Pyruvate kinase-deficiency anemia: Membrane approach

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

Pyruvate Kinase-Deficiency TOMA; Institute

MA~~K,

of Hematology

V~CLAV and Blood

BRABEC, Transfusion, Received

39, 55-63

(1988)

Anemia: Membrane Approach MILAN

KOD~~EK,

U Nemocnice April

AND

PETR

I, 128 20 Prague

JAROL~M 2, Czechoslovakia

6, 1987

Recent progress in the methodology of biochemistry and molecular biology substantially expanded our knowledge on number of genetically determined diseases. It can be well documented in the field of erythrocyte enzymopathies. Much work has been accomplished mainly in the characterization of the enzyme mutants and the genetic analysis of various disorders (1). Pyruvate kinase (PK; EC 2.7.1.40) ranks among the key erythrocyte enzymes of the glycolytic pathway and its deficiency may cause hemolysis of varying severity. It appears evident that the limited metabolic capability and the defective energy production underlie the premature erythrocyte destruction in this type of disorder. Although most cellular PK activity is confined to the cytoplasm, it is likely that also some membrane functions are perturbed in PK-deficiency anemia. Tillmann et al. (2) reported that even a small fraction of PK is localized directly on the membrane though no band in the electrophoretic pattern of membrane proteins has been attributed to PK as yet. These authors also found a parallel decrease of the cytoplasmic and membrane enzyme activity in this disorder (3). Membrane alterations in PK-deficient erythrocytes were further reported by Allen et al. (4) who particularly stressed the necessity of taking reticulocytes into account and described some reticulocyte-dependent membrane changes. Some membrane involvement in the pathophysiology of PK-deficient red cells has been likewise suggested by the diminished red cell deformability (3. Sufficient evidence has been accumulated to claim that the red cell shape and deformability are determined by the state of the membrane-laminating network of skeletal proteins including spectrin, actin, and band 4.1 protein (6). In the light of such observations the search for skeletal abnormalities in PK-deficient red cells is appropriate. Our study deals with the evaluation of the skeletal protein extractability with respect to the elevated reticulocyte count and ATP depletion of PK-deficient red cells. Since changes in the integral membrane components were reported (7) we have also studied membrane fluidity using the fluorescence probe 1,6-diphenyl1,3,5-hexatriene (DPH). We have measured membrane fluidity of ghosts and of intact erythrocyte membranes. 55

0885-4505/88

$3.00

Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

56

MARIK

PATIENTS

ET AL.

AND METHODS

Patients Six patients with PK-deficiency anemia were included in this study. Their diagnosis was established on the basis of hematological and biochemical laboratory examination including enzyme activity measurements and kinetic and electrophoretic studies of PK before and after limited proteolysis with trypsin. Two groups of patients could be distinguished: in three patients the anemia was mild or did not occur at all and hemolysis was compensated while the other three patients suffered from severe uncompensated hemolytic anemia. All relevant blood cell parameters are given in Table 1. Four patients were splenectomized at various times before being subjected to the study protocol. In two cases the splenectomy led to the amelioration of the clinical status, In all subjects studied the decrease of V,,, in the Michaelis-Menten kinetics of PK was observed; in four cases by 20% and in others by 10 and 50%. The details of the clinical and enzymological investigation of all studied subjects were published elsewhere (89). Crude Spectrin Extractability Heparinized blood samples were obtained from PK-deficient patients and, for control, from normal healthy donors. Red cells were isolated from plasma by centrifugation (2OOOg, 10 min) and washed three times with the isotonic (310 mOsm) phosphate buffer, pH 7.6. Buffy coat was carefully aspirated during the washing so as not to disturb the top layer of red cells. “Dodge” pink ghosts were prepared by hypotonic hemolysis followed by five centrifugations (16,OOOg, 20 min) with the hypotonic (20 mOsm) phosphate buffer, pH 7.6. Crude spectrin solution containing residual hemoglobin was obtained by low ionic strength extraction of ghosts with 0.1 mu Na3P04, 1 mu EDTA, 0.1 mu phenylmethylsulfonyl fluoride, 0.2% isopropanol, pH 8.0. The procedure was carried out at 37°C for 30 min. After the extraction the soluble fraction of the skeletal membrane proteins was separated from the ghost debris by centrifugation at 70,OOOg for 1 hr. Ultraviolet spectra of crude spectrin solutions were recorded and the nonheme membrane protein extractability was calculated from the values of absorbance at 280 and 415 nm according to Beutler and Villacorte (10). Using this method we could eliminate the contribution of hemoglobin to the whole protein absorbance peak at 280 nm. In the case of reticulocyte-rich samples the shift of protein peak, orginally found at 280 nm, toward lower wavelengths was observed. This phenomenon is caused by the presence of RNA which was clearly detected by the orcinol test. In the model experiment, we monitored the spectra of crude spectrin extract from the normal subject with the addition of different amounts of RNA. From the set of curves that we obtained we were able to correlate the peak shift with the artificially increased absorbance at 280 nm due to the intensive absorption maximum of RNA at 260 nm. This correlation method was recently described in detail (11).

