- Email: [email protected]
Aged erythrocytes exhibit decreased anion exchange
Mechanisms of Ageing and Development, 39 (1987) 245-250
245
Elsevier ScientificPublishersIreland Ltd.
AGED ERYTHROCYTES EXHIBIT DECREASED ANION EXCHANGE
GRZEGORZ BARTOSZ, MARIA GACZY~SKA, EWA GRZELII~ISKA, MIROS[AW SOSZYI~ISKI,W]~ODZIMIERZ MICHALAKa and ROMAN GONDKOa Laboratory of Biophysics of Development and Aging, Chair of Biophysics and aLaboratory of Bio. logical Sciences, University of l~6d[, 90-237 l]6d~ fPoland}
(Received October 15th, 1986) (RevisionreceivedFebruary 26th, 1987) SUMMARY
The rate of transport of [3~p] phosphate into human and bovine erythrocytes and of a spin-label analogue of phosphate (Tempo-phosphate) into human erythrocytes was found to decrease with increasing erythrocyte age by 15-20% when comparing 20% most dense celis with 20% of lightest cells. The activation energy of Tempo-phosphate transport did not show significant changes upon erythrocyte aging. Key words: Erythrocyte aging; Phosphate transport; Spin label; Electron spin resonance
INTRODUCTION Aging of a mammalian erythrocyte has been demonstrated to involve structural and functional alterations in red cell membrane proteins. They include minor changes in the polypeptide composition [1], progressive cross-linking [2] and aggregation [3], racemization [4], alterations in the physical state of the proteins as evidenced by spin-labeling [5] and fluorescence quenching (submitted), decrease in the number of receptors [6] and enzymatic activities [7] and exposure of a "senescent cell antigen" [8]. It has been proposed [9] that the "senescent cell antigen" is formed by proteolytic degradation in situ of the Band 3 protein of the erythrocyte membrane (the anion exchange protein). Progressive proteolysis of the Band 3 protein was demonstrated to .occur during erythrocyte aging [10]. It seemed therefore worthwhile to examine functional properties of the Band 3 protein in red blood cells of various ages. We have studied the transport of an anionic spin label 2,2,5,5-tetramethyl-3-carboxypyrrolidine-l-oxyl into bovine erythrocytes finding a hampering of its transport in aged cells [11]. This Address all correspondence and reprint requests to: Dr. Grzegorz Bartosz, Laboratory of Biophysics
of Development and Aging, Chair of Biophysics, University of L6dl, Banacha 12/16, PL 90-237 L6d~, Poland. 0047-6374/87/$03.50 Printed and Publishedin Ireland
© 1987 ElsevierScientificPublishers Ireland Ltd.
246 might be due to a decrease in the anion-exchange function of the Band 3 protein since the bovine erythrocytes lack the monocarboxylate transporter [12] and the charged form of the label is expected to be transported by the Band 3 protein in these cells. However, that conclusion could not be definite since the spin-labeled carboxylate may as well penetrate the membrane in an uncharged form. Recently it has been reported [13] that the rate constant for 3sSO2- efflux is higher by 20% in old as compared with young human erythrocytes. It prompted us to reexamine the effect of erythrocyte aging on the anion exchange using [32p]phosphate and a spin-labeled phosphate analogue (Tempo-phosphate). MATERIALSAND METHODS Sodium [32p] phosphate was purchased from the Institute of Nuclear Research (gwierk, Poland) and Tempo-phosphate (4-phospho-2,2,6,6-tetramethylpiperidyl-oxyl) from Syva (Palo Alto, CA). Erythrocytes were obtained from ACD-anti.coagulated human blood provided by a local blood bank and from bovine blood anti-coagulated with citrate, taken from an abbatoir. Erythrocytes were separated by density (and age) by the