich von Hees kindly enabled us to carry out atomic absorption spectrophotometry in the Department of Pharmacology. The work was supported by a grant of the Deutsche Forschungsgemeinschaft.
References I. 2.
C.H. JOINER and P.K. LAUF, J. Physiol.Lond. 283 155-175 (1978). T.A. B ~ Y , R. COI/9@~N and J.B. FINEAN, Biochim.Biophys.Acta 241 752769 (1971).
3. 4.
D.J. HANAHAN and J. EKHOLM, Biochim.Biophys.Acta 255 413-419 (1972). D.J. HANAHAN and J. EKHOLM, Arch.Biochem.Biophys. 187 170-179 (1978).
Vol. 29, No. 4, 1981
5o
6. ?.
.
,
10. 11. 12. 13.
14 15 16 17 18 19 20 21 22 23. 24. 25. 26.
Number and Activity of Red Cell Na+ Pumps
381
J.E. HESKETH and H.W. READING, Biochem.Soc.Trans. 6 918-920 (1978). E. ERDMANN and W. HASSE, J.Physiol.Lond. 251 671-682 (1975). J.C. METCALFE, J.P. BENNETt, T.R. HESKETH, M.D. HOUSLAY, G.A. SMITH and G.B. WARREN, The Structural Basis of Membrane Function, Y. Hatefi and L. Djavadi-Ohaniance, eds. pp. 57-67, Academic Press, New York (1976). H. RASMUSSEN, D.P.B. GOODMAN and E. MAX, Biochemistr~ of Membrane Transport, G. Semenza and E. Carafoli, eds. pp. 470-480, Springer Verlag, Berlin (1977). K.D. NEAME, Anal. Biochem. 80 51-59 (1977). J.T. DODGE, C. MITCHELL and D.J. HANAHAN, Arch.Biochem.Biophys. 100 119130 (1963). J.J. SEDMAK and S.E. GROSSBERG, Anal. Biochem. 79 544-552 (1977). C.L. PENNEY, Anal. Biochem. 75 201-210 (1976). E. CONCUSTELL, M. CORTES, A. FERRAGUT and J. GENER, Clin.Chim. Acta 8_! 267-272 (1977). H.G. ROSE and M. OKLANDER, J.Lipid Res. 6 428-431 (1965). P.F. BAKER and J.S. WIV.LIS, Nature 226 521-523 (1970). R.E. B A R N ~ , Biochemistry 9_ 4644-464-~ (1970). J.D. GARDNER and T.P. CONLON, J.Gen.Physiol. 60 609-629 (1972). G. S C A T S , Ann. N.Y.Acad.Sci. 51 660-672 (1949). E. ERDMANN and W. SCHONER, Biochim.Biophys.Acta 307 386-398 (1973). E. ERDMANN and W. KRAWIETZ, Acta biol.med.germ. 36 879-883 (1977). P.K. LAUF and J.H. JOINER, Blood 48 457-468 (1976). L.J. BEILIN, G.J. KNIGHT, A.D. MUNRO-FAURE and J. ANDERSON, J.Clin. Invest. 45 1817-1825 (1966). J. F 0 ~ E R and J,O. W I ~ , Scand.J.Lab.lnvest. 18 167-180 (1966). F. MARONGIU, H.J. HOLTMEIER, and A. IrON KLEIN-WISENBERG, Klin.Wschr. 444 1405-1412 (1966). M. HOKIN-NEAVERSON, D.A. SPIEGEL, W.A. BURCKHARDT and J.W. JEFFERSON, Clin.Chim.Acta 76 399-401 (1977). C.H. JOINER and P.K. LAUF, Membr.Biochem. ! 187-202 (1978).
