1. phypiol. (1978), 283, pp. 155-175 drug, followed by washing and

.1. Phypiol. (1978), 283, pp. 155-175 155With 7 text-figurewPrinted in Great Britain

THE CORRELATION BETWEEN OUABAIN BINDING AND POTASSIUMPUMP INHIBITION IN HUMAN AND SHEEP ERYTHROCYTES

BY CLINTON H. JOINER AND PETER K. LAUFFrom the Department of Physiology, Duke University Medical Center,

Durham, North Carolina 27710, U.S.A.

(Received 2 March 1978)

SUMMARY

1. [3H]Ouabain binding to human and sheep red blood cells was shown to bespecific for receptors associated with Na/K transport. Virtually all tritium bindingwas abolished by dilution with unlabelled drug. Saturation levels of binding wereindependent of glycoside concentration and were identical to those associated with100 % inhibition of K pumping.

2. [3H]Ouabain binding and 42K influx were measured simultaneously in order tocorrelate the degree of K pump inhibition with the amount of glycoside bound.Results by this method agreed exactly with those obtained by pre-exposing cells todrug, followed by washing and then measuring K influx.

3. Plots of [3H]ouabain binding vs. K pump inhibition were rectilinear for humanand low K (LK) sheep red cells, indicating one glycoside receptor per K pump siteand functional homogeneity of pump sites. High K (HK) sheep red cells exhibitedcurved plots of binding versus inhibition, which were best explained in terms of onereceptor per pump, but a heterogeneous population of pump sites.

4. External K reduced the rate of glycoside binding, but did not alter the relation-ship between binding and inhibition.

5. The number of K pump sites was estimated as 450-500 per human cell and30-50 per LK sheep cell. HK sheep cells had 90-130 sites per cell, of which eighty toninety were functionally dominant. The number of K pump sites on LK sheep cellswas not changed by anti-L, although the maximum velocity of pump turnover wasincreased.

INTRODUCTION

Since Schatzmann (1953) discovered that cardiac glycosides inhibited active(pump) transport ofNa and K in erythrocytes, these drugs have become indispensabletools in the study of the Na/K pump (Post, 1959; Post, Merritt, Kinsolving &Albright, 1960; Tosteson & Hoffman, 1960; Glynn, 1964), its role in cellular volumecontrol (Tosteson & Hoffman, 1960), and its relation to the Na plus K stimulated,Mg-dependent adenosine triphosphatase ((Na + K) ATPase) (Skou, 1960). Thesimilarity of the ouabain sensitivities of the Na/K pump and (Na +K) ATPaseprovided some of the most important evidence that these two processes are physio-logical and biochemical manifestations of the same membrane system (Glynn, 1964;Skou, 1965; Glynn & Karlish, 1975; Schwartz, Lindenmayer & Allen, 1975).

[3H]Ouabain has been employed to measure the number of Na +K pump sites on

C. H. JOINER AND P. K. LAUFhuman erythrocytes (Hoffman, 1966; Hoffman & Ingram, 1969) and on certainpathological red cells (Wiley, Ellory, Shuman, Shaller & Cooper, 1975; Lauf & Joiner,1976), and to study the kinetic characteristics and ionic requirements of cardiacglycoside binding (Bodemann & Hoffman, 1976a, b, c). However, considerablecontroversy remains as to the number of pumps on human red cells, the specificityof [3H]ouabain binding, and the effect of cations on the ouabain/pump interaction.This fact is in part due to a lack of systematic investigations of the correlationbetween [3H]ouabain binding and the inhibition of the Na/K pump.

In this report we present experiments which demonstrate the specificity of [3H]-ouabain binding to human and sheep erythrocytes and firmly establish the tightcorrelation between ouabain binding and the inhibition of active K influx. Toaugment the comparative element of our studies on human and sheep erythrocytes,we were particularly interested in quantitative and kinetic differences of ouabainbinding to HK and LK sheep red cells, and in the effect of anti-L on this process inLK red cells, since HK and LK sheep red cells differ both quantitatively and quali-tatively with respect to active and passive cation transport rates (Tosteson &Hoffman, 1960).The study reported here forms the basis for a subsequent report (Joiner & Lauf,

1978b) in which we explore the modulation of [3H]ouabain binding by internalcations in both human and sheep red cells, and by the L antibody (anti-L) in LKsheep red cells. Studies with [3H]ouabain have revealed that HK sheep red cells havemore pump sites than LK cells (Dunham & Hoffman, 1971; Joiner & Lauf, 1975).Since anti-L stimulated active K transport in LK cells (Ellory & Tucker, 1969; Lauf,Rasmusen, Hoffman, Dunham, Cook, Parmelee & Tosteson, 1970), the questionarose as to whether anti-L increased the number of K pump sites or caused theexisting pumps to translocate ions more rapidly. There were reports that the numberof [3H]ouabain binding sites was increased by anti-L (Lauf et al., 1970; Ellory &Tucker, 1970a, b), but the internal activation kinetics of the LK pump were alsochanged by the antibody (Lauf et al. 1970; Glynn & Ellory, 1972). Subsequently weshowed no increase in the number of [3H]ouabain binding sites in anti-L treated LKsheep red cells (Joiner & Lauf, 1975). Furthermore, in LK goat red cells (Sachs,Dunham, Kropp, Ellory & Hoffman, 1974a, b) anti-L effected only a slight increasein the number of K pump sites, despite a dramatic increase in pump rate due toaffinity changes at the internal cation activation sites. The present paper constitutesa more rigorous study of ouabain binding and K pump inhibition in HK and LKsheep red cells, and the effect of anti-L on these parameters.

