CM rMchl(1992)

13, 658467

0 Lcogman Group UK Ltd 1392

Inhibition of the human erythrocyte calcium pump by dimethyl sulfoxide P.J. ROMERO lnstituto de Siologla Experimental, Fat. Ciencias, Universidad Central de Venezuela, Caracas, Venezuela Abstract - The action of dimethyl sulfoxide on the human red cell Ca*+ pump was studied in inside-out vesicles. In a high-K+ medium at pH 7.6, the organic solvent inhibited both Ca*’ transport and ATP hydrolysis. Half-maximal effect was obtained with about 2% (v/v). At or below 10% dimethyl sulfoxide, the inhibition was overcome by adding inorganic phosphate or oxalate. In the absence of organic solvent, Ca*’ efflux from Ca*+-loaded vesicles consisted of a slow and a fast component whilst in its presence, there appears additionally a leakage component. The size of the iatter depended markedly on dimethyl sulfoxide concentration, being about 3% at that level where Ca*’ uptake was half-maximally inhibited. ATP hydrolysis was more sensitive to dimethyl sulfoxide (10%) when free Ca*+ was increased within the millimolar level than when it was raised within the micromolar range. On the other hand, raising Ca*’ with organic solvent greatly stimulated ATP synthesis through ATP-Pi exchange, without reaching saturation. The results suggest that dimethyl sulfoxide blocks the red cell Ca*+ pump by increasing the affinity of the Ca*+ translocating site at the releasing step. They also show that at high concentrations, this solvent increases Ca*+ permeability.

It is well known that the red cell Ca2’ ATPase is biphasically affected by22r2+. Low concentrations (in the micromolar Ca range) are stimulatory whilst high concentrations (at the millimolar level) become inhibitory 11, 21. This behaviour is not altered by the presence of CaM [3, 41. The reasons for such a biphasic behaviour are still not clear. It is possible that high Ca2’ concentrations may inhibit by facilitating ATPase reversal, as can be inferred Abbreviations : DMSO, dimcthyl sulfoxide; IOVs. inside-out membrane vesicles; Pi, inorganic phosphate; CaM. calmcdulin; SR, sarcoplaamic reticulum.

from data reported in [3]. Recent work on the purified enzyme from human red cells, showed that DMSO acts like CaM increasing both turnover and affinity for Ca2’ and ATP 15, 61. It was tiuther shown that the DMSOactivated ATPase was inhibited by high Ca2+ concentrations 161. Unlike CaM, however, DMSO stimulates ATP-Pi exchange [6, 71. The present investigation tests these findings on IOVs from human erythrocytes. The sidedness of such a preparation is suitable to decide on the {robable assumption that the inhibitory side of the Ca ’ curve (trans side) is similarly affected by DMSO as the 659

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stimulatory (cis) side, namely by an increase of Ca affinity, as suggested by its stimulation of the ATP-Pl exchange reaction.

