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CALCIUM PUMP PHOSPHOENZYME FROM YOUNG AND OLD HUMAN RED CELLS
Cell Biology International 2002, Vol. 26, No. 11, 945–949 doi:10.1006/cbir.2002.0932, available online at http://www.idealibrary.com on
CALCIUM PUMP PHOSPHOENZYME FROM YOUNG AND OLD HUMAN RED CELLS PEDRO J. ROMERO*, VALENTINA SALAS and CONCEPCIO u N HERNA u NDEZ Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo. 47114, Caracas 1041A, Venezuela Received 21 November 2001; accepted 4 June 2002
An increase in intracellular Ca2+ occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca2+ extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca2+ . To test this possibility, Ca2+ pump phosphorylation by [-32P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30 s with Ca2+ , the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca2+ plus La3+ , in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50–90 s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca2+ -dependent radioactive band of about 150 kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca2+ pump units during cell ageing, thus indicating that the reduced Ca2+ pumping activity of aged cells does not arise from pump proteolysis. 2002 Published by Elsevier Science Ltd.
K: human erythrocytes; Ca2+ pump; pump phosphorylation; percoll gradients; senescent red cells.
INTRODUCTION Human red cells show a decrease in Ca2+ pump activity during ageing in vivo (Romero and Romero, 1997). This alteration seems to account for the rise in free Ca2+ found in senescent cells (Romero et al., 1997). The reasons for such a reduced pumping activity are not known at present. Calpain I is a well-recognized proteolytic enzyme in human erythrocytes, which becomes fully activated by micromolar Ca2+ concentrations (Yoshimara et al., 1983). Earlier work has shown that the main calpain target in human red cells is the Ca2+ pump (Salamino et al., 1994). Calpain activation has a biphasic effect on the pump, first inducing a calmodulin-like stimulation, followed *To whom correspondence should be addressed: Tel.: +58(212) 751 0766 ext. 222; Fax: +58(212) 753 7087; E-mail: [email protected] 1065–6995/02/$-see front matter
by inhibition as proteolysis progresses (Wang et al., 1988). The possibility exists that increasing free Ca2+ in ageing cells may cause pump proteolysis and eventually inhibition. This possibility was studied here, using sub-populations of human red cells separated by stringent percoll density gradients. No major differences in relative mass or number of active Ca2+ pump units were found between young and old cells.
MATERIALS AND METHODS All reagents, mainly from SIGMA Chemicals (St Louis, MO, U.S.A.), were of the highest quality available. [-32P]ATP (5000 Ci/mmol) and 45CaCl2 (0.5 Ci/mol) were purchased from New England Nuclear (Boston, MA, U.S.A.). Mouse monoclonal 2002 Published by Elsevier Science Ltd.
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5F10 antibody against the plasma membrane Ca2+ pump was a kind gift of Dr J. T. Penniston (Mayo Clinic Foundation, Rochester, U.S.A.) and Dr H. Adamo (Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, UBA, Argentina). Alkaline phosphataseconjugated antibody and 5-bromo-4-chloro-3indolyl-phosphate (BCIP)/nitroblue tetrazolium (NTB) detection kit were from Bio-Rad Laboratories (Hercules, CA, U.S.A.). Preparation of membranes Fresh human blood (mainly A+ group) from healthy donors was used. Light (L) and dense (D) sub-populations of red cells were prepared using stringent self-formed, percoll density gradients (Romero et al., 1997). The cells were kept overnight in isotonic Na + solutions containing inosine (10 mM), penicillin (104 units/ml) and streptomycin (400 g/ml), as described elsewhere (Romero and Romero, 1997).
