Decreased (Ca2+ + Mg2+)-Stimulated ATPase Activity in Erythrocyte Membranes from Polycythemia Vera Patients 0. SCHARFF & B. FODER Medical Isotope Dept., Finsen Institute, Copenhagen, Denmark

Scharff, 0. & Foder, B. Decreased (Ca*+ + Mg'+)-Stimulated ATPase Activity in Erythrocyte Membranes from Polycythemia Vera Patients. Scand. J . clin. Lab. Invest. 35, 583-589, 1975.

Erythrocytes were hemolyzed in hypotonic phosphate buffer containing 0.5 mmol/l Ca*+ and the membranes subsequently washed twice in hypotonic tris buffer. The centrifugation was performed in a continuous flow system, which was necessary to obtain maximal ATPase activity. The Mga+-dependent Ca*+- stimulated ATPase activity of 14 patients with polycythemia Vera was only 67 per cent (P<O.oOl) of the activity of a control material consisting of 10 donors and 1 1 bank blood specimens. Five patients with secondary polycythemia and four patients with an increased erythrocyte fraction did not differ significantly from the controls. The polycythemia Vera patients with the highest leukocyte count showed the lowest ATPase activity. The apparent calcium dissociation constant of the ATPase in polycythemia Vera was about moI/l, as in controls. The relation between the reduced ATPase activity and the abnormal hemopoiesis of polycy- themia Vera patients is discussed. Key-words: Adenosine triphosphatase; calcium affinity; erythrocyte membrane; leukocyte count; polycythemia 0. Scharif, Medical Isotope Dept., Finsen Institute, Strandboulevarden 49, DK-2100 Copenhagen 0, Denmark

The disease polycythemia Vera implies a disorder of cell proliferation in bone marrow. It has been reported that calcium is involved in the regulation of cell proliferation in mammalian tissues, calcium probably performing its effect inside the cell (6, 12). A disorder of the regulation of the intracellular calcium concentration may therefore exist in polycythemia Vera.

The membrane-bound (Ca2+ + Mg2+)-stimulated ATPase probably participates in the calcium trans- port system that pumps calcium out of the erythro- cyte (1 I ) , maintaining the intracellular Ca2+ con- centration at a very low level (10). In the present work we investigated whether the (Ca2++ Mg2+)- stimulated ATPase activity is changed in the erythrocyte membranes of patients with poly- cythemia Vera.

MATERIAL A N D METHODS

Material

Twenty-three patients who were treated with therapeutic phlebotomy are included in this study. The patients consisted of three groups:

1. 14 patients with polycythemia Vera (male/fe- male = 7 : 7; years since diagnosis = 1 1.4, stan- dard deviation (S.D.) = 6.9). The diagnosis of the polycythemia Vera was in accord with the criteria established by Modan (5) .

2. 5 patients who possibly had secondary poly- cythemia (male/female = 5 :O; years since diagno- sis = 13.0, S.D. = 7.8). The patients had increased erythrocyte volume but not the essential features of polycythemia Vera (e.g. leukocytosis and in- creased leukocyte alkaline phosphatase). Two of

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584 0. Scharff & B. Foder

the five patients had signs of respiratory deficiency and one had indication of renal insufficiency. The remaining tHo patients were brothers with a markedly elevated erythrocyte fraction since early childhocd.

3.4 patients with iccreased erythrocyte fraction during the disease but without demonstrated in- crease of erythrocyte volume (male/female = 3 : 1 ; years since diagnosis = 1.5, S.D. = 1.3).

Most of the patients in groups 1 and 2 had keen treated with 32P or cytostatic drugs, but not within the last 9 months before this investigation.

The control material consisted of blood from I0 healthy persons (male/female = 5 : 5 ) . To supple- ment the control material, erythrocyte membranes were prepared from rccently outdated bank blood (citrate-phosphate-dextrose (CPD) blood). Each tank blood preparation was made from the blood from two individuals.

Both control and rolycythemic blood was col- lected in 14 ml CPD per 100 ml blood and stored overnight at 4 'C before USC.