Pergamon Press
RED CELL OUABAIN BINDING SITES, Na+K+-ATPase, AND INTRACELLULAR Na+
AS INDIVIDUAL CHARACTERISTICS
GGnther Sc~ualzing, Erich Pfaff, and Ursula Breyer-Pfaff Department of Toxicology, University of T~bingen, W-Germany (Received in final form May 26, 1981)
Summary Red cells were used as model cells to assess the relationship between the number of ouabain binding sites and the Na+K+-ATPase activity in humans. (3H)Ouabain binding to intact red cells was found to be a reversible reaction and rectilinear Scatchard plots were obtained. Interindividual comparison revealed identical slopes, whereas the abscissa intercepts differed, indicating variations in the number of ouabain binding sites. The ntm~ber of sites ranged from 330 to 890 (~ : 453 ± I04 SD) per red cell in 73 individuals and was intraindividually stable during the course of 3-12 months. The number of sites was significantly lower in the middle-age females cor~oared to younger ones. A linear relationship (r = 0.96) was found between the total U~K~r of ouabain binding sites of intact red cells and the --ATPase activity of red cell ghosts. The catalytic activity varied little among individuals; a turnover number of 5500 + 250 (SD) molecules ATP bydrolyzed per site and per rain was calculated. A highly significant correlation was established between [Na+li of red cells in vivo and the number of ouabain binding sites. Red cell meznbranes frequently serve as models for studying the properties of membrane-bound enzymes. Among these, the Na+K+-activated ATPase has attracted considerable attention due to its 9~oortance for the maintenance of the interior milieu. Its assessment in human red cells was carried out by measurLug either the number of (3H)ouabain binding sites (for a synopsis, see I) or the enzyme activity in red cell ghosts (2-5). Both procedures resulted in large deviations among the values reported by different groups. In contrast, the interindividual differences were small (I, 6). Joiner and Lauf (I) assumed that the deviations in the number of (3H)ouabain binding sites were mainly due to incorrect estimates of the specific activity of (3H)ouabain. Variations of the Na+K+-ATPase activities of red cell ghosts m y reflect differences in membrane preparation procedures, but such variations were also observed with red cell ghosts prepared from single subjects on successive occasions (5). Differences and alterations in the surrounding lipid organization would provide an explanation of variations+of the catalytic activity of an integral ma~rane protein (7, 8) like the Na+K -ATPase. The present study examines this hypothesis by analysing the relationshi~ between the number of ouabain binding sites per intact red cell and the Na~K~-ATPase activity of red cell ghosts. The number of enzyme molecules and the Na+K+-ATPase activity proved to be intraindividually constant and closely correlated, but they differed among individuals.
0024-3205/81/040371-I1502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Number and Activity of Red Cell Na+ Pumps
Vol. 29, No. 4, 1981
Materials and Methods Chamic~s. (3H)Ouabain (32 Ci/mol) was obtained from Amersham Buchler GmbH (Braunschweig, GFR). The radiochemical purity was 98 %, as determined by thin-layer chromatography and radioscanning. (3H)Ouabain was stored in portions of about 9 uCi in ethanol/benzene (9:1) at -20° C. Under these conditions the substance was stable for at least one year. Just before use the solvent was evaporated under a stream of nitrogen a~d the substance dissolved in the incubation medium. N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes) buffer substance was purchased from Serva (Heidelberg, GFR), ethylphenylpolyethyleneglycol (Nonidet ®) from Fluka (Neu-Ulm, GFR) and silicone oil AP 100 from Wacker Chemie (FCtnchen, GFR). Unisolve I (Zinsser, FrsrAcfurt, GFR) was used as a scintillator. Test reagents from Boehringer (Mannheim, GFR) served to perform hematological analyses. All other substances were analytical grade and obtained through Merck (Darmstadt, GFR). Preparation of blood samples. Blood was drawn from hematologically healthy humans and heparinized. Red cells were washed by repetitive centrifugation and resuspension in a standard medium without K + (concentrations are given in raM: 144 NaC1, 2 MgCI2, 1.2 CaCI2, 3 phosphate buffer of pH 7.4) at room temperature. The buffy coat was carefully removed with attention to minimize the loss of the lightest red cells. Part of the washed erythrocytes were resuspended in an equal volume of the standard medium with I0 mM glucose for ouabain binding experiments. The remaining bulk of red cells were lysed immediately to prepare red cell ghosts. 