Cell Biology International, Vol. 26, No. 11, 2002
100 mM tris-HCl (pH 7.2 at room temperature); 0.5 M [-32P]ATP (500–1500 cpm/mol); 0.5 mM EGTA; 0.2 mM MgCl2, with and without 2 mM CaCl2 (Romero and Rega, 1995). When required, La3+ (0.2 mM final free concentration) was added. Phosphorylation was quenched at different time points by adding a 10% trichloroacetic acid/10 mM H3PO4 solution and membranes were washed 7 by centrifuging and resuspending in 50 vols of the same acid solution at 0C. Membranes were finally resuspended in a small volume of washing medium (usually 20 l) and kept at 0C for both radioactive analyses and protein assays, as described below. The Ca2+ dependent phosphoenzyme level (EP) was calculated as the difference between the amount of 32P incorporated into membrane proteins in a Ca2+ containing medium and that obtained in a medium of identical composition without added Ca2+ (Romero and Rega, 1995). The error between duplicate EP determinations was always less than 10%.
Ca2+ efflux measurements After removal of antibiotics, the cells were loaded with Ca2+ by pre-incubating at 37C in an isotonic Choline medium containing 70–90 mM KCl, 0.3 mM MgCl2, 0.1 mM of 3–5106 dpm/nmol 45 CaCl2, 0.01 mM A23187, and 20 mM tris-HCl, pH 7.2. After 30 min loading, the cells were washed at 4C with 100 vols of isotonic Choline medium containing K + at equilibrium (Romero and Romero, 1997) and 2 mg/ml BSA. The loaded cells were incubated in a similar medium at 37C, but in the presence of 5 mM adenine and 15 mM inosine, with and without 0.2 mM LaCl3. Samples were taken at 2 min intervals and and rapidly separated from the incubation medium by centrifuging through a dibutylphtalate cushion, and further treated as described previously (Romero and Romero, 1997). Membrane phosphorylation Once antibiotics had been removed, the cells were lysed in hypotonic tris (Romero and Weitzman, 1991) or phosphate solutions (Dodge et al., 1963). Haemoglobin was removed by repeated washing with these solutions, the membranes were then frozen and thawed three times in ethanol-CO2 mixtures and kept at 70C until further use. Leupeptin (2 M) was present throughout. Membranes (typically 4–5 mg protein/ml) were phosphorylated for up to 90 s at 0C, in a medium containing 100 mM choline-Cl; 0.1 mM ouabain;
Analytical procedures Electrophoresis was performed essentially as described by Laemmli (1970), using 6% polyacrylamide gels. Polypeptides were electro-transferred onto nitrocellulose membranes (Towbin et al., 1979). The blots were incubated with 5F10 antibody for 1 h and subsequently with secondary antibody for 30 min. Reacting bands were visualized using a BCIP/NTB reaction. For autoradiographic studies, membranes were phosphorylated briefly (10 s) and electrophoresed on 7% polyacrylamide gels under acidic conditions (Sarkadi et al., 1986). Autoradiography was performed on X-ray film for several days at 80C. Liquid scintillation counting assessed the level of radioactivity. Protein concentrations were determined by the Lowry method (Lowry et al., 1951), using BSA as a standard. The Ca2+ influx rate was calculated from the slope of regression curves relating cell Ca2+ content to time. Two-tailed paired Student’s t-tests were performed using GraphPad Instat software. RESULTS Confirming earlier findings (Romero and Romero, 1997), there was a significant difference in Ca2+ extruding capacity between young and old cells. Thus, at pH 7.2, the maximal pumping rate of L-cells was almost double that of D-cells (Table 1).
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Table 1. The action of La3+ on the maximal Ca2+ extrusion capacity of L and D cells Maximal pumping rate (mmoles Ca2+ /l cellsh)
L-cells D-cells P
Without La3+
With 0.2 mM La3+
6.81.56 3.70.82 <0.005
0.280.18 0.150.08 >0.1
Subpopulations of young (L) and old (D) human erythrocytes were loaded with Ca2+ (50 M added externally) in the presence of A23187 (10 M). After ionophore removal, active Ca2+ efflux rate was measured in the presence of 5 mM adenine and 15 mM inosine, with and without 0.2 mM LaCl3. P denotes Student’s t-test probability.