Methcds

Preparation o j erythrocyte membranes. The erythrocytes were washed three times in isotonic phosphate buffer, pH 8.0, as described previously (9). The membranes were prepared as earlier (9), with the following modifications. The erythrocytes were hemolyzed in 9volumes of a buffer containing 6.7 mmol/l inorganic phosphate, 1 .O mmol/l ethyl- eneglycol-bis ((3-aminoethyl ether) N,N'-tetra- acetic acid (EGTA), and 1.5 mmol/l CaCI,, pH 7.4. The hemolysate was stored at 4 "C overnight. The membranes were collected from the suspen- sion by a Szent-Gyorgyi and Blum continuous- flow device at 45,000 g and a flow not exceeding 50 mlsmin-l and washed twice with 9 volumes of 10 mmol/l Tris, pH 7.7. The duration of prepara- tion was 2 days for both patients and controls and 2-3 days for the bank blood. The membranes were stored at -25 "C.

Determination of ATPase. The specific ATPase activity (rate of release of inorganic phosphate) was determined as described previously (9), with slight modifications. The Mg2+-stimulated ATPase activity was determined at 37 "C in a basal medium of 3 mmol/l Tris-ATP, 4 mmol/l MgClp, 1 mmol/l

EGTA, 70 mmol/l Tris, and 0.5-1.0 g dry mem- brane/] medium. The pH was 7.25 at 37 'C. The reaction was started by addition of the meqbrane suspension. For determination of the (Ca2++ Mg2+)-stimulated ATPase activities the basal medium was supplemented with various con- centrations of CaCI, (0-1 80 pmolil Ca2'). From these activities the Ca2+-stimulated activities were calculated by subtracting the Mg2+-stimulated activity. The maximal Calf-stimulated activity (Vmax) is the activity obtained with optimalcon- centration of Caa+ (in the range of 10-80~mol~1-' The ATPase activity unit used is pmol .min-'.g-l, where g means g dry membrane exclusive of hemo- globin (excl. Hb.). The CaZ+ concentration during the incubation, including added calcium and calcium arising from the membranes, was cal- culated as described previously (9).

Methods ofanalysis. The deterniinations of pH, dry matter, hemoglobin, and calcium were per- formed as previously (9), with the exception that calcium was determined by atomic absorption spectrophotometry in a Corning EEL 240 instru- ment with 1.3 per cent LaCI, in the samples. Protein was estimated by the method of Lowry et al. (4).

RESULTS

Laboratory findings in patients and controls

The hematologic data at the time of investiga- tion are shown in Table I. The patients with poly- cythemia Vera showed elevated erythrocyte frac- tion (hematocrit), erythrocyte count, and leuko- cyte count, whereas the thrombocyte count was normal at this time. The group with secondary polycythemia showed only elevated erythrocyte fraction and erythrocyte count. The erythrocyte fraction of the third group of patients had been elevated but was at the time of investigation within the reference interval.

Membrane parameters

There were no significant differences between the parameters of patients and controls. The frac- tion of proteins in membranes was 0.479 g protein excl. Hb. per g dry membrane excl. Hb. (S.D. =

0.034), the membrane-bound hemoglobin was

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Ca*+-ATPae in Polycythemia Erythrocyte3 585

Table 1. Mean values of laboratory findings in patients and control donors at the time of investigation. Common standard deviations are given when possible (Bartlett's test); in the remaining two cases (indicated by an asterisk) standard deviations are given separately in parentheses

Number Erythrocyte Leukocyte Thrombocyte of Age Erythrocyte conc. conc. conc.

Group donors (year) fraction (10l2/1) (100/1) (1 0 ~ 1 )

Control 10 38.6 0.409 4.65 7.01 271 Polycythemia Vera 14 64.6 0.539 6.86 12.73* 299 Secondary polycythemia 5 39.4 0.608 6.30 6.98 163 Increased erythrocyte fraction 4 65.3 0.500 5.60 6.18 271

Standard deviation 10.6 0.036 0.70 1.67 108 (0.088) (6.14)

0.033 g Hb. per g dry membrane incl. Hb. (S.D. =

0.01 5 ) , and the membrane-bound calcium amount- ed to 12.3 ymol per g dry membrane excl. Hb. (S.D. = 4.0).

ATPase activities

The Mg2+-stimulated ATPase activity was 2.00 pmol.min-l.g-l (S.D. = 1.00) in the membrane preparations from both patients and controls. The Ca2+-stimulated ATPase activities are given in Table 11. The activity of the preparations from the polycythemia Vera patients was significantly lower than the activity of the control group. Further- more, the difference between the patients with polycythemia Vera and those with secondary poly- cythemia was significant ( P 0.02). Repeated membrane preparations of blood from the same donor showed a standard deviation of 1.02 ymol .min-l .g-'.