Materials and Methods Analytical grade reagents were used whenever possible. DMSO was obtained from Riedel-de Haen, Germany. Fresh human blood (mainly O(+) group) from healthy donors was supplied by The National Cancer Institute, Brazil. 45Ca2twas purchased from Amershatn, UK. 32Pi obtained from the Brazilian Institute of Atomic Energy, was purified by extraction as phosphomolybdate with isobutanol-benzene, re-extraction to the aqueous phase with ammonia and precipitation as MgNlWO4 [8]. [Y-~+]-ATP was prepared as described by Glynn and Chappell 191. IOVs were prepared as described by Sarkadi et al. [lo] but homogenizing manually by 10 strokes, using a micro-homogenizer equipped with a teflon pestle (A. Thomas A54389). IOVs were finally resuspended in a small volume of a 120 mM KCl, 50 pM ~mercaptoethanol, 1 mM MgC12,50 mM Tris-HCl medium (pH 7.6) to obtain about 15-20 mg protein/ml, divided into 300 pI aliquots and stored under liquid nitrogen. The fraction of inverted vesicles, assessed by acetylcholinesterase measurements [ill, was between 60-W%. CaM (10 @ml) stimulated this preparation by a factor of 1.2-1.5. IOVs were incubated at 37°C in a basal medium consisting of (mM): KCl, 120; MgC12,2; ouabain, 0.1; Tris-HCl, 50 (pH 7.6); plus additions as specified below. 45Ca2t uptake was measured by ultratiltration (0.45 pm Millipore filters), after incubating in basal medium supplemented with 0.05 mM CaC12(with tracer 45Ca added to a specific activity of about 2500 cpm/nmole). The filters were washed 4-times, dried and immersed in standard scintillation liquid for radioactivity measurements, Enzymatic activities were assessed after incubation in basal medium, containing 10-20 mM MgCh, 5 mM Pi-Tris and 0.1-0.5 mM ADP, and either [y-3?P]-ATP (400 cprn/nmole ATP) or [32P]-Pi (10 000 cpm/nmole P$ ATPase activity was assayed by measuring 2Pi release from

I

L

I

I

0

2

C

6

4 6

10

DMSO Concentration (% , v/v 1

Fig. 1 Inhibition of Ca” tmnsport by DMSO and its reversal by addition of Ca” precipitating agents IOVs from human red cells (about 0.4 mg protein) wcm incubated for 1 h at 37°C. with the different DMSO concentrations shown above, in a medium containing (mM): KCI, 120; MgCl2, 2; ATP-Tris, 2; ouabain. 0.1; Tris-HCI, 50 (pH 7.6) and CaCbO.05 (with tracer 45Ca2’ added to a specific activity of about 2500 cpm/mnole). They were incubated without precipitating agents (open triangles) and with 5 (closed circles) and 40 mM Pi (open circles) or with 0.5 (closed squares) and 2 mM oxalate (open squares). 45Ca2’ uptake by IOVs was measured by liquid scintillation counting, after u1trati1ttation through 0.45 pm pore size filters. ‘Ihe results shown are the average value of assays run by duplicate from a typical experiment

[Y-~%]-ATPafter quenching the reaction with 2 vols of activated charcoal in 0.1 N HCl [12]. ATP-Pi exchange was measured by detemrining the amount of 3%‘i incorporated into ATP (in y-position), following extraction of excess 32Pi as phosphomolybdate with an isobutanol-benzene mixture [131. All experiments on ATP-Pi exchange were accompanied by parallel measurements of 45Ca2+ uptake and ATP hydrolysis by the same IOVs batch under similar conditions. Free Ca2’ concentrations were calculated from a computer program based on equations described by Fabiato and Fabiato 1141.Radioactivity was measur-

DIMETHYL

SULFOXIDE

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OF ERYTHROCYTE

661

Ca PUMP

ed by liquid scintillation counting. Protein was estimated by the Lowry method, using bovine serum albumin as standard [151.

increased by 10% DMSO to 3.7 and 1 mM, respectively. The above findings show that DMSO inhibits Ca2’ uptake in a way antagonized by precipitating anions.

Results Effect of DMSO on Ca2+ ejJ?uxfrom IOVs Inhibitionof Ca2’ transportby DMSO In a high K+ medium and without added CaM, Ca2’ uptake was variable, ranging between 500-700 nmoles Ca2’/mg IOV protein in 1 h. As was expected, DMSO inhibited the uptake, half-maximal effect being attained with nearly 2% (v/v) whereas maximal was with about 5-1096 (Fig. 1). The action of DMSO was almost prevented by adding 5 mM Pi or 0.5 mM oxalate (Fig. 1). At higher concentrations of preci itating agents (40 2P mM Pi and 2 mM oxalate), Ca uptake was nearly doubled and the inhibition by DMSO was fully overcome (Pig. 1). DMSO and the precipitating anions appeared as if they were ‘competing’ with each other, as revealed from reciprocal plots (Fig. 2). The amount of Pi and oxalate required for half-maximal effect was 0.26 and 0.63 mM, being