6-i0 ml of blood per person were necessary to carry out the experiments described below. (3H)Ouabain binding. In order to establish Scatchard plots, 200 ul aliquots of red cell suspensions in standard medium with glucose were mixed with 50 ul of (3H)ouabain solutions to achieve final concentrations between 2 and 100 riM. Since the radioactive compound was used without dilution, the added radioactivity ranged between 0.01 and 0.5 ~Ci per sample. An incubation time of 6 h was found to be necessary to reach equilibrium at low ouabain concentrations. Each measurement was done in duplicate. In parallel samples to each assay, the 'nonspecific binding' was determined by measuring the radioactivity in the sediment in the presence of a lOOOfold higher concentration of unlabelled ouabain, using the same amount of radioactivity as in the assay for the specific binding. For the majority of blood samples, the number of binding sites was determined only at an ouabain concentration of about 100 nM by adding 1 uCi of (3H)ouabain dissolved in 100 ~I standard medium to 400 ul of red cell suspension. This volume allowed quadruplicate determination of binding sites. 'Nonspecific binding' was measured in parallel to each assay. The reaction mixtures were analyzed for total hemoglobin content using the cyanohemoglobin method and the number of cells was computed by means of the mean corpuscular hemoglobin content (31 pg per cell). In order to minimize interference of (3H)ouabain in the medium, 100 ~laliquots of the reaction mixture were quickly diluted at the predetermined time with 1.3 ml of standard medium layered over 100 ~I of silicone oil in Eppendorf polypropylene vessels. After centrifugation for 30 s in a Beckman microfuge, the red cells were pelleted below the layer of silicone oil. One-half ml of the supernatant was mixed with 4 ml of scintillator (Unisolve I). The residual supernatant over the layer of silicone oil was discarded. The sides of the vessel were washed twice with methanol without disturbing the red cells below the layer of silicone oil. After washing, the silicone was partially removed, and the cells were hemolyzed with 0.1 ml of water. Decolourization was achieved by the method of Neame (9). The radioactivity was estimated by liquid scintillation spectrometry in a Packard Tricarb® 2660. Counting efficiency, determined using both an internal standard and the channel ratio, was 35 % in 10
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Number and Activity of Red Cell Na+ Pumps
373
ml of Unisolve I and did not change by more than 1 % within one year. The dilution prior to separation of the cells by silicone oil filtration greatly reduced unbound (3H)ouabain between the cells. Nevertheless, the 'nonspecific binding' resulted mainly from entrapped radioactivity. At 100 nM ouabain, it was below 5 % of the specific binding and ranged from 350 to 450 cpm, depending on the hematocrit of the reaction mixture. At a concentration of 2 nM ouabain, 'nonspecific binding' was only slightly above the background. Preparation of red cell ~hosts. Membranes were prepared from washed red cells in analogy to the procedure of Dodge et al. (10), using 10 mM Hepes/NaOH buffer of pH 7.6 as a standard procedure. The ,.~potonically lysed red cells were pelleted in a refrigerated centrifuge at 4 C with a fixed angle rotor at 34 000 g for 20 rain. The membranes were washed 3 times with Hepes buffer containing 1 mM ethylene diamine tetraacetic acid (EDTA) and 2 times with Hepes buffer and 0.1 mM ethyleneglycol-bis(S-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Due to the inclusion of EGTA, the ATPase ~tivities were better preserved during storage (without EGTA traces of Ca~ may activate membrane-bound proteases ). After the final wash the ghost suspensions prepared at pH 7.6 seemed virtually hemoglobin-free. The pellet was resuspended in a small volume of buffer. An aliquot of this ghost suspension was diluted with NaOH (final