METHODS

Media and L antisera

The media used for washing and incubation of cells were prepared from reagent grade materialsdissolved in deionized water. Osmolarity was 300 m-osmole (unless otherwise specified),measured by freezing point depression on an Advanced Instruments Osmette osmometer. WhenTris-HCL or imidazole-HCl buffer was present, it was added as a 1 M solution which had beentitrated so as to obtain pH 7-4 at 37 0C in the diluted concentration. Glucose was always presentat 10 mM.The anti-L antisera used in this study were S-39 and S-44, prepared by Dr Ben A. Rasmusen

156

OUABAIN BINDING TO ERYTHROCYTES(Department of Animal Genetics, University of Illinois) by the injection of heterozygous LKcells into HK sheep. Serum S-39 was absorbed against a panel of HK cells to remove antibodiesnot related to the L system; serum S-44 was not absorbed. Instead this serum was heated to56 0C for 30 min to remove complement components, and the antibody-containing immuno-globulin fraction of the serum was subsequently purified by alternate precipitation with 33 %ammonium sulfate and redissolving in physiological saline (Lauf & Sun, 1976). The precipitatedimmunoglobulin was finally redissolved in saline buffered with 10 mM-Tris-HCl at pH 7-4 andthe concentration of immunoglobulin adjusted to that of native L-antiserum S-44 (about10 mg/ml.). The antibody was kept at -90 0C and dialysed at 40 against incubation mediabefore use. The final concentration of antiserum or immunoglobulin to which cells were exposedwas one sixth that of whole serum. Cell suspensions were preincubated 30 min at 37 00 withantiserum before the beginning of experiments. In some experiments, non-immune HK serumwas used after heating to 56 00 to remove complement. Since this serum had no effect on cationtransport or [3H]ouabain binding, it was usually replaced by 50 mg% bovine serum albumin(BSA, Cohn Fraction V, Sigma Chemical Co., St Louis).

Collection and preparation of erythrocytesBlood was drawn by venepuncture from healthy sheep or human subjects, using heparin as

the anticoagulant, and stored on ice until used later the same day Generally, cells were washedby repetitive centrifugation and resuspension at 4 TC in the particular medium to be used in theexperiment. Care was taken in removing the buffy coat while washing, in order to minimize theloss of the lightest red cells.

Haematocrit was measured, and a suitable dilution of whole blood was haemolysed for opticaldensity determination, using a Gilford 300-N Microsample spectrophotometer. From thesemeasurements the theoretical optical density of packed cells (ODP0) was calculated, and themean corpuscular hemoglobin concentration (MCHC) computed using the extinction coefficientof hemoglobin. The analytical wave-length was either 540 nm for oxyhaemoglobin or 527 nm,the latter being an isosbestic point for oxy- and methaemoglobin; the extinction coefficient ofoxyhaemoglobin at 540 nm is 885 (kgHb/l.)-1 and for oxy- and methaemoglobin at 527 nm is532 (kgHb/l.)-1 (van Assendelft, 1970).

[3H]ouabain[3H]Ouabain was obtained from New England Nuclear Corp. (Cambridge, Mass.) and processed

as described previously (Joiner & Lauf, 1975, 1978a; Lauf & Joiner, 1976). The semi-micromethods employed in these studies required that aqueous solutions of undiluted labelled ouabainbe added to cell suspensions. It was determined that contaminants comprised less than 2% ofcell bound radioactivity, as defined by radioactivity bound in the presence of a 1000-fold excessof unlabelled ouabain.

Because the specific activity of labelled drug varied considerably from batch to batch, it wasessential to determine this parameter for each new shipment. The details of this procedure havebeen given (Joiner & Lauf, 1978a; Joiner, 1977). In brief, the radiochemical purity of thecompound was estimated by thin layer chromatography, while the concentration of chemicallyactive drug was determined by dilution of cell bound radioactivity by unlabelled ouabain.During the 3 years which these experiments spanned, specific activities measured from 11-3 to21-2 c/m-mole (six batches), while the calculated number of ouabain binding sites (based on thesespecific activities) varied only 5% for human and 10% for sheep red cells (Joiner, 1977).

[3H]ouabain bindingThe methods by which [3H]ouabain binding to erythrocytes was measured have also been

published in detail previously (Joiner & Lauf, 1975, 1978a; Lauf & Joiner, 1976). In brief, pre-warmed [3H]ouabain was added at time zero to a 10% (v/v) cell suspension pre-equilibrated inmedium at 37 0C. At desired time intervals, aliquots (0-2-0-4 ml.) were withdrawn and transferredinto ice-cold 103 mM-MgCl2 (pH 7-8) layered over 2 ml. dibutylphthalate. Cells were thenpelleted below the organic layer by centrifugation at 20,000 g and 4 'C. After washing the tubeand aspiration of both overlying liquid phases, cells were haemolysed in 8 ml. ice-cold 10 mm-Tris-Cl, pH 7-5; the ghosts were pelleted by 15 min centrifugation at 28,000 g at 4 'C, and thenwashed twice by resuspension and centrifugation. The supernatant obtained after centrifugation

157

158 C. H. JOINER AND P. K. LAUFof the haemolysate was analysed spectrophotometrically for total haemoglobin content, fromwhich the number and volume of cells counted could be computed on the basis of the meancorpuscular haemoglobin content. This procedure did not result in a loss of labelled ouabainbecause of the extraordinary low dissociation rate of ouabain from its receptor (Joiner & Lauf,1975; Joiner, 1977). The membranes were solubilized in 0.1 -NaOH, then neutralized with HOland fi emission was counted using a Triton-xylene scintillant, in a Beckman Liquid ScintillationCounter (Beckman Instruments, Fullerton, Cal.). The counting efficiency of each sample wasdetermined by external and internal standards and averaged 25% efficiency. The number ofouabain molecules bound per cell was computed according to a previously published equation(Joiner & Lauf, 1975).

Measurement of K influxUnidirectional K influx (iMp) was estimated using 42K as tracer. 42K2CO3 was obtained from

the Nuclear Services Center of the Department of Nuclear Engineering at North Carolina StateUniversity and had specific activity of 0.5 c/g. The K2C03 obtained was titrated with 0-07 N-HC1to give a solution of 150 mM-KCl and pH 7-8. After equilibration of cells to 37 'C, isotope wasadded and samples taken at desired time intervals. Cells were separated from medium bycentrifugation through phthalate, as described above. After haemolysis, samples were countedfor 42K in an Intertechnique (Dover, N.J.) gamma spectrophotometer, with correction for decaywhen appropriate. The hemoglobin concentration in the haemolysate was measured spectro-photometrically, from which the number of cells could be calculated. [3H]Ouabain could also bemeasured on the pelleted cell membranes if desired (see also Joiner & Lauf, 1975). From thesemeasurements the cellular content of radioactivity of each sample ((R,)t) was calculated andplotted vs. time.