An apparent inhibition of Ca2’ uptake may arise from an increased Ca2’ permeability elicited by DMSO. To test this possibility, IOVs were previously loaded with 45Ca2t by incubating with ATP. Thereafter, Ca2’ efflux was assessed in isotonic KC1 medium containing various DMSO concentrations, with and without addition of precipitatin anions. !+ Without DMSO, the amount of Ca taken up during the loading period apparently leaves IOVs from two different compartments (Fig. 3). The ‘fast’ component comprising about 25% of the total, has a rate constant (mean + 1 SD of 4 experiments) of -23.8 + 0.38 (x 10m3min-I). The ‘slow’ component having a rate constant of -3.7 It 0.65 (x 1O-3 mm-‘) (see Table 1). In addition to these compartments, there is another from which Ca2’ leaks out within mixing time in the presence of DMSO (Fig. 3). Its

al Reciprocal Efg. 2 Antagonism IOVs were incubated

between precipitating as described

I

1

a

1

Anion Concentration

2

(m Id-‘)

agents and DMSO

in the caption to Figure 1, either in the presence

(right panel), with and without the following

DMSO additions

(closed circles). The graph shown is 8 reciprccal

of various Pi (left panel) or oxalate concentrations

(v/v): none (open circles); 2 (open triangles);

plot of mean values from two experiments

5 (open hexagons)

and 10%

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1

I

lo

'0

I

I

I

20

30

40

Rg. 3 EBlux kinetics from Ca2+-loaded vesicles and action of DMSO IOVs (1.8 mg/ml) were preloaded with 4sCa2+by incubating for 30 min at 37°C in 0.5 ml of a medium containing (n&I): KCl, 120; Tris-HCI, 20 @H 7.5); MgClz 2; CaClz. 0.1 (with tracer 4sCa2+ added to a specific activity of 3 x lo6 cpm/mnole); ATP.Na2, 2; creatine phosphate, 10; and creatine kinase (10 units/ml). After washing (54imes with 1 ml of 150 mM KCl t 20 mM Tris-HCl @H 7.5) medium), the vesicles (0.8-1.2 mg/ml) were incubated at 37’C in 0.1 ml of fresh washing medium, which contained no (circles) or 10% (v/v) DMSO (triangles). Aliquots were taken at time zero and then at regular intervals, ultrafiltered and the radioactivity assessed as described in the caption to Figure 1. Both ‘fast’ and ‘slow’ components of efflux were obtained from graphical analyses, as shown above for a single representative experiment Notice presence of a thhd compartment in the presence of DivfSO, from which Ca2’ leaks out within mixing time. The Ca2’ efflux rate constant was calculated from linear regression analysis of the log (efflux) versus time. Without DMSO. the two di&rent compartments having rate constants of -23.8 f 0.38 and -3.7 f 0.65 (x 10V3min-‘) (mean It 1 SD of 4 experiments)

Time 1 min 1 Table 1 The effect of DMSO on Ca2’ permeability of IOVS DMSO concentration

Fraction of “Ca2’ retained ajier loadng (90)

(%) (v/v)

[or C(12’ eflux rate constant (Inin-’ x lcr”)] NO Pi

0

100 (4)

5mMPi 98.5 f 2.5 (4) [-3.1 f 0.501

lo&Pi

100 (2) f-3.31

NO Pi I 10 Mg2’

-

r-3.7 f 0.651 -

2

97.3 f 3.1 (4)

98.7 f 1.7 (4)

5

80.6 f 8.3 (4) [-3.1 f 0.861

78.5 f 7.8 (4) [-1.7 f 0.40]

83.7 (2) (-2.41

45.4 f 8.8 (4) [-2.0 f 0.671

55.9 f 5.4 (4) I-l.6 f 0.441

67.1 (2)