The mean ages of the groups of patients and the

controls differed considerably (Table I). Fig. 1 shows the Ca2f-stimulated ATPase activity versus the age of the polycythemia Vera patients and the controls. There was no correlation between the age of patients and the activity, nor did the two remaining groups of patients show any correlation between age and activity. However, the age of the controls was positively correlated to the activity (Y = 0.70, P -: 0.05), but the material was too small to make allowance for this correlation when the activities werecompared. A possible correction would actually enhance rather than decrease the difference between controls and polycythemia Vera patients.

Fig. 2 shows a Lineweaver-Burk plot of the Ca2f-stimulated ATPase activity versus the Ca2+ concentration. Straight lines fitted both the poly- cythemia Vera group and the control group when the Ca2 + concentration was varied between 2.5 and 30 pmol.1-l. Each point in Fig. 2 represents a mean of 11-12 membrane preparations. A diagram

Table 11. Ca2+-stimulated ATPase activity (Vmax) and apparent calcium dissociation constant (Kd). The Vmax values are the means of the observed maximal activities of each group. The K d values are the means of the calculated dissociation constants (see text). The means were compared by Student's f test

Vm ax K d

Number of pmol. Comparison Comparison Group preparations min-l. g with control pmo1.l-l with control

Control 10 14.20 - 0.70 -

Bank blood I I 14.44 P > 0.5 I .09 P > 0.05 Polycythemia Vera 14 9.45 P<0.001 0.83 P > 0.4 Secondary polycythemia 5 12.17 P>O.l - -

Increased erythrocyte fraction 4 13.96 P=.0.5 - -

Standard deviation 1.988 0.441

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586 0. Scharff & B. Foder

20 I- ,

"t 0

20 40 60 80 0 0.2 0.4 0.6 0

Reciprocal of Ca2' ,om ( I prnd-' I Age (year)

Fig. 1. Dependence of Caa+-stimulated ATPase activity on donor age. Open circles: control donors; filled circles: polycythemia Vera patients. Each point indicates the activity (Vmax) of a single membrane preparation. The regression lines are shown. For control donors, the correlation is significant (r =0.70, P<0.05); for polycythemia Vera patients, the correla- tion is not significant (r=-O.l3, P>0.5).

Fig. 2. Lineweaver-Burk plot. Reciprocal of Caa+- stimulated ATPase activity versus reciprocal of Ca*+- concentration. Open circles: control donors; each point represents the mean of the activities of 11 mem- brane preparations. Filled circles : polycythemia Vera patients; each point indicates the mean of 12 mem- brane preparations. The vertical bars represent standard errors of the mean.

like that in Fig. 2 was plotted for each preparation, mechanism is not completely known (13), for a n d a straight line was fitted by themethodof least which reason the constant, in this article, will be squares in the appropriate conczntration interval. referred to as the apparent dissociation constant The Michaelis constants obtained from the di- (Kd). No significant differences between the means agramsarerelated to the truedissociationconstant of the K d of the three groups could be detected of theenzyme-metal complex in a way that depends (Table 11). on the mechanism of calcium activation. This The deviation from linearity in Fig. 2 at the

Table 111. Mean values of leukocyte count and Caz+-stimulated ATPase activity of controls and polycythemia Vera patients. The leukocyte counts were determined by Coulter counter at the time of investigation. The means are compared by Students t test

Caz+ -st imulated ATPase

Group

Number of Leykocyte conc. Vm ax Comparison with pmol patients without

-1 leukocytosis g donors ( 100m

Control 10 Patients without leukocytosis 7 Patients with leukocytosis 7

Standard deviations

7.01 14.20 P<O.OI 7.41 10.60 -

18.04 8.30 P<0.05

1.84 1.958 1.81 3.55

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CaP'-ATPase in Polycythemia Erythrocytes 587

- 15 c I .

0- 0 20 40 60

Leukocyte conc I lo9, 1-l )

Fig. 3. Dependence of Caz+-stimulated ATPase activity of polycythemia Vera patients on maximal leukocyte count demonstrated during the disease. Each point indicates the activity (Vmax) of a single membrane preparation. The regression line is shown. The correlation is significant (r==-0.73 P<O.Ol).

reciprocal of Ca2+ concentration above 0.5 was due to a sigmoid course of the curve depicting the activity versus Ca2' concentration when the latter was below 2 Pmolil. At high Ca2+ concentrations the ATPase activity was inhibited (Fig. 2).