Calculations were made using equations derived from the general flux equation

d(R1)t/dt = 'MK (XJ) -0MK(Xi) (1)where d(Ri)t/dt is the rate of change of cellular radioactivity at any given time, and X0 and Xiare external and intracellular specific activities of isotope, respectively; 'MK = unidirectionalK influx; 0MK = unidirectional K efflux. Simplifying assumptions were made according to theexperimental conditions. For cells which could be assumed to be close to 'steady state', it wasassumed that 'MK = "MK, and eqn. (1) could be 'integrated' to allow for the calculation ofK uptake by

Kptk = R1 (2)X0-XJ2assuming X1/2 represented the average intracellular specific activity of 42K from time zeroto time t.For cells in which active transport was stimulated, by anti-L for example, this steady-state

assumption did not apply. The back flux of isotope was then approximated by using the totalinflux of 'steady state' cells to estimate efflux. The equation was

Kuptak = (R)t +'MK ((X1)t/2)xtuptake = XO ~~~~~~~~~~~~~~(3)where 'MK = total rate of influx in 'steady state' cells, measured separately; t = length oftime over which the measurement was made.The ouabain-insensitive uptake of42K was measured in the presence of 10'4M-ouabain (Sigma

Chemical Corp., St Louis). K uptake could be calculated from cellular radioactivity contentwithout the correction for back flux of isotope in these cells because intracellular specific activitiesremained insignificantly low relative to external specific activities of42K over the time courseof these experiments.The rates of K uptake, both total, 'MK, and ouabain-insensitive, MiM, were obtained by

plotting the appropriate values of K uptake versus time. The slope of the best line (by least-squares analysis) was taken to represent these parameters. The ouabain-sensitive component ofinflux, which defines the K pump rate, 'M', was then calculated as

JMLIMPK = IMK- K' (4)

OUABAIN BINDING TO ERYTHROCYTES 159Since 'MI has been shown to be linearly proportional to the external K concentration withinshort ranges (Sachs & Welt, 1968), any small differences between the K. of the ouabain treatedand untreated suspensions could be normalized by means of the ratios of the concentrations.In some experiments, partially inhibited K pump fluxes were measured (see below) in order tocorrelate binding of the drug with a particular level of inhibition. The percent inhibition of asample was calculated as follows

IMp uninhibited-MM sample 100. (5)

imp uninhibited

The value of 'MI uninhibited (as well as the 'MI value required for both pump calculations)was determined on cells which had experienced the same conditions (time of preincubation,cation alteration, etc.) as the partially inhibited sample (see below).

Correlation of [3H]ouabain binding with K pump inhibitionOne of the major goals of this study was to correlate the binding of [3H]ouabain with its

pharmacological effect in red cells, the inhibition of active K transport. Two methods wereemployed to achieve this end. The first, described by Hoffman (1969), involved preincubatingcells with labelled glycoside, washing to remove unbound extracellular ouabain, and thenmeasuring the uptake of 42K to estimate the residual K pump activity. This procedure offeredthe advantage of allowing the binding of ouabain under conditions which are not favourable tothe normal operation of the Na/K exchange pump. The second 'simultaneous' method, whichwas developed for this study, involved the simultaneous determination of [3H]ouabain bindingand 42K uptake.The 'washing' method consisted of preincubating a 10% suspension of cells at 37 0C with

[3H]ouabain, usually at several different concentrations; a parallel flask with control cells wassimilarly incubated, only without glycoside. At each of several time points (usually three),2 ml. of the [3H]ouabain suspension and 4 ml. of the control suspension were placed in centrifugetubes containing 35 ml. ice cold medium. The cells were centrifuged and then washed again, andtransferred to glass tubes and resuspended at 10% haemotocrit in the flux medium. The controlcells were divided into two samples and ouabain was added to one at 10-' M final concentrationto allow the determination of the ouabain-insensitive K influx (1M'). After warming to 37 'C,42K was added to the three tubes and the incubation allowed to proceed 45-60 min. Triplicatesamples were taken and prepared for counting. The various K flux values and percent inhibitionwere calculated as described, without efflux correction. The samples incubated with [3H]ouabainwere assayed for the amount of drug bound. Control experiments demonstrated that less than1% of the bound [3H]ouabain was lost during the incubation with 42K (see also Joiner & Lauf,1975).For the simultaneous estimation of 42K uptake and [3H]ouabain binding, cells were prepared

in 10% suspension and warmed to 37 'C. At zero time, aliquots of the suspension were trans-ferred to three prewarmed tubes: one contained only 42K; the second, 42K and unlabeled ouabainsufficient for 10-4M final concentration; the third tube contained 42K and [3H]ouabain to give0-2-10 x 10-7 M final concentration. The high concentration of unlabelled drug inhibited activeK transport instantly relative to the time period of the experiment and allowed the measurementof the passive component of K influx ('ML). At the low concentrations of the tritiated drug,however, binding and K pump inhibition proceeded over the time course of the experiment,and the intent was to follow and correlate the temporal development of these two parameters.Duplicate or triplicate samples were taken from the third tube at eight or ten time points overa 90 min period and prepared for 42K counting and ['H]ouabain binding assay as describedpreviously. Triplicate samples at 30, 60, and 90 min were taken from tubes number one and twofor estimation of iMK and MiM. From the first tube, which contained only 42K, the uninhibitedrate ofK uptake (AMK) was calculated (eqn. (3)) and from this value the uninhibited pump rate('M' uninhibited) for these cells could be estimated (eqn. (4)).For the cells incubated with [3H]ouabain and 42K, the cellular content of radioactivity (R,)t,

was calculated for each time point and plotted v8. time, as shown in Fig. 1A. A curve wasdrawn by eye, assuming that the slope of the curve could not increase with time. Next, tangentswere drawn at each time point and the instantaneous rate of change of cellular radioactivity,

160 C. H. JOINER ANVD P. K. LAUF

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.