10

I-o.61

-

60.8 (2) [-3.31

20

38.9 f 5.7 (4) [-2.9 f 0.311

-

-

-

40

32.3 f 9.6 (4) [-3.4 i 1.131

-

-

-

?he vesicles (0.8-1.2 mg/ml) were incubated as dwcni in the caption to Figure 3, in fresh washing medium cont&ing the additions stated in the Table. ‘lbe Cap efflux mte constant was calculated fmm linear regremion analysis of the log (eRlux) vuaus tims. ‘lbe fkcticn of tica* (%) mnkning in the vesicles atIer loading (referred to thcee loaded witbout DMSO) and the ‘abw’ component late constanta (in min-’ x lt?, shown enclosed in brackets) are given as the mean value f 1 SD of the number of experiments shown in parenthesis

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Ca PUMP

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Fig.4 The timecourse of DMSO effects on Ca2’ uptake, ATP hydrolysis and associated ATP-Pt exchange IOVs (about 0.446

mg protein) were incubated at 37°C for the

length of time indicated in abscissae, (n&l):

in a medium containing

KCl, 120; ATP, 2; ADP, 0.1; MgClz,

10; Pi-T&,

ouabain, 0.1; Tris-HCl, 50 @H 7.6) and Cat& without

tracer 45Ca2’, added to a specific

5;

0.5 (with and

activity

of 5000

cpmlnmole

Ca2+), in the presence of 0 (open circles); 5 (open

triangles);

10 (open squares) and 20 % (v/v) DMSO (closed

circles).

At the time indicated by arrows, 5 pM A23187

added. 45Ca2+ uptake by IOVs ultraWation,

as mentioned

(Part

a) was

measured

was by

in the caption to Figure 1. ATP

hydrolysis (Part b, upper graph) and ATp-& exchange (Part b. lower graph) were assessed by incubating in the above medium, but containing [y3?‘]-ATP Time

(10 000

(h)

cpm/nmole

assayed by measuring

4).

(400 cpmlnmole respectively.

[3P]-R

ATP) and [32P]-pi

ATPase

release

activity

was

from [y-32P]-ATP after

quenching the reaction with 2 vols of activated charcoal in 0.1 N HCl.

ATP-R

incorporation extraction

exchange

was

measured

of [32P]-PI into ATP

of excess

isobutanol-benzene

by

determining

(in y-position),

[32P]-Pi as phosphomolybdate

the

following with an

mixture. Results fmm a typical experiment are

presented using the same preparation

0

1

2

3

Time (hl

size increased with DMSO concentration, being about 55 and 68% at 10 and 40 % (v/v) DMSO, respectively (Table 1). Regardless of the various DMSO concentrations tested, the ‘slow’ efflux component was always present (see brackets in Table 1). By contrast, the size of the ‘fast’ component was correspondingly reduced upon raising DMSO as

illustrated in Figure 3, where it was decreased roughly from 25 to 19% as DMSO was raised from 0 to 10%. The ‘slow-component’ rate constant was little affected by adding 5-40 mM Pi (Table 1) or 2 mM oxalate (results not shown), with and without DMSO. On the other hand, at 10 % DMSO and high Pi concentrations there was a trend for reducing slightly the leaky fraction (see Table 1). A similar trend seems to occur when 10 mM Mg2+ was subStitUkd for Pi. The results show that DMSO increases IOVs’ Ca2’ permeability in a way scarcely affected by precipitating anions. ATP hydrolysis and ATP-Pi exchange The effect of DMSO on the time course of Ca2+ transport and associated ATPase activity was studied under conditions favouring ATP-Pi exchan e 9+* that is, in the presence of (ITIM): Ca2+, 0.5; Mg , 10; ATP, 2; ADP, 0.145; and Pi, 5.