As expected, addition of La3+ almost completely inhibited Ca2+ efflux, thus reducing it by about 95% in both cell types. After a 30 s incubation with Ca2+ , the EP produced by L-cell membranes was almost half that of the D-cell membranes (Fig. 1). Unexpectedly, addition of La3+ dramatically increased (about three-fold) the EP of L-membranes, whilst having practically no effect on D-cells. On the other hand, almost the same EP level was attained in both preparations in the presence of La3+ . Thus, EP (in pmoles 32P/mg protein) was about 1.8 and 1.6 in L- and D-cell membranes, respectively (Fig. 1). By increasing the phosphorylation time to 50 s, the amount of EP formed with La3+ was correspondingly increased to 2.6 and 2.3, levelling off to about 2.8 in both membrane preparations after 90 s. However, in another similar experiment, the EP value reached nearly 2.5 in L and D-cell membranes after 90 s. Various 32P-labelled polypeptides were demonstrated by acidic gel electrophoresis (Fig. 2). However, only a single band appeared upon addition of Ca2+ , having an apparent MWr of 145 and 150 kDa in L- and D-cell membranes, respectively (lanes 2 and 4 in Fig. 2). The radioactive signal of this band in the L-cell (lane 2) was stronger than in the D-cell membranes (lane 4). To determine whether the large difference in EP detected by autoradiography in young and old erythrocytes could be due to the short incubation time used for phosphorylation, membranes were phosphorylated for 10 s in the presence of Ca2+ plus La3+ . As expected, a statistically significant difference in EP levels was found (P<0.05). Thus, the EP value (mean1 SD of three experiments)
Fig. 1. Effect of La3+ on Ca2+ -dependent phosphoenzyme formation by light- (L) and dense-cell membranes (D). The membranes were phosphorylated for 30 s at 0C and the amount of Ca2+ -dependent phosphoenzyme formed (EP) was calculated as indicated in the text. La3+ at a final free concentration of 0.2 mM was present where indicated. Results from a single experiment are shown as mean value (1 SE) of three replicate determinations. Bars represent the standard error.
was 0.170.05 and 0.380.12 pmoles 32P/mg protein in D and L-cell membranes, respectively. In close agreement with the autoradiographic results, Western blots showed a single reactive band, whose mobility in L-cell membranes was slightly greater than in D-cell membranes, giving an apparent MWr of 145 and 150 kDa, respectively (Fig. 3).
DISCUSSION The effect of ageing on Ca2+ pump activity was studied here, using sub-populations of human red cells that had been separated into light (L or young) and dense (D or old) fractions by stringent density-gradient centrifugation (Romero et al., 1997). Pump activity was assessed by measuring both active Ca2+ efflux and the amount of Ca2+ dependent phosphoenzyme formed with and without La3+ . This ion is a well-known inhibitor of the plasma membrane Ca2+ pump (Quist and Roufogalis, 1975) that presumably blocks the E1P–E2P interconversion step (Luterbacher and Schatzmann, 1983). In agreement with previous findings (Romero and Romero, 1997), there was a statistically significant difference in pump extruding capacity between young and old cells. Thus, the maximal pumping
948
Fig. 2. Autoradiography of L- (lanes 1, 2) and D-cell membranes (lanes 3, 4) after 7% polyacrylamide acidicelectrophoresis. Phosphorylation was performed for 10 s at 0C in the presence of La3+ , with (even lanes) and without 2 mM Ca2+ (uneven lanes). Similar protein amounts (10 g) were loaded into each well. Molecular weight markers (from top to bottom): 200, 116, 97 and 66 kDa.
rate of young cells was nearly twice that of old ones. As was expected in both cell types, the pump was almost completely blocked by La3+ under conditions similar to those employed for membrane phosphorylation. An important result of the present work is that, while the EP level increased with time in the presence of La3+ , it did not differ significantly between L- and D-cell membranes after 30–90 s phosphorylation, where an equilibrium was apparently reached. Thus, in spite of the fact that the maximal pump rate of L-cells is greater than that of D-cells, a similar phosphorylation level is attained at equilibrium with La3+ in both membrane types. With La3+ present, all pump units become arrested in a phosphorylated state, so the amount of EP formed at equilibrium gives a valid measure of the number of active pumps. This finding indicates that young and old cells have equal numbers of active pump units. An apparent inconsistency in these results is that the autoradiographic signal of the Ca2+ -dependent band from L-cell membranes was stronger than that from the D-cell ones. However, this finding
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Fig. 3. Western blot of L- and D-cell membranes, separated by 6% polyacrylamide gel electrophoresis. The blots were incubated with anti-Ca2+ pump 5F10 monoclonal antibody, followed by incubation with an alkaline phosphatase-conjugated antibody. A typical result from three experiments is shown.