method of Murphy [14]. From the stratified cell column, the upper 20% ("young" cells), middle 20% and bottom 20% ("old" cells) were withdrawn and used for the experiments. For experiments with [32p]phosphate, the cells were washed with 205 mM sucrose in 28 mM sodium citrate (pH 7.4) [15]. The solution used for the final wash and cell resuspension contained, moreover, 5 mM glucose and 2 mM adenosine. For experiments with Tempo-phosphate, the erythrocytes were washed with Eagle's minimal essential medium (Medium 1959; WSS Lublin, Poland, pH 7.35). The cells were brought to the hematocrit of 0.50, equilibrated at appropriate temperature (18°C for [32p]phosphate studies and usually 37°C, or 18-37°C for Tempo-phosphate studies) and added with an 0.01 vol. of [a2P]phosphate in 100 mM sodium phosphate (pH 7.4) or 10 mM Tempophosphate in phosphate-buffered saline (145 mM NaC1 in 10 mM phosphate, pH 7.4) and mixed thoroughly. The cell suspensions were incubated with gentle shaking. At appropriate time intervals (usually 10, 20, 40, 60, 90, 120, 180,240 and 300 min)aliquots were withdrawn, centrifuged, and radioactivity of 32p was measured in the supernatants by Cerenkov radiation in a Beckman LS 7800 scintillation counter. Alternatively, electron spin resonance (ESR) spectra of the suspensions containing Tempo-phosphate were taken [16]. RESULTSAND DISCUSSION Up to at least 180 min the semi-logarithmic plots of the fraction of [32p]phosphate remaining in the extracellular medium were linear. The absolute values of these slopes were taken as permeation rate constants Kp for the [32p]phosphate influx into the ceils under the experimental conditions employed. Similarly, the semi-logarithmic plots of the intensity of the middle peak of ESR spectra of Tempo-phosphate in human
247 erythrocyte suspensions were linear within the time-range of the experiments (Fig. 1). It has been demonstrated [16] that the spin label is transported into the erythrocyte by the Band 3 protein and that the transport of that spin label into the cells is the ratelimiting step in the decay of its ESR spectral intensity in erythrocyte suspensions. Therefore, absolute values of the slopes of lines from these plots were taken as permeation constants Ktp of Tempo-phosphate into erythrocytes under the conditions employed. Interestingly, no decrease in the ESR spectral intensity of Tempo-phosphate was observed in bovine erythrocyte suspensions (not shown). It might be due to the label either not penetrating into the cells or not being reduced inside. The latter possibility seems less likely as other spin labels are reduced by suspensions of bovine red blood cells [5,11,17]. It can be hypothesized that the anion transport site of the bovine erythrocyte Band 3 protein differs from that of the human in being unable to bind and transport the tempo analogue of the phosphate anion. Comparison of the permeation constants revealed a consistent decrease with advancing erythrocyte age of the phosphate transport rate into human and bovine erythrocytes (Table I) and of the transport rate of Tempo-phosphate into human red blood cells (Table II). The obtained values of activation energy for the Tempo-phosphate transport are in good agreement with those found by Ross and McConnell [16] and were not subject to significant changes during erythrocyte aging (Table II).
1.0 0.9 0.8 _
0.7
~-
0.6
? "7,
"6 0.5
!
~6 0.4
"
Ia-
0
2b
4'0
60
8'0
100
12b t (~i..)
"
Fig. 1. Time-course of the decrease in the height of the middle peak of ESR spectra of Tempo-
phosphate in suspensionsof variousage fractions of human erythrocytes.