RED

CELL

MEMBRANE

IN PK-DEFICIENT

ANEMIA

57

58 Sodium

MARIK

dodecyl sulfate-Polyacrylamide

ET AL.

Gel Electrophoresis

(SDS-PAGE)

SDS-PAGE was carried out in 5% polyacrylamide gel tubes using the buffer system of Maize1 (12). The samples for electrophoresis were prepared by diluting the membrane suspension or extract with the solubilization solution of 8 M urea, 2% SDS, 2% 2-mercaptoethanol (1: 1, v/v) followed by a 3-min incubation at 100°C to destroy all proteolytic activity. Fluorescence

Labeling

with DPH

Intact cells and ghosts were labeled with DPH as follows: 2 x 10e4 M solution of DPH in acetone was diluted 1: 100 (v/v) with an appropriate buffer, heated to 60°C and vigorously stirred for 1 hr. The labeling solution prepared in this manner was then mixed 1: 1 with the erythrocyte ghosts suspension (20 pg protein/ml) or with intact erythrocytes (10’ cells/ml) and incubated at 37°C for 1 hr in a continuously shaken water bath. After incubation, the sample was washed once with an appropriate buffer (final concentration of ghosts 10 pg protein/ml and of erythrocytes 5 x lo6 cells/ml) and measured within 1 hr. All procedures were performed under diminished light and completed on the day of blood withdrawal. Fresh DPH stock solution in acetone was prepared every month and stored under refrigeration. For each measurement a new labeling solution was prepared. Fluroescence measurements were made with an SLIM-Aminco 4800 spectrofluorometer, the excitation and emission wavelengths being 365 and 450 nm and excitation and emission slit widths 8 and 4 nm, respectively. Samples were placed in the OP-02T double cuvette thermostatic turret connected to a circulatory water bath set at 37°C. Average fluorescence anisotropy r was calculated with corrections according to Azumi and McGlynn (13). Maximum error of r was 2 0.002. We have measured membrane fluidity of ghosts as described in (14). Further, we measured the DPH fluorescence anisotropy in intact erythrocytes and corrected it for MCHC of the cells. Normalized anisotropy values were calculated from the equation rnorm = 0.2r/(0.00263 MCHC + 0.113), where r is the measured anisotropy, the denominator is the theoretical r value for given MCHC, and factor 0.2 is the theoretical anisotropy for MCHC = 33.1%. RESULTS The electrophoretic patterns of the red cell membrane proteins in PK-deficiency anemia are shown in Fig. 1. We failed to observe any alteration in their composition compared to the normal. Crude spectrin extraction of ghosts with low ionic strength buffer yielded a solution largely of spectrin. Hemoglobin, actin, and some other minor constituents were also permanently present in the extract. In the case of the reticulocyte-rich PK-deficient blood samples the typical reddish button was always found at the bottom of the cuvette following the high-speed centrifugation after the ghosts had undergone the extraction. The extract composition was identical in both the normal and the PK-deficient red cells (Fig. 2). The spectrin dimer was the prevailing species as revealed by gel chromatography and nondenaturing gel electrophoresis (data not shown). Figure 3 shows uv spectra of the extracts which were used for calculation of crude spectrin ex-

RED CELL MEMBRANE

IN PK-DEFICIENT

ANEMIA

FIG. 1. Electrophoretic profiles of the erythrocyte membrane proteins (A) control, (B) PKdeficiency anemia, low reticulocytosis (
tractability. The typical feature of extract spectra from PK-deficient subjects with extreme reticulocytosis was the shift of peak at 280 nm toward lower wavelengths caused by the intensive absorption of ribonucleic acid at 260 nm. The protein peak shift was proportional to the reticulocyte count in the blood sample. Using the correction method for calculation of crude spectrin extractability in these samples we conclude that the spectrin extractability from the reticulocyterich PK-deficient samples is elevated in comparison with control and PK-deficient blood samples with low reticulocytosis. Since the character of the metabolic defect in this anemia implies the disturbances in the energy supply we also studied the PK-deficient erythrocyte membrane under the conditions of ATP depletion. Figure 4 demonstrates that the PKdeficient red cell membranes with low reticulocytosis are little affected as for

FIG. 2. Crude spectrin extract composition from control (A), PK-deficient red cell membranes, low reticulocytosis (B), and PK-deficient red cell membranes, extreme reticulocytosis (C)

60

MABIK

ET AL.