concentration 0.5 raM) and incubated at 37° C for 30 rain. Bovine serum albumin was handled in the same way. Protein was measured using the Coomassie method (11). According to this protein determination, the ghost suspension was adjusted to 1 mg protein per ml with buffer and dithioerythritol (final concentration 2 raM). A second protein determination served as a control of the adjustment. ATPase ass~v. ATPase activities were assayed in terms of the liberation of inorganic phosphate b~ conventional procedures giving maximal activities in pilot tests. Mg ~+, Na+K -ATPase activity was determined in a total volume of 1 ml containing (n/N): 2 MgC12, 2 Na2-ATP , 10Q NaC1, 20 KC1, 0.1 EGTA and 100 mosM Tris/HCl of pH 7.4 at 37° C; for the M~+-ATPase activity KC1 was omitted and 0.1 mM ouabain was added. The reaction was started by the addition of 0.5 ml of red cell ghosts (0.5 mg protein) to the prewarmed incubation medium. After 5 and 20 m/n, 0.2 ml aliquots were transferred into 0.4 ml Beckman polypropylene vessels containing 0.15 ml of CHC13. The two phases were quickly intermixed to stop the reaction (the whole procedure requires about 10 s). After short centrifugation in a Beckman microfuge, the precipitated protein was found at the water/CHC13 interface. The samples were stored on ice until phosphate was measured The Na+KV-A~Pase activity was calculated as ouabain inhibitable activity by subtracting Ng ~ -ATPase activity from Mg 2+, Na+K+-ATPase activity. Inorganic phosphate assa~. Phosphate was measured spectrophotometrically in duplicate by a modification of the Penney method (12). The reaction was carried out in a semi-microcuvet. To 650 ~l of the acid-molybdate reagent (13.5 g ammonium molybdate/1 1.25 N HC1), 20 -50 ul of the san~ple or the phosphate standard and subsequently 50 ~i of malachite green were added (600 mg malachite green/1 aqua bidest, with 0 . 1 % Nonidet®, v/v). The dye solution was thoroughly mixed by means of the pipette. At a wavelength of 624 r~n the absorbance was observed to increase rapidly, reaching a plateau within 30 s, followed by a slow decline. Analysing samples with residual ATP, this colour loss occurred markedly slower. The cause presumably was an acid hydrolysis of ATP. The hydrolysis proceeds still after reaching maximal absorbance and new colour will be formed delaying the decrease. This behaviour did not impair the accuracy of the determination, when the absorbance was read within 1-2 rain after adding the dye solution. During the increase of the absorbance, the acid hydrolysis was negligible, since the period of colour development was very short. The addition of Nonidet® (13) was necessary for increasing the accuracy of the assay. Moreover, the standard curve was linear up to an absorbance >1. The various quantities
374
Number and Activity of Red Cell Na+ Pumps
Vol. 29, No. 4, 1981
of phosphate liberated by the ATPases at the different times could be measured with sufficient sensitivity and accuracy, when the volume of the sample was varied between 20 and 50 ul. P~spholipid-P of intact red cells and red cell ~hosts. Intact red cells were washed 3 times with I00 mM MgC12. Lipids were extracted according to the method of Rose and 0klander (14). After evaporation of the solvent, the residue was digested with 0.5 ml of perchloric acid (70 %, w/v) at 180° C. In the case of red cell ghosts, an aliquot of the suspension corresponding to 0.1 mg of protein was mixed directly with the perchloric acid. Phosphate standards were handled in the same way. The colourless and cooled digest was diluted with 2 ml of aqua bidest. Phosphate was measured using malachite green as described above Na + and K + concentrations of red cells. One-tenth ml of whole blood was mixed'with iO ml of 100 mM MgC12, layered over 0.3 ml of silicone oil in conical polycarbonate tubes. Red cells were pelleted below the silicone oil layer by centrifugation at 3000 g for 8 rain. After discarding the supernatant, the sides of the tubes were washed twice with aqua bidest, without affecting the red cells below the oil layer. A Beckman atomic absorption spectrophotometer was used to estimate the Na + and K + content of the red cells after lysis with 5 ml of 0.I % Nonidet® in aqua bidest. The volume of the cells was computed from the hemoglobin concentration of whole blood.