OUABAIN BINDING TO ERYTHROCYTES 161

(dR1Idt)t, was estimated. The rate of K uptake at each point, (WMK)t could then be calculatedby the following relation, derived from eqn. (1):

('MK)t = (d.RIdt)t+ 1MK (Xi)t (6)

where 'MK (Xj)t defined the correction for efflux of the isotope from the cells at each time point.Such a correction was essential in these calculations, since K pumping occurred early in theexperiment, loading the cells with isotope. As inhibition supervened with continued exposure to[3H]ouabain, the efflux of isotope became significant in relation to the rate of change of cellularradioactivity. 'MK of the [3H]ouabain treated cells was approximated by using 'MK of theuntreated cells, since K efflux is passive and equivalent of K influx in cells at steady state.Although the [3H]ouabain treated cells were not at steady state, K efflux is only negligiblyaffected by ouabain (Bodemann & Hoffman, 1976a; Simons, 1974), so that iMK measured inuninhibited cells provided a reasonable estimate of 0MK in inhibited cells. From the total rate ofK uptake at each time point ('M0)0, the residual pump rate of the [3H]ouabain treated cellscould be obtained by subtraction of the passive component of K influx ('M') measured inother cells. The percent K pump inhibition of the [3H]ouabain treated cells could then becalculated at each time point according to eqn. (5). Thus for each sample on which [3H]ouabainbinding was measured, an estimate of K pump inhibition was also obtained to allow correlationof the two parameters.The kinetics of [3H]ouabain binding to normal human red cells at two [3H]ouabain concen-

trations are shown for this experiment in Fig. 1 B. The standard errors of the triplicate samplesare representative of numerous similar experiments, and give an indication of the precision withwhich [3H]ouabain binding could be measured. At the higher concentration of [3H]ouabain(filled circles), binding proceeded rapidly for the first 60 min, giving the appearance of theexponential curve which would be expected for the saturation of a single population of receptors.However, after that time, binding proceeded almost linearly for the next 3 hr until saturationwas reached at approximately 500 molecules per cell. This type of binding curve, which was alsoseen with sheep red cells, suggested that heterogeneity existed in receptor affinities for [3H]-ouabain in the cell sample. A more detailed discussion of this point will be presented later inrelation to the specificity of binding.The tight correlation between [3H]ouabain binding and K pump inhibition is readily demon-

strated in the direct plot, illustrated for the foregoing data in Fig. 1 C. The fact that the pointsfor the two different [3H]ouabain concentrations coincide over the range of 30-55 % inhibitionindicates that the relationship between binding and K pump inhibition was independent of theglycoside concentration and the time required for binding. For example, 39 % inhibition wasobtained at the lower drug concentration (open circle) after 2 hr incubation; 45 % inhibitionoccurred within 16 min exposure to the higher concentration. It should be borne in mind thatthe variable of time remains 'hidden' in such plots, unless the specific conditions of the experi-ment are recalled.

Fig. 1. Correlation of ouabain binding with K pump inhibition in human red cells bythe simultaneous measurement of [3H]ouabain binding and 42K uptake. The experimentdepicts cells incubated with 42K at two different [3H]ouabain concentrations (indicatedin panel B). A, time course of 42K uptake. Points are the means of triplicates, with barsdepicting + S.E. of mean. Interrupted lines were drawn by eye connecting the datapoints; heavy solid lines are representative of tangents used to estimate the uni-directional K influx (see Methods). B, time course of [3H]ouabain binding. Means oftriplicates + S.E. ofmean are depicted as for panel A. Curves were drawn by eye. C, [3H]-ouabain binding versus K pump inhibition. K pump inhibition was calculated fromthe data in panel A as described in the text. Each value was then plotted versus theconcomitantly measured amount of bound [3H]ouabain. Open and filled circles againrefer to the glycoside concentrations depicted in panel B. The asterisk (*) indicatesthe maximal amount of [3H]ouabain which could be bound to these cells (5-5 x 10-7 M-[3H]ouabain at 4 hr incubation).

6 PHY 283

C. H. JOINER AND P. K. LAUF

RESULTS

Specificity of [3H]ouabain bindingIn order to interpret data on [3H]ouabain binding to erythrocytes and to draw

inferences regarding its functional relation to the Na/K pump, it was essential toascertain the specificity of the interaction between drug and cells. First, virtually alltritium binding to human and sheep red cells was abolished by dilution of [3H]-ouabain by unlabelled drug (see Methods), indicating that tritiated contaminants didnot contribute to the measured radioactivity of cell samples. Secondly, external K(10 mM) was capable of completely blocking [3H]ouabain binding at glycoside concen-trations which resulted in relatively rapid binding and K pump inhibition in the

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Fig. 2. Effect of external K and two experimental methods on the correlation of[3H]ouabain binding and K pump inhibition in normal human erythrocytes. Circlesare for data derived from cells preincubated with [3H]ouabain, washed, and thenincubated with 42K, along with appropriate control cells. Open symbols (0) representcells preincubated in low K. (0-5 mM) medium and [3H]ouabain at 1 0 and 5 0 x 10-8 M.Filled circles (@) are from cells preincubated at Ko = 5 mM; [3H]ouabain = 1P5 and5-0 x 10-7 M. 42K influx was subsequently measured at 5*5 mm K.. Data derived fromthe same cells employing the simultaneous measurement of [3H]ouabain binding and42K uptake (as in Fig. 1) are depicted in this figure as asterisks (*) to allow a comparisonof the two different experimental methods.

absence of K0. In addition, although the time course of binding exhibited a 'linear'portion as the incubation progressed (Fig. 1B), saturation levels, when achieved,were independent of [3H]ouabain concentration and the time required to achievesaturation. Furthermore, the number of binding sites at saturation correlatedexactly with the numbers obtained from the functional correlation of binding withK pump inhibition (see Fig. 1 C). Also, for a given number of glycoside moleculesbound, an identical degree of inhibition was obtained, regardless of whether binding

162

OUABAIN BINDING TO ERYTHROCYTES

was achieved by incubation with high [3H]ouabain concentration for a short time orlow concentration for a longer period (Fig. 1 C). This excluded a time or concen-tration dependent component of non-specific binding.

Correlation between [3H]ouabain binding and K pump inhibitionThe strongest evidence for the specificity of [3H]ouabain binding was its strict

correlation with K pump inhibition in both human and sheep red cells. Fig. 2demonstrates this relationship for human red cells in two experiments employingthe two different methods described in Methods. The data represented by asteriskswere obtained by the 'simultaneous' measurement of P3H]ouabain binding and 42Kinflux, the method illustrated in Fig. 1. The other experiment (circles) employed the'washing' method. There was clearly no difference between the two experimentalprocedures in so far as the relationship between binding and inhibition is concerned.In addition to validating the comparison of data derived by the two differentmethods, these results strongly suggested that the binding of a molecule of ouabainto its receptor caused the immediate inhibition of the associated K pump, without alag period. This confirmed a similar inference drawn from work on microsomal(Na + K) ATPase preparations (Schwartz, Matsui & Laughter, 1968; Barnett, 1970).