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Effect ofCa2+and DMSO on ATPase cycle

0

10

20

DMSO Concentration

30

co

Upon increasing free Ca2+ from 0.1 jrh4 to 1 mM, ATP hydrol sis showed the usual biphasic behaviour, CaSt king activatory in the micromolar range and inhibitory at the millimolar level (Fig. 6). Concomitantly, under ATP-Pi exchange conditions, ATP synthesis was about lOOO-timeslower than hydrolysis and was scarcely affected by raising Ca2+ in the above concentration range. Above this level, however, ATP synthesis was increased lo-fold by raising Ca2+to 2 mM. The activation by Ca2’ in the micromolar range was not affected by the presence of 10% DMSO. However, within the millimolar level, the enzyme seemed to become inhibited by much lower Ca2+ concentrations, as can be inferred from the amount of Ca2’ required for half-maximal inhibition (Fig. 6). In the presence of dimethyl sulfoxide, ATP-Pi exchange was greatly stimulated without showing signs of saturation. On the other hand, addition of CaM (10 pg/ml) affected neither the extent of the exchange reaction nor the activation of this reaction by DMSO (results not shown).

(%, v/v 1

Parallel changes of Ca’+ uptake, ATP hydrolysis and ATP-R exchange during incubation with DMSO IOVs were incubated for 2 h at 37°C with the different DMSO concentrations shown in the graph, under conditions stated in the caption to Figure 3. Collected results from two experiments are given

Fig. 5

Ca2’ uptake and ATP hydrolysis were both inhibited by raising DMSO (Fig. 4a,b). However, high concentrations were required as could be expected for the simultaneous presence of 5 mM Pi and the organic solvent. Hydrolysis was affected to an extent similar to that of uptake, being half-maximally inhibited with about 25% DMSO (Pig. 5). On the other hand, ATP-Pl exchange was increased from 0 to 12 nmoles ATP/mg protein in 2 h as DMSO was raised to 40%. Addition of A23187 (5 @I) during Ca2’ uptake halted both hydrolysis and exchange whilst uptake could not be sustained and Ca‘+ leaked out from the vesicles (Fig. 4a.b).

A 1.6 .

Ng. 6 The effect of DMSO on the Ca2+-ATPase cycle IOVs were incubated with (circles) and without 10% (V/V) DMSO (triangles) and the different free Cr*’ concentrations shown above. as described in the caption to Figure 3. The extent of both ATP hydrolysis (open symbols) and ATP-Pt exchange (closed symbol.9 were estimated alter 2 h incubation. Results are given as mean values from two experiments

DIMETHYL SULFOXIDE INHIBITION OF ERYTHROCYTE Ca PUMP

Discussion A dual action of DMSO on IOVs Ca2’ transport was found in the present work. At low concentrations (2-S%), DMSO inhibited active uptake whereas at higher concentrations it also increased Ca2+ permeability. As an apparent inhibition of transport may arise from an increased prmeability and IOVs showed a low transport capacity, some aspects of Ca2+ transport in untreated IOVs must be discussed first. Pump activity was about one-fifth of reported values when measured through Ca2+ uptake [lo] whereas it was a half of that expected when assessed from ATPase activity. These findings suggest that our preparation either contained a small fraction of sealed vesicles, consisted of vesicles with a high Ca” permeability or both. In addition, vesicular size may be so small that the ump becomes backinhibited very rapidly as Ca!+ is taken up. These possibilities arc considered below, Permeability of IOVs to Ca2+

Without DMSO Ca2’ efflux from Ca2+-loaded IOVs consisted oi two components. The ‘slow’ one, representing the least permeable vesicles, comprised about 75% of the total population. It showed a rate constant of about -4 x 10m3mid’. almost identical to that reported for the ‘slow’ component of Ca2’ efflux in ATP-depleted intact cells [16]. Although rate constants cannot be used for direct comparison of intrinsic membrane permeabilities in systems with widely different area/volume ratios, the results show that the ‘slow’ vesicular compartment has an abnormally low Ca2’ permeability by having a ‘normal’ rate constant. These observations indicate that most of the IOVs presently used are sealed and do not show a high Ca2+-permeability. It therefore seems probable that the low-capacity of IOVs for Ca2+ transport mainly arises from a reduced vesicular size. Modification of Ca2+ permeability by DMSO and pump inhibition