Result s (3H) 0uabain binding. The time course of (3H)ouabain binding was measured at different (3H) ouabain concentrations in order to define equilibrium conditions (Fig. 1). For promoting ouabain binding, external K + was omitted (15-17).
o 600C free
~. 500"tD
e99 nMI e34 nM
-~ 400-
---e 13 nM
.~ 200" 100L
L
L
I
L
L
1
2
3
4
5
6
L
7
8 t~ne (h)
FIG. I Time and concentration d e p ~ e n c e of (3H)ouabain.binding. H ~ a n red cells were incubated in a K free medzum at various concentrations of (3H)ouabain and the amount of bound (3H)ouabain was measured at the times indicated. According to the Scatchard plots constructed from the equilibrium data presented here and shown in Fig. 2 with the same symbols (e), the total number amounted to 525 ouabain binding sites per red cell. Cfree means: final concentration of unbound ouabain.
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Number and Activity of Red Cell Na+ Pumps
375
Because of the concentration dependence of the binding rate, incubation times as long as 6 h were required at the lowest ouabain concentration of about 2 nM; at I00 nM, 2.5 h were sufficient. In a series of experiments, (3H)ouabain binding was terminated by the addition of excess non-radioactive ouabain (0.i mM), and the subsequent release of bound (3H)ouabain was monitored. Sen/logarithmic plots of these data vs. time showed a linear decline. A half-life time of dissociation of 9 h at 37° C was calculated. Straight lines were obtained when the equilibrium data ware plotted according to Scatchard (18), as shown in Fig. 2. The extrapolated number of ouabain binding sites on the abscissa intercept differed among individuals. Irrespective of the total number of sites, the slopes of these plots, calculated from least squares regression lines, yielded dissociation constants between 2.6 and 3.2 oM (E = 2.8 + 0.2 SD) for red cells from 8 subjects. Therefore, the majority of ouabain binding experiments was carried out at a nearly saturating ouabain concentration (100 nM). According to the law of mass action, this concentration labels 97 % of the total number of binding sites at a Kd of 2.8 nM. The corrected values agreed well with those obtained by Scatchard plots. In quadruplicate determinations of binding sites, the confident interval (p = 0.05) was found to be + 1.4 % of the mean.
JD les
100
200
300
400
500
600
700
oust~n modec~es bound per red ca~
FIG. 2 Scatchard plots for (SH)o~bain binding to intact red cells. Human red cells were incubated at 37° C with a range of (SH)ouabain concentrations between 2 and 100 nM for 6 h. The ratio bound/ free is the number of ouabain molecules bound per red cell divided by the f~ee ouabain concentration in nM. Accordirg to the intersections on the abscissa, the following total numbers of ouabain binding sites were calculated: (o) 355; (e) 525; (m) 612. Open symbols represent data fram a female, filled symbols data from 2 male volunteers.
376
Number and Activity of Red Cell Na + Pumps
Vol. 29, No. 4, 1981
The total number of ouabain binding sites per red cell from 6 individuals did not change during the course of one year (Fig. 3). Moreover, such a constancy was observed with red cells from additional subjects, in whom binding sites were determined two or more times during the course of several months. An exception was observed in the case of a 26 a old woman: during an interval of 5 months the number of ouabain binding sites declined from 550 (5 baseline determinations within one year) to 380 (3 determinations within half a year). Sin~ltaneously, the Na+K+-ATPase activity of red cell membranes decreased and the Na + content of erythrocytes increased. I I
I"
I
i •
3O0
•
o
J
i
I
i
2o~
l
I i
[
L lgTg Jan.