Figs. 3 and 4 represent our experiments with HK and LK sheep red cells respec-tively. For HK red cells both the 'washing' (Fig. 3A, B) and 'simultaneous'(Fig. 3 C, D) methods were used, while for LK red cells only the simultaneous methodof ouabain binding and K influx determination was used. From Fig. 3 the compar-ability of the two methods is again evident. In contrast to LK sheep red cells (Fig. 4,open symbols) the relationship between binding and K pump inhibition was non-linear in HK cells. Despite this fact, 100 % K pump inhibition in HK cells was neverachieved without the occupation of the entire complement of receptor sites and nofurther binding took place beyond the number of sites indicated at the 100 %inhibition intercept. Hence, as in LK cells, [3H]ouabain binding to HK sheep erythro-cytes was always correlated with K pump inhibition.Some of the data points presented in Figs. 2 and 3A, B were from ouabain binding

experiments in presence of 5 or 10 mM-Ko (filled circles). It is well known that KOdepresses the rate of glycoside binding (Hoffman, 1966, 1969; Sachs, 1974; Glynn,1957; Baker & Willis, 1970, 1972; Beauge & Adragna, 1971; Gardner & Conlon,1972; Gardner & Kiino, 1973; Gardner & Frantz, 1974), and this is reflected in thefact that to achieve equivalent levels of binding and inhibition within the timeperiod of the experiment, higher [3H]ouabain concentrations were required in thepreincubation media containing K than in K-free media. It can be seen that for agiven level of inhibition, the number of [3H]ouabain molecules bound per cell was thesame, regardless ofwhether binding occurred in the presence or absence of external K.Thus, there was no difference in the two preincubation conditions as to the functionalrelationship between [3H]ouabain binding and K pump inhibition. Hence, the effectofKo on the rate of [3H]ouabain binding was to prevent interaction between glycosideand receptor sites which were associated with the K pump; that is, Ko preventedBpeciftc ouabain binding.

Fig. 3B also shows an additional condition: the presence of 50 mm-CsCl replacingsome of the NaCl in the medium. The effect of Cs was investigated since it had been

6-2

163

164 C. H. JOINER AND P. K. LAUFclaimed that Cs was especially effective in reducing 'non-specific' [3H]ouabainbinding (Hoffman, 1969; Dunham & Hoffman, 1971). Again, the relationshipbetween K pump inhibition and [3H]ouabain binding was independent of theexternal ionic environment in which the binding took place.

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D52II I I I820 40 60 80

Fig. 4

Fig. 3. Correlation of [3H]ouabain binding and K pump inhibition in HK sheeperythrocytes by two experimental methods. A, washing method, HK 103. Open circlesrepresent cells preincubated with [3H]ouabain (0-9-9-0 x 10- M) at low external K(04 M); filled symbols (@) are for preincubation at 10 mM-K0, [3H]ouabain = 0-4-1.4 x 10-7 M. 42K influx was measured at 9 mM-Ko. B, washing method, HK 100. Opencircles (0) represent cells preincubated at low K0 (0-4 mM), filled circles (0) at 10 mM-K.; asterisks (*) represent preincubation in a medium in which 50 mM-CsCl replacedpart of the NaCl. [3H]ouabain in the preincubation solutions was 3-5 x 10'8 M; 42Kinflux was measured at 9 mM-Ko. C, simultaneous method, HK 129. [3H]ouabainbinding (4-6 x 10- and 7-6 x 10-7 M) and 42K influx were measured at Ko = 6-8 mm.D, simultaneous method, HIK 122. [3H]ouabain concentrations were 3-0 and 5-0 x 10-7 M;K0 was 6-0 mM.Fig. 4. Correlation of [3H]ouabain binding and K pump inhibition in LK sheep erythro-cytes with and without anti-L. All experiments involved the simultaneous measurementof 42K uptake and [3H]ouabain binding. Open circles (0) represent control cells; filledsymbols (-), cells treated with anti-L (839). [3H]ouabain was 3-0-5-0 x 10-7 M; K,,was 5-6 mM.

a)

6-

C.)

(na)

0

EC

.0

0m

)_

1 V%

_-


Stoicheiometric relationship between glycoside receptors and K pump sitesThe linearity of the relationship between [H]ouabain binding and K pump

inhibition for human red cells (Figs. 1C and 2) provided strong evidence that one andonly one ouabain molecule bound to and inhibited each K pump site. If two glycosidemolecules were required to inhibit a functional transport moiety, then plots such asFig. 2 would have been convex, with considerable binding of drug occurring beforeK pump inhibition began. Conversely, if one ouabain molecule inhibited transport,but was later followed by the binding of another molecule to the inhibited pump site,concave plots of binding versus inhibition would be expected. Although such stoi-cheiometries cannot be rigorously excluded, additional assumptions about the natureof the system, for which no data exist, would be required to explain the straight lineplots obtained. The results were therefore more consistent with the association of oneglycoside receptor with one K pump moiety. On this basis (one to one stoicheio-metry), the fact that a straight line adequately fitted the data points of Fig. 2indicated that the K pump sites of human cells were functionally homogeneous tothe extent that they each carried an equivalent portion of the total K pump activity.The data for HK sheep red cells were more complex. The plots of [3H]ouabain

binding vs. K pump inhibition in Figs. 3 and 6 were clearly curved: binding of thefirst ouabain molecules caused proportionally greater inhibition than those bindingtoward the end of the incubation. Non-specific binding was excluded as mentionedpreviously by the lack of binding tritiated contaminants, the independence ofsaturation levels of binding on [3H]ouabain concentration and exposure time, and thesensitivity of binding to external K. Furthermore, under all circumstances theoccupation of all 120-130 receptor sites was required to achieve 100 % inhibition ofactive K influx.Although a stoicheiometric relationship of two glycoside receptors per pump site

cannot be rigorously excluded (see also Discussion), the most reasonable inter-pretation of the plots of [3H]ouabain binding versus K pump inhibition for HK sheepcells is that the curvature resulted from a functionally heterogeneous population ofK pumps. That is, the sixty to seventy ouabain binding sites associated with the first80 % inhibition carried proportionally more of the K pump flux of the cells than didthe forty to fifty receptors involved in the inhibition of the final 20 % K pumpactivity.