Unlike in SR membranes [17], DMSO elicits Ca2+ leakage in IOVs. The vesicles, however, do not

665

behave homogeneously since a resistant fraction persists. This fraction is about 97% at a DMSO concentration that reduced Ca2’ uptake halfmaximally, namely 2% v/v (see Table 1). These findings clearly show that 2% DMSO. genuinely inhibits active transport whilst having a negligible effect on permeability. At concentrations higher than 5%, however , Ca2’ permeability is progressively increased. Pump inhibition by low DMSO concentrations is antagonized by Pi. As oxalate led to similar findings, a direct pump effect is highly unlikely to be involved. Pi presumably acts via its precipitating capacity. Ca2+ precipitation may increase uptake through two mechanisms. First, by preventing pi inhibition due to an elevated intravesicular Ca level. Secondly, by reducing a non-specific Ca2+ leak. As permeability was scarcely affected by the above anions, a high free Ca2’ concentration within vesicles must be inhibitory, such as occurs with the SR Ca2’ ATPase [13, 181. It is known that at low concentrations, DMSO substitutes for CaM, increasing affinity of the high-affinity site of the purified red cell enzyme [6]. The possibility exists that a similar action of DMSO may be present at the low-affinity site. In a system such as IOVs where the sidedness of the pump has been preserved, the native enzyme must expose alternatively the low and high affinity Ca2+ binding sites (transport sites) to different compartments to accomplish ion translocation. Therefore, it is clearly expected that the raised affinity of both binding sites promoted by increasing amounts of DMSO would lead to Ca2+ uptake inhibition, as presently found. This is well supported by the marked stimulation of ATP-Pi exchange elicited by DMSO, as discussed below. The above suggestion also may explain the dual effect of DMSO recently reported on the purified enzyme, with and without CaM [5,6]. To our knowledge, this is the first systematic study of the action of DMSO on active Ca2’ tTiiI.iSport in human red cells. Previous work with IOVs showed a marked reduction of Ca2+ uptake by 0.5% DMSO, that could be overcome by increasing the amount of vesicles above 0.4 mg protein/ml assay media [19]. At comparable protein concentrations, the present work has shown inhibition by DMSO of both transport and hydrolytic activities. The

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simultaneous inhibition of Ca” uptake and ATP hydrolysis by DMSO is not exclusive to IOVs as this effect was originally reported in SR vesicles [17]. In both systems, DMSO is equally effective on the two pump expressions and seems to have the same inhibitory potency. ATP-Pi exchange At a Ca2+ concentration that sahrrates only the high affinity binding site (about 0.1 r&I), the enzyme exclusively catalyzes ATP hydrolysis. The affinity of this site is barely affected by DMSO (Fig. 6). This result may be expected from observations on the CaMcontaining purified enzyme 161, if it is assumed that endogenous CaM is present in IOVs. The latter is a likely possibility as IOVs were scarcely stimulated by added CaM and they were prepared without EDTA. On the other hand, increasing Ca2+ to the millimolar range resulted in both inhibition of ATPase activity and activation of ATP synthesis through the ATP-Pi exchange reaction. This shows that blockade of the overall reaction of the ATPase cycle arises from an activation of reversal. Such an idea is consistent with a recent proposal that Ca2+ at high concentrations, inhibits ATP hydrolysii by preventing the dephosphorylation of the Ca2+-ATPase complex 131. Our findings on the ATP-Pi exchange reaction agree well with recent observations on the purified preparation [6]. Lack of participation of the Nat pump in the ATP-Pi exchange Without monovalent cations, the Nat pump can be phosphorylated by Pi and affinity for this anion is increased by DMSO 1201. However, ATF-Pi exchange exclusively ensues in the presence of Na+ [20, 211. In addition, the exchange reaction is blocked by ouabain and low Kt concentrations [21-231. Because in the present experiments IOVs were prepared in the absence of Nat and both ouabain and a high Kt concentration were used, it is highly unlikely that Nat pump activity may have contributed to the results.