Blood parameters and erythrocyte ATPase

Within the group of polycythemia Vera patients there was no relation between the Caz+-stimulated ATPase activity and erythrocyte volume, erythro- cyte count, erythrocyte fraction, thrornbocyte count, and leukocyte alkaline phosphatase. The material was too small to permit detection of dif- ferences between males and females with respect to the hematologic data and the ATPase activities.

Table I11 shows that patients with leukocytosis a t the time of investigation had a significantly lower ATPaseactivity than patients without leuko- cytosis. Nevertheless, the patients without leuko- cytosis still had a significantly lower ATPase activity than the control group. Fig. 3 shows that there is also a significant correlation (r = - 0.73, P -c 0.01) between the maximal leukocyte count observed during the disease and the Ca2+-stim- ulated ATPase.

Table 1V. Effect of type of centrifugation. Blood from three patients and two bank blood specimens were each prepared by two different types of centrifugation. The meansof thefivepreparationssubjected to thesame type of centrifugation are given. The effect of centri- fugation was tested by a Student's t test applied to the five paired observations

ATPase activity pmol min-' g-I

~~

Type of centrifugation Mga+- Caa+- stimulated stimulated

Continuous-flow system

Closed centrifuge tubes (n=5) 1.67

( n = 5 ) 1.15

Difference 0.52

Standard deviation 0.633

~~

1.30

3.54

7.76

1.657

Significance P > 0 . 2 P<O.001

Effect of treatment

Most of the polycythemia Vera and secondary polycythemia patients had been treated with the cytostatic drug busulfan (bis-(methanesulfonyl- oxy)-1,4-butane) and some patients in addition with szP. The material did not indicate any effects of treatment on the Caz+-stimulated ATPase, but more experiments are needed.

Effect of cmtrijic6.ation

Table IV shows that the activities of Ca2+-stim- ulated ATPase depended on the type of centrifuga- tion during the preparation of the erythrocyte membranes. The above-mentioned results had all been obtained by centrifugation in a continuous- flow system. This system involves a shear stress on the membranes when the suspension passes the border between the stationary and the rotating parts of the flow system, in contrast to ordinary centrifugation using closed centrifuge tubes. The differences between the Ca2+-stimulated ATPase activities caused by the centrifugations were the same in patients and controls.

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DISCUSSION

Comparison of donor groups

The only difference between the investigated membrane preparations was the significantly lower Ca2+-stimulated ATPase activity (Vma) of the polycythemia Vera membranes (Table 11). The similarity of the membrane parameters between the groups of donors justifies a comparison of activities on the basis of membrane dry weight. I t is interesting that the group with secondary polycythemia differs significantly from the poly- cythemia Vera group. However, the number of patients in the former group is small, and further experiments are needed to confirm this result. The group with secondary polycythemia may be too heterogeneous and might be divided into various groups with different values of Vmax.

A TPase and hemopoiesi5

The synthesis of the erythrocyte membrane probably occurs in the bone marrow, and the ATPase is most likely incorporated into the membrane at an early stage of cell proliferation. It is possible that the increased erythropoietic activity of the bone marrow in the polycythemia Vera patients occurs together with a diminished incorporation of ATPase molecules into the membrane, which could explain the reduced ATP- ase activity in these patients.

In addition, the ATPase activity is lower in patients with leukocytosis than in polycythemia Vera patients with normal leukocyte count at the time of investigation (Table 111). This suggests that the enzyme activity of the erythrocyte membrane is also related to other hemopoietic activities. However, the correlation between the ATPase activity that we observed and the maximal leukocyte count observed during the disease (Fig. 3) indicates that the level of ATPase activity may be influenced by the individual progress of the disease and that the activity is not related to the hemopoietic activity in a simple manner.

I t has been suggested that the calcium ho- meostatic system constitutes a main regulator of cell proliferation in the bone marrow of rats and that a brief increase of the intracellular calcium

concentration may trigger DNA synthesis (12). An influence of the reduction of Caa+-stimulated ATPase activity in polycythemia Vera may be an increased intracellular concentration of calcium ions at appropriate points in the cell proliferation cycle caused by diminished function of the Ca2f- pump.

However, it has been reported that the life span of the erythrocytes in polycythemia Vera patients decreases as the disease progresses (8). Moreover, it is known that biochemical and enzymatic changes accompany the erythrocyte aging (e.g. Refs. 2,7), so one cannot preclude that the reduc- tion of ATPase activity in polycythemia Vera takes place when the erythrocytes are in circulation.