Estimation of the number of Na/K pump sitesAssuming specific binding of [3H]ouabain to one population of receptors, each

associated with a single K pump site, the aggregate data from human cells of sixindividuals are presented in Fig. 5. The intercept of the straight line obtained byleast-squares analysis was 470 pump sites per cell. (The estimated s.E. of mean of+ thirty sites per cell was obtained from the variance analysis of the least-squaresfit.)The data for HK sheep red cells (Fig. 6) yielded an estimate of 90-130K pump sites

per cell, assuming one ouabain receptor per pump site. However, it will be recalledthat sixty to seventy pump sites were functionally associated with approximately80 % of the active K influx in these cells, indicating eighty to ninety functionallydominant K pumps per HK red cell.

165

C. H. JOINER AND P. K. LAUFFor LK cells, the 100 % inhibition intercept of Fig. 6 indicated thirty to fifty K

pump sites per cell, and this number correlated well with saturation binding levels.However, several homozygous (LL) LK sheep had red cells (not shown in Fig. 6)with more [3H]ouabain binding sites; these were (see Fig. 7C, F) sheep LL 98 withfifty-five sites, LL 71 with sixty sites and LL 115 with sixty-five sites per cell. Thesehigher numbers were reproducible and not due to non-specific binding as definedabove. They may represent simple biologic variability or additional complexity inthe genetic control of the HK/LK phenotype.

40 60% K pump inhibition

700

600

onn_Jvv _

C.)c._M

400 -a

3000)

a)r-

200 w

100

Fig. 5. Correlation of [3H]ouabain binding and K pump inhibition in the erythrocytesof five individuals. Binding and inhibition were determined simultaneously as describedfor the experiment depicted in Fig. 1. Values for K. ranged from 5 to 7 mM; [3H]ouabainwas 4 x 10-8-8 x 10-v m. The continuous line was derived from least-squares analysis ofthe data. The intercept is 470 molecules per cell at 100% K pump inhibition. Dashedlines represent the 68 O/o confidence interval of the data, analogous to a standard errorof the mean.

The effect of anti-L antiserum on LK sheep red cellsWe have previously shown that anti-L did not increase the numbers of [3H]-

ouabain binding sites on LK sheep red cells despite a two to six-fold stimulation ofactive K transport (Joiner & Lauf, 1975). Using the simultaneous method to estimateouabain binding and K pump inhibition, identical data were obtained as with thewashing procedure (Joiner & Lauf, 1975). The kinetics of [3H]ouabain binding tothe erythrocytes of five LK sheep (Fig. 7) again revealed stimulation by anti-L of

166


120 1

100

en6-

CDC

._

0o

m

80

60

40

20

% K pump inhibition

Fig. 6. Correlation of [3H]ouabain binding and K pump inhibition in the normalerythrocytes of six HK and seven LK sheep. These data were obtained by both ofthe experimental methods described in the text for the correlation of binding withinhibition.

60 LM128 A 60 LM112 B 60 LL7

50 50 -50 C

40 40 -40

=30 30 3020 K0=0-3 mm 2 K0=0-3 mm 2 K,0=0-3 mm2-01 <0 20 20 0 m10 6.6x10-8 M 1 66X10 M 9.9x10-8 M

a, 10 t ~~D 118 10 D1 18 1 D 116

1 2 3 1 2 3 1 2 3o Hours Hours Hours3-60 D 60 E 60 LL98 F

~50 *50 - 500~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0~40 40 403o- ~~~30 30

20 K0=5.2 mM 20 Ko=0 3 mM 20 K0=0 2 mM1.1 x10-6 M 7-2x10 8 M 9 7x10 8 M

10 D 114 10 D 114 10 D 116

Fig. 7. Effect of anti-L on [3H]ouabain binding to the erythrocytes of two LM andthree LL sheep. In all of these graphs open circles (0) represent control cells, filledcircles (@) anti-L treated cells. The antibody was partially purified from antiserumS44. Note that panels D and E depict [3H]ouabain binding to cells of LL 116 at K. of5-2 and 0 3 mm, respectively. Other figures represent binding at low K.. Symbolsrepresent the average of duplicate samples.

167


the rate of glycoside binding without a change of the maximum number of receptorsites. Whether the experiment was performed in presence (Fig. 7D) or absence(Fig. 7E) of K0, the saturation levels of binding were identical, indicating no effectof K0 on this parameter. (Different concentrations of glycoside (11 x 10-6 M VS.7-2 x 10-8 M) were required to achieve similar time courses of binding, due to thedepression of binding rate by Ko).

Fig. 4 also contains the correlation between [3H]ouabain binding and K pumpinhibition for LK red cells in the presence of anti-L. As compared to the data pointsin absence of anti-L (see above) there was no increase in the number of functionalK pump sites on LK red cells after treatment with antibody. Thus, the effect ofanti-L to stimulate active K influx cannot be due to recruitment of new transportsites or conversion of inactive to active pumps, but rather must result in an increasedrate of transport of the existing population of K pumps of the LK cells.

DISCUSSION

The principle thrust of this report has been the correlation of PH]ouabain bindingto human and sheep erythrocytes with the drug's pharmacological effect in thesecells, the inhibition of active K transport. The specificity of binding of the labelleddrug was established by demonstrating the lack of bound radioactive contaminants,the independence of saturation levels of binding on external cations and [H]ouabainconcentration (up to 5 x 10-7 M), and by the strict interdependence of binding andK pump inhibition.

Other investigations have focused on the interaction of cardiac glycosides withintact cells, and most have demonstrated that K pump inhibition was achieved bythe bound drug. Dunham & Hoffman (1971) presented plots of [3H]ouabain versusK pump inhibition for sheep erythrocytes, and Sachs et al. (1974b) published similardata for goat red cells. However, these studies, as well as those of Hoffman (1966,1969) and Hoffman & Ingram (1969) on human cells, were encumbered by a com-ponent of nonspecific binding of glycoside which was felt to be inhibited by externalK or Cs. Gardner & Conlon (1972) presented a rectilinear plot of [3H]ouabain bindingto human red cells versus residual active K influx. However, their results are diffi-cult to interpret because they are based on a kinetic analysis which assumes reversi-bility of the ouabain/receptor interaction. Our work (see also Joiner & Lauf, 1975;Lauf & Joiner, 1976) as well as that of Hoffman (1966, 1969), Dunham & Hoffman(1971), and Sachs (1974), has shown that ouabain binding to intact red cells is, forpractical purposes, irreversible, as evidenced by the lack of submaximal steady statelevels of binding and the extremely slow elution of bound drug.Our experimental approach also permitted a careful analysis of the specific effect

of K0 on the relationship between binding and K pump inhibition in intact cells. Ithas been proposed that K0 and Cs0 inhibit non-specific (i.e. non-pump associated)glycoside binding (Hoffman, 1969; Hoffman & Ingram, 1969; Dunham & Hoffman,1971). However other investigators have found that, while K0 reduced the rate ofouabain binding, the effect was on specific, pump-associated receptors (Beauge &Adragna, 1971; Baker & Willis, 1972; Gardner & Conlon, 1972). The results of thisstudy provided conclusive evidence (Figs. 2 and 3) that the relationship between