Action of A231 87 A puzzling result is the cessation of ATPase and ATP-Pl exchange after ionophore treatment. If IOVs were perfectly coupled5+ the addition of A23187 should have released Ca (which in fact it did) and increased ATP hydrolysis. This result was not found IOVs were incubated with enough A23187 to render them leaky (5 pM) and with a rather high Ca2’ concentration (0.5 mM). It seems probable, therefore that the equilibrium Ca2+ concentration reached &er ionophore addition, is just enough to keep a steady state between ATP hydrolysis and synthesis due to Ca2’ binding to an intravesicular inhibitory low-affinity site (see below). In the presence of DMSO a new steady state level is to be expected. Pump inhibitionby activationof reversal Addition of DMSO largely increased the extent of ATP-Pi exchange and seemed to make the enzyme more susceptible to inhibition by a high Ca2’ concentration, thus showing that DMSO increases Ca2’ aflinity at the low affinity binding site. Such a behaviour is clearly distinct from that found with SR vesicles, where DMSO does not alter affinity of either site [18]. On the other hand, with and without DMSO, CaM (up to 10 pg/ml) did not change the extent of the ATP-Pl exchange reaction. This is to be expected from the maintenance of a steady state of phosphorylation, arising from the simultaneous increase by CaM of both phosphorylation and dephosphorylation rates [3,24]. As the inhibition of both transport and hydrolytic activities is paralleled by an increase in the ATP-Pi exchan e DMSO may be increasing the affinity of the Cati+? translocating site at the releasing step. These findings lend support for the view that high Ca2’ concentrations may become inhibitory by facilitating ATPase reversal [3] and further suggest, that the bell-shape dependence on Ca2+ of the ATPase arises from interaction at two different binding sites. The enzyme apparently displays sequentially two Ca” binding sites of different affinities during its catalytic cycle, so that occupancy of the low-affinity site would lead to the inhibitory effect.

DlMIXHYL

SULFOXIDE

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OF ERYTHROCYTE

Acknowledgements The author is grateful to Dr Leopold0 de Meis for providing optimal facilities in his laboratory, encouraging help and critically reading the manuscript. He also wishes to acknowledge financial support from the Conselho National de Desenvolvimento Cientifico e Tecnologico, Brazil and to both the Consejo de Desarrollo Cientifico de la Universidad Central and Consejo National de Investigaciones Cientfficas y TecnoQicas, Venezuela, for a traveliing fellowship. He is also indebted to Dr Vivian Rumjanek from The National Cancer Institute, Brazil, for kindly providing the blood and to Mr Isaltino Soarcz and Valdecir

Suzano

for skilled

technical

12.

13.

14.

15.

aid. 16.