248 TABLE I PERMEATION RATE CONSTANTS OF [a2P]PHOSPHATE INTO HUMAN AND BOVINE ERYTHROCYTES OF VARIOUS AGE FRACTIONS Temperature: 18.03C;P with respect to young cells Age fraction
Kp
Human cells (n = 5] Young Middle-age Old Bovine cells (n = 6) Young Middle-age Old
P
(6.77 -+0.32) X 10-3 min-1 = 100% 90.0 _+ 1.8% P < 0.001 83.9 _+ 4.4% P < 0.01 (2.45 -+0.12) × 10-3 min-~ = 100% 88.9 _+ 9.2% P < 0.05 85.3 _+10.6% P < 0.05
Our results evidence a decrease in the transport efficiency of the Band 3 protein with advancing erythrocyte age. The consistency o f data obtained under two different experimental conditions is an argument against an artifactual nature o f the changes observed. These results contradict those o f Zanner and Galey [13] who observed an increase in the rate constant for ~SO~- efflux when comparing the 10% most dense ( " o l d e s t " ) with 10% lightest ( " y o u n g e s t " ) human erythrocytes fractionated by the same method. The reasons for this discrepancy are unclear. They may be related to different experimental attitudes (measuring influx vs. efflux) and studying various anions but in principle this should not be a probable source o f qualitative differences. The temperature of transport studies was different in experiments with sulfate [13] and phosphate (this paper) but the transport of Tempo-phosphate was monitored at 37°C and no significant celt-age related alterations in the value of activation energy for the transport of this anion were found (Table II). Lately a paper appeared [18] stating a considerable impairment o f sulfate exchange in aged rat erythrocytes measured under Donnan equilibrium conditions (a more than two-fold decrease of maximal flux and a three-fold increase of the apparent Michaelis-
TABLE I1 RATE CONSTANTS AND ACTIVATION ENERGY E FOR TEMPO-PHOSPHATE TRANSPORT INTO HUMAN ERYTHROCYTES OF VARIOUS AGE FRACTIONS Ktp values for 37.0°C; E for the range of 18.0-37.0°C; n = 5; P with respect to young cells (Student's t-test) Age fraction
Ktp
E
Young Middle-age Old
(16.2 _+0.9) X 10-3 min -1 = 100% 87.2 -+8.2% P < 0.05 82.6 -+7.3% P < 0.01
102 _+9 kJ/mol = 100% 91.1 _+12.2% NS 96.1 _+10.6% NS
249 Menten constant in old vs. middle-aged ceils). Our data confirm this conclusion qualitatively, though a direct comparison is hardly possible because of employment of various quantities (Krp vs. Vmax), apparently the magnitude of cell-age related changes in anion transport was much higher in the study of Kay etal. [18] than in ours. This may be due to a lower efficiency of the method of Murphy [14] of cell separation according to age as compared with centrifugation on Percoll density gradient, [18], to comparison of large fractions of cells which may attenuate changes occurring in the few per cent of very oldest cells and/or to species differences in the extent of alterations related to erythrocyte aging. A decrease in the anion transport rate in aging erythrocytes is more comprehensible than its increase since: (i) Progressive proteolysis of the Band 3 protein occurs during erythrocyte aging [10] and proteolysis of this protein is known to decrease, if any, the anion exchange [19]. (ii) Calcium which accumulates in aged erythrocytes is known to inhibit the anion exchange [20,21]. (iii) Moreover, anion exchange through the erythrocyte membrane depends on membrane potential [16,22]. A decrease in membrane potential diminishes the rate of Tempo-phosphate uptake by erythrocytes [16]. Our preliminary studies indicate a decrease in the value of membrane potential in aged erythrocytes (in preparation). During erythrocyte aging, cell volume decreases without apparent changes in the cellular shape. This indicates an increase in the cell surface