168


binding and inhibition was unaffected by the ionic environment in which bindingoccurs. There was no component of K-sensitive, non-specific glycoside binding tohuman and sheep cells. Thus, the effect of Ko (and Cs.) was simply to slow the rateof[3H]ouabain binding to its specific, pump associated receptors on the cell membrane.The rectilinear relationship between ouabain binding and K pump inhibition in

human and LK sheep red cells was interpreted as indicating a one-to-one ratio ofreceptors to K pumps. The concavity of such plots for HK sheep cells was consistentwith this stoicheiometry coupled with a functionally heterogeneous population oftransport sites. This evidence is important in light of the ambiguity which persists asto the molecular relationship between the cardiac glycoside receptor and the(Na + K) ATPase of microsomal preparations. There is evidence to suggest that theouabain receptor is associated with, or in close proximity to, the large chain (85,000to 135,000 Daltons) of the ATPase enzyme which is labelled by y-labelled (32p) ATPin the presence of Na and Mg (Ruoho & Kyte, 1974; Hegyvary, 1975). Most reportshave indicated a one-to-one ratio of [3H]ouabain binding to 32p incorporation (Albers,Koval & Siegel, 1968 (for mammalian tissues); Erdmann & Schoner, 1973; Pitts &Sawartz, 1975; Kaniike, Lindenmayer, Wallick, Lane & Schwartz, 1976; Hegyvary,1975, 1976). However, other investigators have found ratios varying from 1:2(J0rgenson, 1974) to 2: 1 (Kyte, 1972; Albers et al. 1968, for electroplax) and up to4:1 (Erdmann & Schoner, 1973, for guinea-pig kidney microsomes). Against thisbackground of uncertainty, our indirect evidence for one-to-one receptor/pumpstoicheiometry in human and sheep erythrocytes takes on added significance.For human red cells the number of K pump sites was found to be 450-500. This

result is compared to the findings of others for human red cells in Table 1, whichillustrates the over-all variability of this measurement in different laboratories. Suchestimates have ranged from 100 pump sites per cell (Hoffman, 1969) up to 1200 percell (Gardner & Conlon, 1972). Earlier reports agreed on approximately 200 glycosidereceptors per cell (Ellory & Keynes, 1969; Hoffman & Ingram, 1969; Baker &Willis, 1972); however, later investigations have found higher numbers (Wiley et al.1975; Knauf, Proverbio & Hoffman, 1974; Gardner & Conlon, 1972).For sheep red cells fewer estimates of the numbers of ouabain binding sites have

been made, but they were no less discordant. Dunham & Hoffman (1971) found 42and 7-6 sites for HK and LK sheep cells respectively, although with a second batchof [3H]ouabain, the numbers were 50 % higher. Ellory & Tucker have reported both8-4 (1970b) and 37 (1970a) ouabain binding sites for LK sheep cells. Estimates in thestudy by Lauf et al. (1970) ranged from twenty-seven to ninety-seven moleculesbound per LK cell.The possible reasons for these discrepancies are numerous: tritium counting

techniques, extraction of labelled drug from cells, incorrect specific activity of[3H]ouabain. This study has attempted a thorough determination of [3H]ouabainspecific activity and extensive correlation of drug binding with K pump inhibition.Accordingly, the values reported here of 450-500 glycoside receptors per humanerythrocyte, 90-120 for HK sheep red cells and thirty to fifty for LK cells shouldweigh heavily among the efforts to reach a correct estimate of this cellular parameter.Apart from curiosity and a positivistic belief in the 'correct' answer, the primary

interest in determining absolute (rather than relative) numbers of K pump sites

169

C. H. JOINER AND P. K. LAUFderived from their use in the calculation of the molecular turnover of the K transportsystem. K pump turnover was determined in this study for human cells to be2600-2900 ions/site. min, as is shown in Table 1 in comparison with turnover numberscalculated by others. Since it is known that 2 K ions are pumped for each ATPhydrolysed by the human erythrocyte transport system (Sen & Post, 1964; Garrahan& Glynn, 1967), the K pump turnover numbers for human cells correspond to an

TABLE 1. The number of [3H]ouabain binding sites on human erythrocytes.See text for discussion.

Turnover numbers are calculated for active K influx of 1-5 m-mole/l.cell.hr and cellvolume of 87 /Sm3.

b This estimate was made from the loss of scillaren from incubation media.c Cs+ was reported to decrease the 'non-specific' binding which occurred in Na media.d The author(s) cite the Ph.D. Dissertation of C. J. Ingram, Yale Univ., 1971.e This calculation is based on a protein content per single ghost of 2 x 10-9 g (Bodemann &

Hoffman, 1976a).I Extrapolated to 100% inhibition of (Na + K) ATPase of ghost membranes.Numbers as high as 360 appear in the kinetic studies of this work.

h Calculated from Schatchard plots using ghost membranes and very low ouabain concen-trations (10-810-10 M).

' The authors reported that the use of Bray's for extraction resulted in the removal of onlytwo thirds of the radioactivity removed from cells by extraction with the triton/toluenescintillant.