Feb
March April
May
J u n e July
Aug Se~ od
Nov
Dec
Jaa
l~e. 3 Ouabain binding sites per red cell during the course of one year in different subjects between 20 and 45 years of age ( o m A males, oa females). Confident interval (p = 0.05) + 1.6 % in 2 subjects (oo). Three of the subjects were identical with those in Fig. 2, and their data were depicted with the same symbols.
The number of ouabain binding sites ranged from 330 to 890 in red cells from 73 subjects. In all cases, serum K + was within the normal range. The mean value of ouabain binding sites was significantly (p < 0.01) lower in the middleage (>40 a, ~ = 403, N = 16) than in young females (<40 a, E = 508, N = 20). The number of males above 40 years was too small to permit statistical comparison. In red cells from men below 40 a, a mean value of 443 ouabain binding sites was obtained (N = 29). Rh null cells of 4 subjects in our series were found to have 350, 386, 482, and 504 ouabain binding sites. ATPase activity. In order to check whether the nLm~0er of ouabain binding sites per intact red cell correlates with the Na+K+-ATPase activity, leaky red cell ghosts were prepared. However, hemolysis in a solution of low ionic strength may result in significant alterations of lipid-protein interactions. Therefore, different procedures for the preparation of red cell ghosts were reinvestigated. An increase of the pH value of the hypotonic lysis and washing buffer led to a decrease of protein content (including hemoglobin) relative to phospholipid-P of red cell ghosts. In parallel, ATPase activities increased. Red cell ghosts prepared with 10 mM buffer of pH 7.6 were y'i~ually hemoglobinfree and exhibited maximal ATPase activities. The lower Na'K -ATPase activities of membranes prepared at a pH below 7.2 were probably caused by a restricted
Vol. 29, No. 4, 1981
Number and Activity of Red Cell Na+ Pumps
377
accessibility of the hydrolysis sites for ATP (2); this could be demonstrated by increasing the activity by procedures which are thought to increase permeability, viz freeze-thawing, sonication, and addition of deterrents. In contrast, these procedures did not influence or even decreased the Na+KW-ATPase activity of untreated pH 7.6 membranes. From an individual's red cells, ghosts were prepared alternatively by initial isosmotic (310mosM imidazole buffer of pH 7.6) an~+hypotonic lysis. Both procedures yielded nearly identical Na+K+-ATPaseand Mg~ -ATPase activities provided that the washing was carried out with a l O m M buffer of pH 7.6; the nature of the buffer substance (Hepes, Tris, or imidazole) did not influence the results. A highly significant correlation (p < 0.001) was established between the number of ouabain binding sites of intact red cells and the Na+K+-ATPase activity of erythrocyte membranes in 50 subjects (Fig. 4). In contrast, the Mg 2+ATPase activity of red cell ghosts did~not depend on the Na+K+-ATPase activity and ranged between 590 and 480 r~ol PO~- liberated per ~mol phospholipid-P per hour. The protein content rarg~d from 0.84 to 0.92mgproteinper ~nol phospholipid-P in differe~ series of me~rane preparations within one year. Repeated measurements of Mg ~ -ATPase and Na K~-ATPase activities in a number of individuals revealed intraindividual stability of ATPase activities in relation to phospholipid-P. The extraction of phospholipids from intact red cells in several cases allowed to relate the amount of bound ouabain to the quantity of phospholipid-P. Since the Na+K+-ATPase activitywas usually referredto phospholipid-P, the number of ATP molecules hydrolyzed per m in and per site could be calculated (Table I). In II subjects, a mean value of 5500 z 250 (SD) molecules per min and per site was obtained.
~
u
÷to z
1£ o
O.c
o
o females ( n , 2 5 ) • maJes ( n . 2 5 ) oeo •
O~
~5 °
a,
oOe o
0.~
o o
Q(
o
% o
o
f
Q.,
Q~
O~
y-0.00148 x - 0.008 r-0.96
a21
Qll
ooaba~ ~
s~es ~
~
c~
FIG. 4 Relationship between the ntm~0er of ouabain binding sites and Na+K+-ATPase activity in 25 females (o) and 25 males (oI age 20-57 years. ATPase activities a r e expressed as ~nol PO~liberated per ~Bol phospholipid-P per hour.