resulted from a large difference in EP levels produced after a short incubation period, as shown by brief (10 s) phosphorylation experiments with Ca2+ plus La3+ . The maximum EP level attained in this study was higher than that of the 1.9 pmoles/mg protein reported for non-fractionated human red cells, incubated without La3+ but otherwise under similar conditions (Romero and Rega, 1995). This discrepancy may not be surprising since the latter value was obtained under steady-state conditions. Both autoradiography and immunoblotting revealed a single molecular species for the Ca2+ pump on both membrane preparations. However, the apparent Ca2+ pump mass was consistently 5 kDa higher in D-cell membranes than in the L-cell ones. In 3 experiments, this apparent increase in size was statistically significant (P<0.025). This finding clearly rules out proteolysis as a likely explanation for the reduced pumping activity of older cells. Non-enzymatic glycosylation of the Ca2+ pump has previously been reported in human red cells (Gonzalez-Flecha et al., 1990, 1993). Such a covalent modification leads to significant pump inhibition. Therefore, it seems likely that the pump may become glycosylated as the red cell ages,
Cell Biology International, Vol. 26, No. 11, 2002
thus increasing its apparent MWr and reducing its activity. Although our results are compatible with this view, only five to six moles of glucose are maximally incorporated per Ca2+ pump mole (Gonzalez-Flecha et al., 1993). This would cause 1 kDa rise in molecular mass and, unless the glycosylated pump had an anomalous electrophoretic migration, glycosylation by itself is insufficient to explain the MWr increase found in aged cells. More work is needed to clarify this point. In summary, these results demonstrate that circulating young and old red cells have both similar molecular mass and number of active Ca2+ pump units. In addition, they show that no major proteolytic degradation of the Ca2+ pump occurs during circulation of senescent cells. The differences in pumping rate found between young and old cells, therefore, cannot be attributed to pump proteolysis. ACKNOWLEDGEMENTS This work was supported by grants from CONICIT (S1-97000369) and CDCH de la UCV (No. 03.10.3502.95, 03.10.0369.97, and 03.10.3836.99). REFERENCES D JT, M C, H DJ, 1963. The preparation and chemical characteristics of haemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100: 119– 130. G-F FL, B MC, C NV, G JJ, R JPFC, 1990. Decreased Ca2+ ATPase activity after glycosylation of erythrocyte membranes in vivo and in vitro. Diabetes 39: 707–711. G-F FL, C PR, C AJ, G JJ, R JPFC, 1993. The erythrocyte calcium pump is inhibited by non-enzymatic glycation: studies
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in situ and with the purified enzyme. Biochem J 293: 369–375. L UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. L OH, R NJ, F AL, R RJ, 1951. Protein measurements with the Folin phenol reagent. J Biol Chem 193: 265–275. L S, S HJ, 1983. The site of action of La3+ in the reaction cycle of the human red cell membrane Ca2+ -pump ATPase. Experientia 39: 311–312. Q EE, R BD, 1975. Determination of the stoichiometry of the calcium pump in human erythrocytes using lanthanum as selective inhibitor. FEBS Lett 50: 135–139. R PJ, W C, 1991. The effect of cholera toxin on the human red cell CaATPase. Biochem Biophys Res Commun 181: 208–212. R PJ, R AF, 1995. Effects of magnesium plus vanadate on partial reactions of the Ca2+ -ATPase from human red cell membranes. Biochim Biophys Acta 1235: 155–157. R PJ, R EA, 1997. Differences in Ca2+ pumping activity between sub-populations of human red cells. Cell Calcium 21: 353–358. R PJ, R EA, W BMD, 1997. Ionic calcium content of light and dense human red cells separated by percoll density gradients. Biochim Biophys Acta 1323: 23–28. S F, S B, M E, M M, V PL, P S, C E, 1994. The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte. Cell Calcium 15: 28–35. S B, E A, F-P Z, G´ G, 1986. Molecular characterisation of the in situ red cell membrane calcium pump by limited proteolysis. J Biol Chem 261: 9552–9557. T H, S T, G J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354. W KKW, V A, R BD, 1988. Activation of the Ca2+ -ATPase of human erythrocyte membrane by an endogenous Ca2+ -dependent neutral protease. Arch Biochem Biophys 260: 696–704. Y N, K T, S T, K A, H M, M T, 1983. Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney. J Biol Chem 258: 8883–8889.