I\

0.5

‘\

e f :

1 mo

350

\

\ I I

I

330

I 350

.I

.-

1. a00

I

1. J *a0

1(4

3. Ultraviolet spectra of crude spectrin extracts from normal (full line) and PK-deficient red cell membranes, low reticulocytosis (dotted line) and extreme reticulocytosis (dashed line) FIG.

protein composition by the incubation in glucose-free medium unlike PK-deficient reticulocytes where the metabolic depletion leads to the adsorption of the cytoplasmic protein MW 50,000 to the membrane. Crude spectrin extractability from the ATP-depleted ghosts was reduced in all studied subjects, while the extract composition remained unchanged.

FIG. 4. Electrophoretic profiles of the PK-deficient red cell membrane proteins prepared from fresh blood samples of low and extreme reticulocytosis (A,B) and ATP-depleted red cells of low and extreme reticulocytosis (A,‘B)‘, respectively. The arrow indicates the position of the MW 50,000 protein.

RED

CELL

MEMBRANE

IN PK-DEFICIENT

ANEMIA

61

All measured and calculated fluorescence anisotropy values for the group of our patients with PK deficiency and normal blood donors are shown in Table 2. For both groups the mean values and standard deviations are calculated. DISCUSSION The close relationship between the proper function of red cell membrane skeleton and the effective cell deformability has been widely accepted. In some membrane hemolytic disorders, e.g., hereditary spherocytosis and hereditary elliptocytosis, the reduced red cell deformability has been conclusively reported and the suspected membrane skeletal abnormalities have in many cases been revealed and characterized (1.5). In pyruvate kinase-deficiency anemia, on the other hand, it is believed that the metabolic impairments rather than membrane defects dominate the pathogenetic mechanism and by affecting cell flexibility they may shorten the life span of the red cells. This is, however, questioned by the finding that no correlation exists between the severity of anemia or reticulocytosis and the degree of the cell deformability reduction; this observation was confirmed by ektacytometry performed in our group of patients (16). The role of the membrane skeleton in the pathological mechanism of this disorder thus remains unclear. Obviously, any skeletal defect could be considered directly responsible for the diminished red cell deformability. Therefore, here we examined membrane skeleton and its release from the membrane of fresh and ATP-depleted red cells. Our results do not point out any particular skeletal defect but are consistent with the importance of considering reticulocytes typically occurring in PK-deficiency anemia after splenectomy. We found direct correlation between crude spectrin extractability and the reticulocyte count but this relation could be observed in reticulocytes from any other source (11). No alterations could be attributed exclusively to this particular enzyme deficiency. Membrane protein pattern provided by SDS-PAGE was identical with the pattern of control sample as well as the composition of the skeletal protein extract. The adsorption to the membrane of cytoplasmic proteins of PK-deficient reticulocytes upon ATP depletion may be considered to play some role in the mechanism of the selective PK-deficient reticulocyte destruction. The cytoplasmic origin of the MW 50,000 protein is suggested by the generally unchanged red cell membrane protein pattern in the high molecular weight region which rules out the origin of this protein as proteolytic digest. Allen et al. (4) consequently associated protein adsorption with the increased specific gravity of PK-deficient red cell membranes. We failed to observe meaningful changes in the integral domain of the membrane. If we compare the mean uncorrected r values we find a 3.5% decrease (increase in fluidity) in the group of PK-deficient patients which is statistically significant at the 5% significance level. Nevertheless, after correction for MCHC which is higher for healthy blood donors we find only 0.7% difference in r which is rather insignificant. This apparent discrepancy is due to resonance energy transfer from DPH to hemoglobin. This transfer increases with increasing MCHC and leads to the increase in fluorescence anisotropy values. Consequently, only the normalized

32.5 33.4 32.6 35.0 30.3 34.4 31.8 33.6 32.95 1.49

MCHC

0.193 0.200 0.195 0.208 0.188 0.204 0.198 0.200 0.1983 0.0063

r 0.194 0.199 0.1% 0.203 0.195 0.201 0.201 0.199 0.1985 0.0032

~.orm

anemia

Erythrocytes

PK-deficiency

of DPH in Intact Erythrocytes

Note. SD, standard deviation.