378
Number and Activity of Red Cell Na+ Pumps
Vol. 29, No. 4, 1981
TABLE I The Hydrolysis of ATP per Ouabain Binding Site in il Individuals Subject
Males
Binding sites Amount of per red cell bound ouabain
Na+K+-ATPase activity
Turnover _i number (min )
26 26 29 31 31
a a a a a
401 490 510 540 612
1.77 1.99 2.20 2.40 2.66
0.57 0.70 0.72 0.75 0.90
5300 5900 5400 5200 5700
Females 45 33 27 20 32 33
a a a a a a
355 425 480 490 495 5O0
1.47 1.82 2.07 2.33 2.23 2.00
0.48 0.57 0.66 O. 81 0.79 0.68
5500 5300 5300 5800 5500 55OO
The amount of bound ouabain (in ~mol) is referred to 1 ~mol phospholipid-P of ~ntact red cells. Na+K÷-ATPase activity is expressed as ~mol PO~- liberated per ~ o l phospholipid-P of red cell ghosts per hour. Turnover number means: ATP hydrolyzed per min and per ouabain binding site.
Na + and K + concentrations. The Na + content per liter of red cells varied by a factor of 2 and was negatively correlated (p <0.001) with the number of ouabain binding sites (Fig. 5) and the Na+K+-ATPase activity. •
I% °
°° ~
°o oo eo
+~ z E
~ females,
n-24
•
n-27
males,
o
o
y- - 0.0086 X + 10.8 r- 0.94
ouabain b~qdlng sites per red cell
FIG. 5 Relationship between the intracellular Na + concentration and the number of ouabain binding sites in red cells from 24 females (o) and 27 males (e).
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Number and Activity of Red Cell Na+ Pumps
379
Red cells' Na + concentration was significantly (p <0.05) higher in middle-age females (>40 a, ~ = 7.2 meq/l of cells, N = 16) than~in young ones (<40 a, = 6.3 meq/l of cells, N = 20). The intracellular K- concentration ranged from 96 to 104 meq/l of red cells and did not correlate with the number of ouabain binding sites. During the course of several months, constant levels of both cations were measured in red cells From the 6 individuals, in whom the number of ouabain binding sites remained constant (Fig. 3).
Discussion As with the Na+K+-ATPase of other sources (15, 16, 19), ouabain binding to intact human red cells was found to be a reversible reaction, although dissociation proceeds very slowly with a half-life time of 9 h. This is in agreement with the ouabain binding to human red cell ghosts reported by Erdmann and Hasse (6), but in contrast with Joiner and Lauf (1), who termed ouabain binding to human red cells as "irreversible for practical purpose", because they did not observe subnsximal steady state levels upon decreasing the ouabain concentration. The discrepancy is due to the facts that they incubated for 3 h only and that their lowest ouabain concentration was still nearly sufficient for saturation of the binding sites; this results frcm a comparison with the dissociation constant found here (Fig. 2). On the other hand, the small variation in the number of binding sites during the course of years and the mean value they reported (E = 470 ± 30 SE, N = 6) agree well with data presented here (Fig. 3; = 453 ± 104 SD, N = 73). Presumably because they investigated red cells from 6 subjects only, Joiner and Lauf (1) did not report interindividual differences. Hypokalaemia as a possible reason for an increased number of binding sites (20) could be excluded in our series. In contrast to a report of Lauf and Joiner (21), the number of ouabain binding sites was found to be not increased in Rh null cells. •
.
.
.
.
.
.
.