CALCIUM PUMP PHOSPHOENZYME FROM YOUNG AND OLD HUMAN RED CELLS PEDRO J. ROMERO*, VALENTINA SALAS and CONCEPCIO u N HERNA u NDEZ Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, UCV, Aptdo. 47114, Caracas 1041A, Venezuela Received 21 November 2001; accepted 4 June 2002
An increase in intracellular Ca2+ occurs during ageing of human erythrocytes in vivo. The aged cells show a reduced capacity for active Ca2+ extrusion. Such a defect may arise from pump proteolysis, due to calpain activation by the raised intracellular Ca2+ . To test this possibility, Ca2+ pump phosphorylation by [-32P]ATP was studied on percoll-separated young and old human erythrocytes. After phosphorylation for 30 s with Ca2+ , the amount of phosphoenzyme produced by the young cell membranes was 50% that of the old cells. With Ca2+ plus La3+ , in contrast, the phosphoenzyme level was nearly the same in both preparations. After a prolonged phosphorylation period (50–90 s), the phosphoenzyme reached almost identical equilibrium levels in both membrane preparations. On the other hand, a single Ca2+ -dependent radioactive band of about 150 kDa was apparent in both preparations after acidic electrophoresis. Likewise, Western blotting using 5F10 monoclonal antibody also detected a single band of similar molecular weight. These results demonstrate that there is no alteration in either molecular mass or number of active Ca2+ pump units during cell ageing, thus indicating that the reduced Ca2+ pumping activity of aged cells does not arise from pump proteolysis. 2002 Published by Elsevier Science Ltd.
K: human erythrocytes; Ca2+ pump; pump phosphorylation; percoll gradients; senescent red cells.
INTRODUCTION Human red cells show a decrease in Ca2+ pump activity during ageing in vivo (Romero and Romero, 1997). This alteration seems to account for the rise in free Ca2+ found in senescent cells (Romero et al., 1997). The reasons for such a reduced pumping activity are not known at present. Calpain I is a well-recognized proteolytic enzyme in human erythrocytes, which becomes fully activated by micromolar Ca2+ concentrations (Yoshimara et al., 1983). Earlier work has shown that the main calpain target in human red cells is the Ca2+ pump (Salamino et al., 1994). Calpain activation has a biphasic effect on the pump, first inducing a calmodulin-like stimulation, followed *To whom correspondence should be addressed: Tel.: +58(212) 751 0766 ext. 222; Fax: +58(212) 753 7087; E-mail: [email protected] 1065–6995/02/$-see front matter
by inhibition as proteolysis progresses (Wang et al., 1988). The possibility exists that increasing free Ca2+ in ageing cells may cause pump proteolysis and eventually inhibition. This possibility was studied here, using sub-populations of human red cells separated by stringent percoll density gradients. No major differences in relative mass or number of active Ca2+ pump units were found between young and old cells.
MATERIALS AND METHODS All reagents, mainly from SIGMA Chemicals (St Louis, MO, U.S.A.), were of the highest quality available. [-32P]ATP (5000 Ci/mmol) and 45CaCl2 (0.5 Ci/mol) were purchased from New England Nuclear (Boston, MA, U.S.A.). Mouse monoclonal 2002 Published by Elsevier Science Ltd.