ATPase and preparation procedure

The fact that the Ca2+-stimulated ATPase activ- ity depends on the type of centrifugation (Table IV) indicates that the accessibility of the enzyme sites is decisive for the enzymic determination (see also Ref. 3). The effect of the centrifugation by use of the continuous-flow system is probably a demasking of the enryme sites provoked by a shear stress, as the site of the Caz+-stimulated ATPase is located on the inside of the membrane (I I) .

it is possible that the detected difference between the CaZ1-stimulated ATPase activities of the poly- cythemia Vera patients and the control donors is caused by different responses by the two types of membranes to the preparation procedure, caused, for instance, by a difference between polycythemic and control blood with respect to fragility of the erythrocytes, which has been demonstrated (1).

An important factor during membrane prepara- tion is the calcium concentration. As previously shown (9), the CaZi--stimulated ATPase activity is reduced and the affinity towards Ca2’- decreased if Caa+ is removed by EGTA during the hemolysis. But since the amount of membrane-bound calcium and the apparent calcium affinity of the ATPase are not changed in the polycythemic membranes, the lower Ca2+-stimulated ATPase activity found here is not likely to be a result of a changed calcium binding during membrane preparation or ATPase assay, nor a result or a diminished access to the calcium site.

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Ca2’-ATPase in Polycythemia Erythrocytes 589

Conclusion

It may be concluded that the Ca2+-stimulated ATPase activity of the erythrocyte membranes from polycythemia Vera patients is depressed to about two thirds of the activity of control donors. Among the patients with the highest leukocyte count the reduction of ATPase activity is even more distinct. These facts indicate a relation between the ATPase activity of erythrocytes and the bone marrow activity, but further experiments are needed to confirm this.

ACKNOWLEDGEMENT

We thank Jytte Moller for valuable technical assistance.

REFERENCES 1 . Ben-David, A. & Gavendo, S. Osmotic fragility

changes of ACD blood units from polycythemic donors. Transfusion 13, 320, 1973.

2. Bessis, M. pp. 97-98 in Living Blood Cells and Their Ultrastructure. Springer-Verlag. Berlin, 1973.

3. Hanahan, D. J., Ekholm, J. & Hildenbrandt, G. Biochemical variability of human erythrocyte membrane preparations, as demonstrated by sodium-potassium-magnesium and calcium ade- nosine triphosphatase activities. Biochemistry 12, 1374, 1973.

4. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265, 1951.

5. Modan, B. pp. 4 1 4 in The Polycythemic Dis- orders. Charles C Thomas, Publisher, Springfield, IIJ., USA, 1971.

6. Perris, A. D. The calcium homeostatic system as a physiological regulator of cell proliferation in mammalian tissues. pp. 101-131 in Nichols, G. & Wasserman, R. H. (eds.) Cellular Mechanisms for Calcium Transfer and Homeostasis. Academic Press, New York, 1971.

7. Phillips, G . B., Dodge, J. T. & Howe, C. The effect of aging of human red cells in vivo on their fatty acid composition. Lipids 4, 544, 1969.

8. Pollycove, M., Winchell, H. S. & Lawrence, J. H. Classification and evolution of patterns of erythro- poiesis in polycythemia Vera as studied by iron kinetics. Blood 28, 807, 1966.

9. Scharff, 0. The influence of calcium ions on the preparation of the (Caa++ Mg2+)-activated mem- brane ATPase in human red cells. Scand. J. clin. Lab. Invest. 30, 313, 1972.

10. Schatzmann, H. J. Dependence on calcium con- centration and stoichiometry of the calcium pump in human red cells. J. Physiol. 235, 551, 1973.

1 1 . Schatzmann, H. J. & Vincenzi, F. F. Calcium movements across the membrane of human red cells. J. Physiol. 201, 369, 1969.

12. Whitfield, J. F., Rixon, R. H., MacManus, J. P. & Balk, S. D. Calcium, cyclic adenosine 3’,5’-mono- phosphate, and the control of cell proliferation: a review. In Vitro 8, 257, 1973.

13. Wolf, H. U. Studies on a Caz+-dependent ATPase of human erythrocyte membranes. Effects of Caa+ and H+. Biochim. biophys. Actu (Amsr.) 266, 361, 1972.

Received 13 January 1975 Accepted 8 April 1975

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