+
Concermng the questlon of nnterlndiwdual varlatlon of Na -pump molecules, one can refer to previous studies on the variation of intracellular Na + concentration, since both values were closely correlated in the present series. Considerable interindividual differences were reported: The subject-to-subject variation of [Na+]i was significantly greater than the day-to-day variation for an individual (22) and amounted to a factor of 2.5, whereas the intracellular K + levels were similar (22-24). Accordingly, the interindividual differences (Fig. 3) are not likely to be a special feature of the population investigated here. As in previous studies (22, 25), the red cells of the older fsnales contained more Na" (p <0.05); this agrees well with the observation of HokinNeaverson et al. (25), who found a higher active sodium efflux from erythrocytes of 18-39 a old females compared with the cells of 40-70 a old females. The significantly lower nt~nber of ouabain binding sites in the older group supports these findings. In our study the cause was not an over-all decrease in the number of sites, but a lack of females with a very high number of sites, as they were found in the group of the younger ones (i.e. 4 females <30 a with 650 to 890 binding sites). The true value of Na+-pumps per single cell renmins unknown, since the measured number of ouabain binding sites per erythrocyte represents a mean of scme 10 y cells. The cell population may consist of two types of cells with either a high or a low number of sites. This hypothesis would explain interindividual differences as being due to varying fractions of the two erythrocyte types. These may mature from two different types of stem cells in the bone marrow. The switchoff of stem cells which produce erythrocytes with a high number of Na+-pu~ps may explain the lower average number of sites in the group of the older females and the decrease observed in the case of the 26 a old female. Differences in
380
Number and Activity of Red Cell Na+ Pumps
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the number of ouabain binding sites between light and heavy red cells (26) were correlated with the age of the cells with a decline of binding sites in heavier (older) red cells. However, these findings may also be brought about by a parallel partial separation of subpopulations of red cells that differ in their specific weights and number of binding sites, but not in their age. The results on properties of membranes prepared by different procedures confirm and extend findings of other working groups. Dodge et al. (I0) showed that ghosts became hemoglobin-free when prepared in 10-20 mosM phosphate buffer at pH 7.4-8.0. At this pH, non-hemoglobin nitrogen was minimal. Bramely et al. (2) recognized that ghosts prepared at low ionic strength at pH 7.4 with accordingly low hemoglobin content exhibited high ATPase activities, which were not increased by sonication or detergents. They proved that low ATPase activities are principally due to the impermeability of the preparation to ATP. Such findings were confirmed by Hanahan and Ekholm (3), but later these authors proposed initial lysis in an isotonic buffer with saponin (4) as the most suitable method. However, we found that ATPase activities of leaky ghosts prepared in 10 mosM buffers at pH 7.6 remained unchanged or were reduced when saponin or other detergents were added. Saponin may be stimulatory when not all ghosts are leaky The correlation between Na+K+-ATPase activity and the total number of ouabain binding sites could only be obtained indirectly, since the first parameter was measured on red cell ghosts and the second on intact red cells. However, [Na+]i in vivo and the number of ouabain binding sites were also closely correlated. Therefore, the highly significant correlation between enzyme activity and the number of ouabain binding sites seems to indicate that the activity reported here is representative of a physiological parameter and not a fortuitous value produced as an artifact. Even if bound ouabain is referred to the phospholipid-P content of intact red cells, individual differences in the catalytic activity were small (Table I). This additionally demonstrates that the relation of binding sites to hemoglobin in healthy subjects is correct as well. A turnover number of 5500 ATP molecules hydrolyzed per ~i~e and per min agrees well with previously reported hydrolysis numbers of Na-K--ATPase from different sources (6, 19; for a synopsis, see 1). The physiological or pathophysiological significance of the interindividual differences in the intracellular sodium concentration depends on the question whether red cells are representative as model cells and whether interindividual differences can be expected in excitable cells.
Acknowled~nt We thank Ms. Petra Kutschera for excellent technical assistance and Prof. Dr. Winne for a valuable discussion. Mr. Ulrich von Hees kindly enabled us to carry out atomic absorption spectrophotometry in the Department of Pharmacology. The work was supported by a grant of the Deutsche Forschungsgemeinschaft.
References I. 2.
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