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5F10 antibody against the plasma membrane Ca2+ pump was a kind gift of Dr J. T. Penniston (Mayo Clinic Foundation, Rochester, U.S.A.) and Dr H. Adamo (Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, UBA, Argentina). Alkaline phosphataseconjugated antibody and 5-bromo-4-chloro-3indolyl-phosphate (BCIP)/nitroblue tetrazolium (NTB) detection kit were from Bio-Rad Laboratories (Hercules, CA, U.S.A.). Preparation of membranes Fresh human blood (mainly A+ group) from healthy donors was used. Light (L) and dense (D) sub-populations of red cells were prepared using stringent self-formed, percoll density gradients (Romero et al., 1997). The cells were kept overnight in isotonic Na + solutions containing inosine (10 mM), penicillin (104 units/ml) and streptomycin (400 g/ml), as described elsewhere (Romero and Romero, 1997).
Cell Biology International, Vol. 26, No. 11, 2002
100 mM tris-HCl (pH 7.2 at room temperature); 0.5 M [-32P]ATP (500–1500 cpm/mol); 0.5 mM EGTA; 0.2 mM MgCl2, with and without 2 mM CaCl2 (Romero and Rega, 1995). When required, La3+ (0.2 mM final free concentration) was added. Phosphorylation was quenched at different time points by adding a 10% trichloroacetic acid/10 mM H3PO4 solution and membranes were washed 7 by centrifuging and resuspending in 50 vols of the same acid solution at 0C. Membranes were finally resuspended in a small volume of washing medium (usually 20 l) and kept at 0C for both radioactive analyses and protein assays, as described below. The Ca2+ dependent phosphoenzyme level (EP) was calculated as the difference between the amount of 32P incorporated into membrane proteins in a Ca2+ containing medium and that obtained in a medium of identical composition without added Ca2+ (Romero and Rega, 1995). The error between duplicate EP determinations was always less than 10%.
Ca2+ efflux measurements After removal of antibiotics, the cells were loaded with Ca2+ by pre-incubating at 37C in an isotonic Choline medium containing 70–90 mM KCl, 0.3 mM MgCl2, 0.1 mM of 3–5106 dpm/nmol 45 CaCl2, 0.01 mM A23187, and 20 mM tris-HCl, pH 7.2. After 30 min loading, the cells were washed at 4C with 100 vols of isotonic Choline medium containing K + at equilibrium (Romero and Romero, 1997) and 2 mg/ml BSA. The loaded cells were incubated in a similar medium at 37C, but in the presence of 5 mM adenine and 15 mM inosine, with and without 0.2 mM LaCl3. Samples were taken at 2 min intervals and and rapidly separated from the incubation medium by centrifuging through a dibutylphtalate cushion, and further treated as described previously (Romero and Romero, 1997). Membrane phosphorylation Once antibiotics had been removed, the cells were lysed in hypotonic tris (Romero and Weitzman, 1991) or phosphate solutions (Dodge et al., 1963). Haemoglobin was removed by repeated washing with these solutions, the membranes were then frozen and thawed three times in ethanol-CO2 mixtures and kept at 70C until further use. Leupeptin (2 M) was present throughout. Membranes (typically 4–5 mg protein/ml) were phosphorylated for up to 90 s at 0C, in a medium containing 100 mM choline-Cl; 0.1 mM ouabain;
Analytical procedures Electrophoresis was performed essentially as described by Laemmli (1970), using 6% polyacrylamide gels. Polypeptides were electro-transferred onto nitrocellulose membranes (Towbin et al., 1979). The blots were incubated with 5F10 antibody for 1 h and subsequently with secondary antibody for 30 min. Reacting bands were visualized using a BCIP/NTB reaction. For autoradiographic studies, membranes were phosphorylated briefly (10 s) and electrophoresed on 7% polyacrylamide gels under acidic conditions (Sarkadi et al., 1986). Autoradiography was performed on X-ray film for several days at 80C. Liquid scintillation counting assessed the level of radioactivity. Protein concentrations were determined by the Lowry method (Lowry et al., 1951), using BSA as a standard. The Ca2+ influx rate was calculated from the slope of regression curves relating cell Ca2+ content to time. Two-tailed paired Student’s t-tests were performed using GraphPad Instat software. RESULTS Confirming earlier findings (Romero and Romero, 1997), there was a significant difference in Ca2+ extruding capacity between young and old cells. Thus, at pH 7.2, the maximal pumping rate of L-cells was almost double that of D-cells (Table 1).