Experiment No.

Fluorescence Anisotropy

0.207 0.220 0.211 0.198 0.201 0.196 0.211 0.209 0.2074 0.0076

r

Ghosts

I 2 3 4 5 6 7 8 Mean SD

Experiment No. 37.3 34.5 33.6 34.9 34.4 36.4 35.5 34. I 35.09 1.24

MCHC

TABLE 2 and Ghosts from the Patients with PK-Deficiency Donors

0.214 0.205 0.203 0.205 0.206 0.205 0.204 0.200 0.2053 0.0040

r

rmml 0.203 0.201 0.202 0.200 0.202 0.1% 0.198 0.197 0.1999 0.0026

Erythrocytes

Healthy donors

0.211 0.200 0.197 0.199 0.201 0.198 0.202 0.203 0.2014 0.0044

r

Ghosts

Anemia and from Healthy Blood

RED CELL

MEMBRANE

IN PK-DEFICIENT

ANEMIA

63

fluorescence anisotropy value should be considered as a correct measure of intact membrane fluidity. We can conclude that there are no significant differences in DPH fluorescence anisotropy of intact erythrocyte membranes between the groups of healthy blood donors and patients with PK deficiency. With the ghosts, it is difficult to estimate the concentration of residual hemoglobin in the ghosts. While we were able to prepare white ghosts from all normal erythrocytes, some of the ghosts from PK-deficient cells were slightly pink, indicating a higher amount of bound hemoglobin. This hemoglobin may again increase the measured fluorescence anisotropy which may explain 3% difference, i.e., an apparent decrease in fluidity of PKdeficient ghosts compared to ghosts from normal erythrocytes. Since it is difficult to find a reliable correction for resonance energy transfer in the case of ghosts we cannot make any conclusions from their measurements. SUMMARY

Low ionic strength extraction (37”C, .30 min) of ghosts from PK-deficient erythrocytes provided crude spectrin extract. No significant differences in the extract composition compared to normal donors were observed. The reticulocytedependent spectrin extractability was found among the subjects with PK-deficiency anemia. Likewise ATP-depletion affects spectrin extractability and also leads to the adsorption of cytoplasmic protein MW 50,000 to the reticulocyte membrane. The measurement of membrane fluidity using the fluorescence probe DPH did not reveal significant alterations in the moiety of integral membrane constituents. REFERENCES 1. Beutler, E., In “Hemolytic Anemia in Disorders of Red Cell Metabolism” (M. M. Wintrobe, Ed.). Plenum Medical, New York/London, 1979. 2. Tillmann, W., Cordua, A., and Schriiter. W., Biochim. Biophys. Acta 382, 157 (1975). 3. Lakomek, M., Tillmann, W., Scharnetzky. M., Schriiter, W., and Winkler, H., Enzyme 29, 189 (1983). 4. Allen, D. W., Groat, J. D., Finkel. B.. Rank, B. H., Wood. P. A., and Eaton, J. W., Ameri. J. Hematol. 14, II (1983). 5. Leblond, P. F., Lyonnais, J., and Delage, J. M., Brit. J. Haematol. 39, 63 (1978). 6. Marchesi, V. T., Blood 61, 1 (1983). 7. Zanella, A., Brovelli, A., Mantovani. A., Izzo. C., Rebulla, P., and Balduini, C., Brit. J. Haematol. 42, 101 (1979). 8. Brabec, V., Jacobasch, G., Janele, J., and Friedmann, B., VnitJni Le’k. 29, 463 (1983). 9. Jacobasch, G., Brabec, V., Janele, J., and Friedmann, B., VnitFnni L&k. 29, 471 (1983). 10. Beutler, E., and Villacorte, D., Transfusion 21, 96 (1981). 11. Maffk, T., KodXek, M., and Brabec, V., C/in. Chim. Acta 153, 15 (1985). 12. Maize], J. V., Jr., Science 151, 988 (1966). 13. Azumi, T., and McGlynn, S. P., J. Chem. Phys. 37, 2413 (1962). 14. Jarolim, P., and MirEevovB, L., Biochim. Biophys. Acta 688, 460 (1982). 15. Palek, J., and Lux, S. E., Semin. Hematol. 20, 189 (1983). 16. PlBSek, J., Maiik, T., and Brabec, V., Biomed. Biochim. Acta 42, 132 (1983).