References 1. Klinger R. Wetzker R. Fleischer I. Frunder H. (1980) Effect of calmodulin, Ca” and Mg2+ on the (Ca’++Mg”)-ATPase of erythrocyte membranes. Cell Calcium, 1,229-240. 2. Vincenzi FF. Hinds TR. Raes BU. (1980) Cahnodulin and the plasma membrane calcium pump. Ann. NY Acad. Sci., 356,232-244. 3. Allen BG. Katz S. Roufogalis BD. (1987) Effects of Ca”, M$+ and calmodulin on the formation and decomposition of the phosphorylated intermediate of the erythrocyte Ca2+-stimulated ATPase. B&hem. J., 244,617-623. 4. Schatzmann HJ. (1982) The plasma membrane calcium pump of erythmcytes and other animal cells. In: Carafoli E. (ed.) Membrane Transport of Calcium. New York, Academic Press, pp 41-107. 5. Benaim G. de Meis L. (1989) Activation of the purified erythrocyte plasma membrane Ca”-ATPase by organic solvents. FEBS Lett., 244.484486. 6. Benaim G. de Meis L. (1990) Similarities between the effects of dimethyl sulfoxide and calmodulin on the red blood cell Ca’+-ATPase. B&him. Biophys. Acta, 1026, 87-92. I Chiesi M. Zurini M. Carafoli E. (1984) ATP synthesis catalyzed by the purified erythrocyte Ca-ATPase in the absence of calcium gradients. Biochemistry, 23,25%-26(X1. 8 Kanazawa T. Boyer PD. (1973) Occurrence and characteristics of a rapid exchange of phosphate oxygens catalyzed by sarcoplasmic reticulum ATPase. J. Biol. Chem., 248.3163-3172. 9. Glynn IM. Cba pell JB. (1964) A simple method for the R preparation of P-labelled adenosine triphosphate of high specific activity. B&hem. J., 90,147-149. 10. Sarkadi B. Szasz I. Gardos G. (1980) Characteristics and regulation of active calcium transport in insideout red cell membrane vesicles. Biochim. Biophys. Acta, 598, 326-338. 11. Steck TL. Kant JA. (1974) Preparation of impermeable

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21.

22.

23.

24

ghosts and inside-art vesicles from human erythrocyte membranes. Meth. Enzymol., 31A, 172-180. Grubmeyer C. Penefsky HS. (1981) The presence of two hydrolytic sites on beef heart mitochondrial adenosine triphosphatase. J. Biol. Chem., 256.3718-3727. de Meis L. Carvalho MGC. (1974) The role of the Ca” concentration gradient in the AT&Pi exchange reaction catalyzed by sarcoplasmic reticulum. Biochemistry, 13, 5032-5038. Fabiato A. Fabiato F. (1979) Calculated programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris), 75, 463-505. Lowry OH. Rosebmugh NJ. Farr AL. Randall RJ. (1951) Protein measumments with the Folin phenol reagent. J. Biol. Chem., 193,265-275. Schatzmann HJ. Vincenzi FF. (1%9) Calcium movements across the membrane of human red cells. J. Physiol., 201, 368-395. The R Hasselbach W. (1977) Stimulatoty and inhibitory effects of dimethyl sulfoxide and ethylene glycol on A’lPase activity and calcium transport of sarcoplasmic membranes. Eur. J. B&hem.. 74,611-621. Makinose M. Hasselbach W. (1965) Der einfluB von oxalat auf den calcium-transport isolierter vesikel des sarkoplasmatischen reticulum. B&hem. 2.. 343. 360-382. Romem PJ. OrtIz CE. (1988) Electmgenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes. J. Membr. Biol., 101,237-246. Goncalves de Momes VL. de Meis L. ( 1987) ATP synthesis by the (Na++K+)-ATPase in the absence of an ionic gradient: effects of organic solvent. FEBS Lett., 222. 163-166. Goncalves de Momes VL. de Meis L. (1982) Exchange between inorganic phosphate and adenosine triphosphate in (Na++K+)-ATPase. Biochim. Biophys. Acta, 688.131-137. Fahn S. Koval GJ. Albers RW. (1%6) Sodium-potassium activated adenosine triphosphatase of electrophorus electric organ. I. An associated scdium-activated tmnsphosphorylation. I. Biol. Chem., 241.1882-1889. Tanimhi K. Post RL. (1975) Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem., 250, 3010-3018. Rega AF. Garmhan PJ. (1980) Effects of calmodulin on the phosphoenzyme of the Ca2+-ATPase of human red cell membranes. Biochim. Biophys. Acta, 596.487-489.

Please send reprint requests to : Dr Pedro J. Romero, Institute de Biologia Experimental, Fat. Ciencias, Universidad Central de Venezuela, Apartado 47114, Caracas 1041-A, Venezuela Received : 6 April 1992 Revised : 4 June 1992 Accepted : 5 June 1992