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Table 1. The action of La3+ on the maximal Ca2+ extrusion capacity of L and D cells Maximal pumping rate (mmoles Ca2+ /l cellsh)
L-cells D-cells P
Without La3+
With 0.2 mM La3+
6.81.56 3.70.82 <0.005
0.280.18 0.150.08 >0.1
Subpopulations of young (L) and old (D) human erythrocytes were loaded with Ca2+ (50 M added externally) in the presence of A23187 (10 M). After ionophore removal, active Ca2+ efflux rate was measured in the presence of 5 mM adenine and 15 mM inosine, with and without 0.2 mM LaCl3. P denotes Student’s t-test probability.
As expected, addition of La3+ almost completely inhibited Ca2+ efflux, thus reducing it by about 95% in both cell types. After a 30 s incubation with Ca2+ , the EP produced by L-cell membranes was almost half that of the D-cell membranes (Fig. 1). Unexpectedly, addition of La3+ dramatically increased (about three-fold) the EP of L-membranes, whilst having practically no effect on D-cells. On the other hand, almost the same EP level was attained in both preparations in the presence of La3+ . Thus, EP (in pmoles 32P/mg protein) was about 1.8 and 1.6 in L- and D-cell membranes, respectively (Fig. 1). By increasing the phosphorylation time to 50 s, the amount of EP formed with La3+ was correspondingly increased to 2.6 and 2.3, levelling off to about 2.8 in both membrane preparations after 90 s. However, in another similar experiment, the EP value reached nearly 2.5 in L and D-cell membranes after 90 s. Various 32P-labelled polypeptides were demonstrated by acidic gel electrophoresis (Fig. 2). However, only a single band appeared upon addition of Ca2+ , having an apparent MWr of 145 and 150 kDa in L- and D-cell membranes, respectively (lanes 2 and 4 in Fig. 2). The radioactive signal of this band in the L-cell (lane 2) was stronger than in the D-cell membranes (lane 4). To determine whether the large difference in EP detected by autoradiography in young and old erythrocytes could be due to the short incubation time used for phosphorylation, membranes were phosphorylated for 10 s in the presence of Ca2+ plus La3+ . As expected, a statistically significant difference in EP levels was found (P<0.05). Thus, the EP value (mean1 SD of three experiments)
Fig. 1. Effect of La3+ on Ca2+ -dependent phosphoenzyme formation by light- (L) and dense-cell membranes (D). The membranes were phosphorylated for 30 s at 0C and the amount of Ca2+ -dependent phosphoenzyme formed (EP) was calculated as indicated in the text. La3+ at a final free concentration of 0.2 mM was present where indicated. Results from a single experiment are shown as mean value (1 SE) of three replicate determinations. Bars represent the standard error.
was 0.170.05 and 0.380.12 pmoles 32P/mg protein in D and L-cell membranes, respectively. In close agreement with the autoradiographic results, Western blots showed a single reactive band, whose mobility in L-cell membranes was slightly greater than in D-cell membranes, giving an apparent MWr of 145 and 150 kDa, respectively (Fig. 3).
DISCUSSION The effect of ageing on Ca2+ pump activity was studied here, using sub-populations of human red cells that had been separated into light (L or young) and dense (D or old) fractions by stringent density-gradient centrifugation (Romero et al., 1997). Pump activity was assessed by measuring both active Ca2+ efflux and the amount of Ca2+ dependent phosphoenzyme formed with and without La3+ . This ion is a well-known inhibitor of the plasma membrane Ca2+ pump (Quist and Roufogalis, 1975) that presumably blocks the E1P–E2P interconversion step (Luterbacher and Schatzmann, 1983). In agreement with previous findings (Romero and Romero, 1997), there was a statistically significant difference in pump extruding capacity between young and old cells. Thus, the maximal pumping
948
Fig. 2. Autoradiography of L- (lanes 1, 2) and D-cell membranes (lanes 3, 4) after 7% polyacrylamide acidicelectrophoresis. Phosphorylation was performed for 10 s at 0C in the presence of La3+ , with (even lanes) and without 2 mM Ca2+ (uneven lanes). Similar protein amounts (10 g) were loaded into each well. Molecular weight markers (from top to bottom): 200, 116, 97 and 66 kDa.