Reference

Glynn (1957)Ellory & Keynes (1969)

Hoffman (1969)

Hoffman & Ingram (1969)Hoffman (1973)Knauf, Proverbio & Hoffman(1974)

Bodemann & Hoffman(1976a, b, c)

Baker & Willis (1972)Gardner & Conlon (1972)

Erdmann & Hasse (1975)Wiley et al. (1975)

This study

Maximumbindingsites/cell

< 1,200200

200(100 with Cs+)c

200250d

225'275d3609200

1,200

230h340

450-500

Turnovernumber

K ions/site. min"

6,500

6,500(13,000)6,5005,2002,700

5,8004,8003,600

[3H]ouabaincounting method

b

Solubilized ghosts in Bray'ssolution

Extraction from cells or

ghosts with Bray's solutionAs aboveAs aboveAs above

As above

6,500 Solubilized ghosts1,100 Extraction from cells with

perelloric acid; triton/toluene scintillant

5,700 Solubilized ghosts3,800 Extraction from cells

with triton/toluenescintillant'

2,600-2,900 Solubilized membranes

ATP hydrolysis rate of 1300-1500 molecules/site.min for cells pumping ions at a

'normal' rate. This number is considerably lower than the ATP hydrolysis turnovercalculated for microsomal or 'purified' (Na + K) ATPase of 6500 to 11,000/ouabainbinding site per min (Barnett, 1970; Hansen, 1970; Lane, Copenhaver, Lindenmayer& Schwartz, 1973; Jorgenson, 1974; Pitts & Schwartz, 1975). However, there are

170

OUABAIN BINDING TO ERYTHROCYTESseveral reasons to believe that the enzyme turnover rates are compatible with lowerturnover numbers found in erythrocytes in this study. Enzyme turnover rates wereusually measured under conditions where membrane asymmetry was lost and at highNa/K ratios, so that these values represent maximal reaction rates (see referencesabove). In constrast, the turnover numbers reported for human cells were calculatedfor 'normal' pump rates. Experiments in the following paper (Joiner & Lauf, 1978b)and by Garay & Garrahan (1973) indicate that K pump rates may be stimulated3-4 times the normal rate by internal cation alterations.The total number of ouabain binding sites on HK sheep red cells was estimated

from Fig. 6 to be 9-120 per cell, giving turnover numbers of 200}-2600 ions/site. mibased on an average K pump activity of 0 788 + 0 050 m-mole/l. cells. hr. However,it was noted that sixty to seventy pump sites carried the bulk (about 80 %) of theactive K influx of these cells. Turnover numbers calculated for these functionallydominant sites ranged from 2700 to 3200, which was close to those of human cells.The average value of active K influx for LK cells in this study was 0-183 +

0*014 m-mole/l. cells. hr. Using the value of thirty to fifty K pump sites per cell(Fig. 6), turnover numbers of 1100-1800 ions/site. min were obtained. Thus underphysiological conditions, the transport sites of LK cells operated at a rate of onehalf to one third that of HK sites. It is known that the cation pump of LK cells ismore sensitive to inhibition by internal K than that of HK cells (Lauf et al. 1970;Hoffman & Tosteson, 1971), and this characteristic may be responsible for some of thedifference in turnover numbers between the two cell types. However, differences inthe maximum velocity ofK pump turnover also appear to be important. Hoffman &Tosteson (1971) determined by kinetic analysis a maximum pump capacity of2 8 m-mole K/l. cells. hr for HK cells, giving a maximum turnover number of 10,000(assuming eighty-five dominant sites). Unfortunately, the PCMBS treatment used toalter internal cations inhibited LK pump flux (more than HK), so that they believedtheir estimate of maximum pump rate for these cells to be low. Our estimate ofthe parameter was taken from LK cells made essentially K free by nystatintreatment (see Joiner & Lauf, 1978b) and was approximately 0-5 m-moles/l. cells. hr,giving a maximum turnover number of 4000 ions/site . min. Thus, LK pumps werequalitatively different from HK pumps in two ways: their kinetic response to internalcation activation and their maximum velocity of pump turnover. However, at theirnormal internal cation concentrations, both HK and LK cation pumps operated atapproximately one third of their maximum rate.Our previous report (Joiner & Lauf, 1975), and present results (Figs. 4 and 7),

have established conclusively that anti-L does not change the number of [3H]ouabainbinding sites or functional K pumps on LK sheep red cells. Thus the stimulation ofactive K transport by the antibody must result from increased turnover of eachpump site. In this regard, it should be noted that for goat red cells, anti-L stimulatedK pump rate to a much greater extent than it increased the apparent number of Kpump sites (Sachs et al. 1974a, b). Thus in these cells anti-L also increased K pumpturnover.An estimate of the maximum K pump rate for anti-L treated LK cells of 1.2

m-mole/l. cells. hr comes from experiments reported in the following paper (Joiner &Lauf, 1978b). This value yields a turnover number of 9000-10,000 ions/site. min.

171


Thus, if the value of 4000 represents the true maximum turnover rate of normal LKcells, it would appear that anti-L increases this parameter. In contrast, Sachs et al.(1974b) reported kinetic studies which suggested that the maximum pumping rate ofLK goat red cells was unchanged by anti-L. Similar results were obtained in studiesof goat red cell membrane (Na + K) ATPase (Cavieres & Ellory, 1975, 1977). Resolu-tion of these discrepancies, as well as evaluation of the significance of the similaritybetween the maximum K pump turnover of HK and anti-L treated LK red cells,awaits further study of the kinetic behaviour of the cation pump at very low intra-cellular K concentrations.Although anti-L did not alter the number of [3H]ouabain binding sites on LK cells,

its effect on the rate of glycoside binding was as dramatic as its stimulation of activeK transport (Fig. 6; also Joiner & Lauf, 1975). This increase was correlated with theincreased rate of K pumping brought about by the antibody. This phenomenongenerated the hypothesis that ouabain binding might be modulated by interactionsat the internal surface of the membrane and related to the control ofK pump activity.This concept is explored in the following report (Joiner & Lauf, 1978b).

We are happy to acknowledge the technical assistance of Elizabeth Herndon, Barbara Stiehl,and William Sun. We are also grateful to David Shoemaker for suggestions on the manuscriptand to Ms Patti Muck for expert typing. This work was supported by the National Institutes ofHealth (grant USPHS 2 P01-12,157) and a predoctoral fellowship (to C.H.J.) from the InsuranceMedical Scientist Scholarship Fund and the Massachusetts Mutual Life Insurance Company.

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BODEMANN, H. H. & HOFFMAN, J. F. (1976a). Side-dependent effects of internal versus externalNa and K on ouabain binding to reconstituted human red blood cell ghosts. J. gen. Physiol.67, 497-525.

BODEMANN, H. H. & HOFFMAN, J. F. (1976 b). Comparison of the side-dependent effects of Naand K on orthophosphate-, UTP-, and ATP-promoted ouabain binding to reconstitutedhuman red blood cell ghosts. J. gen. Physiol. 67, 527-545.

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