rate of young cells was nearly twice that of old ones. As was expected in both cell types, the pump was almost completely blocked by La3+ under conditions similar to those employed for membrane phosphorylation. An important result of the present work is that, while the EP level increased with time in the presence of La3+ , it did not differ significantly between L- and D-cell membranes after 30–90 s phosphorylation, where an equilibrium was apparently reached. Thus, in spite of the fact that the maximal pump rate of L-cells is greater than that of D-cells, a similar phosphorylation level is attained at equilibrium with La3+ in both membrane types. With La3+ present, all pump units become arrested in a phosphorylated state, so the amount of EP formed at equilibrium gives a valid measure of the number of active pumps. This finding indicates that young and old cells have equal numbers of active pump units. An apparent inconsistency in these results is that the autoradiographic signal of the Ca2+ -dependent band from L-cell membranes was stronger than that from the D-cell ones. However, this finding
Cell Biology International, Vol. 26, No. 11, 2002
Fig. 3. Western blot of L- and D-cell membranes, separated by 6% polyacrylamide gel electrophoresis. The blots were incubated with anti-Ca2+ pump 5F10 monoclonal antibody, followed by incubation with an alkaline phosphatase-conjugated antibody. A typical result from three experiments is shown.
resulted from a large difference in EP levels produced after a short incubation period, as shown by brief (10 s) phosphorylation experiments with Ca2+ plus La3+ . The maximum EP level attained in this study was higher than that of the 1.9 pmoles/mg protein reported for non-fractionated human red cells, incubated without La3+ but otherwise under similar conditions (Romero and Rega, 1995). This discrepancy may not be surprising since the latter value was obtained under steady-state conditions. Both autoradiography and immunoblotting revealed a single molecular species for the Ca2+ pump on both membrane preparations. However, the apparent Ca2+ pump mass was consistently 5 kDa higher in D-cell membranes than in the L-cell ones. In 3 experiments, this apparent increase in size was statistically significant (P<0.025). This finding clearly rules out proteolysis as a likely explanation for the reduced pumping activity of older cells. Non-enzymatic glycosylation of the Ca2+ pump has previously been reported in human red cells (Gonzalez-Flecha et al., 1990, 1993). Such a covalent modification leads to significant pump inhibition. Therefore, it seems likely that the pump may become glycosylated as the red cell ages,
Cell Biology International, Vol. 26, No. 11, 2002
thus increasing its apparent MWr and reducing its activity. Although our results are compatible with this view, only five to six moles of glucose are maximally incorporated per Ca2+ pump mole (Gonzalez-Flecha et al., 1993). This would cause 1 kDa rise in molecular mass and, unless the glycosylated pump had an anomalous electrophoretic migration, glycosylation by itself is insufficient to explain the MWr increase found in aged cells. More work is needed to clarify this point. In summary, these results demonstrate that circulating young and old red cells have both similar molecular mass and number of active Ca2+ pump units. In addition, they show that no major proteolytic degradation of the Ca2+ pump occurs during circulation of senescent cells. The differences in pumping rate found between young and old cells, therefore, cannot be attributed to pump proteolysis. ACKNOWLEDGEMENTS This work was supported by grants from CONICIT (S1-97000369) and CDCH de la UCV (No. 03.10.3502.95, 03.10.0369.97, and 03.10.3836.99). REFERENCES D JT, M C, H DJ, 1963. The preparation and chemical characteristics of haemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100: 119– 130. G-F FL, B MC, C NV, G JJ, R JPFC, 1990. Decreased Ca2+ ATPase activity after glycosylation of erythrocyte membranes in vivo and in vitro. Diabetes 39: 707–711. G-F FL, C PR, C AJ, G JJ, R JPFC, 1993. The erythrocyte calcium pump is inhibited by non-enzymatic glycation: studies
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