to volume ratio (by over 2% for bovine red blood cells separated according to age under the same conditions, as found by recalculation of our previous data [22] ). This effect would compensate for the phenomenon of decrease in the transport rate, so if to express the anion flux/cell, the actual magnitude of the diminution in the transport rate would be even higher than that given here in relation to unit cell volume (Tables I and II). ACKNOWLEDGEMENTS We are indebted to Prof. W. Leyko for stimulating discussions. REFERENCES 1 M. Kadlubowskiand J.R. Harris, The appearance of a protein in the human erythrocyte membrane during agein_g.FEBS Lett., 47 (1974) 252-254. 2 S.K. Jain and P. Hochstein, Polymerization of membrane components in aging red blood cells. Bioehem. Biophys. Res. Commun., 92 (1980) 247-254. 3 M. Gaczyflska and G. Bartosz, CrossUnking of membrane proteins during erythrocyte aging. Int. J. Bioehem., 18 (1986) 377-382. 4 J.R. Barber and S. Clarke, Membraneprotein carboxyl methylation increases with human erythrocyte age. EvidenCefor an increase in the number of methylable sites, d. Biol. Chem., 258 (1983) 1189-1196. 5 G. Bartosz, Spin-label studies of membrane lipids, proteins and permeability. Bioehim. Biophys. Aeta, 664 (1981) 69-73. 6 R.F. Dons, L.M. Corash and P. Gorden, The insulin receptor is an age-dependent integral component of the human erythrocyte membrane.J. Biol. Chem., 256 (1981) 2982-2987. 7 N.S. Cohen, J.E. Ekholm, M.G. Luithra and D.L Hanahan, Biochemical characterization of density-separatedhuman erythrocytes. Biochim. Biophys. Acra, 419 (1976) 229-242.
250 8 M.M.B. Kay, Role of physiologic autoantibody in the removal fo senescent human red cells. J. Supramol. Struct., 9 (1978) 329-341. 9 M.M.B. Kay, Localization of senescent cell antigen on Band 3. Prec. Natl. Acad. ScL U.S.A., 81 (1984) 5753-5757. 10 M.M.B. Kay, Band 3, the predominant transmembrane polypeptide, undergoes proteolytic degradation as cell age. Monogr. Dev. Biol., 17 (1984) 245-253. 11 G. Bartosz and K. Gwo~dzifiski, Aging of the erythrocyte. 23. Changes in the permeation of spinlabeled electrolytes. Am. J. Hematol., 14 (1983) 377-379. 12 B. Deuticke, I. Rickert and E. Beyer, Stereoseleetive, SH-dependent transfer of lactate in mammarian erythrocytes. Biochim. Biophys. Acta, 507 (1978) 137-155. 13 M.A. Zanner and W.R. Galey, Aged erythrocytes exhibit increased anion exchange. Biochim. Biophys. Acta, 818 (1985) 310-315. 14 J.R. Murphy, Influence of temperature and method of centrifugation on the separation of erythrocytes. J. Lab. Clin. Med., 82 (1973) 334-341. 15 J.D. Craik and R.A.F. Reithmeier, Inhibition of phosphate transport in human erythrocytes by water-soluble carbodimides. Biochim. Biophys. Acta, 778 (1984) 429-434. 16 A.H. Ross and H.M. McCormell, Permeation of a spin-label phosphate into the human erythrocyte. Biochemistry, 14 (1975) 2793-2798. 17 K. Gwo~dzifiski, G. Bartosz and W. Leyko, Effect of gamma radiation on the transport of spinlabeled compounds across the erytkrocyte membrane. Radiat. Environ. Biophys., 19 (1981) 275-283. 18 M2~.B. Kay, G.J.C.G.M. Bosman, S.S. Shapko, A. Bendich and P.S. Bassel, Oxidation as a possible mechanism of cellular aging: vitamin E deficiency causes premature aging and IgG binding to erythrocytes. Prec. Natl. Acad. ScL U.S.A., 83 (1986) 2463-2467. 19 M.L. Jennings and M.F. Adams, Modification by papain of the structure and function of Band 3, the erythrocyte anion transport protein. Biochemistry, 20 (1981) 7118-7123. 20 P.L. LaCeUe, F.H. Kirkpatrick, M.P. Utkow and B. Arkin, Membrane fragmentation and Ca ~membrane interaction: potential mechanism of shape change in senescent red cells. Nouv. Rev. Fr. Hematol., 12 (1972) 789-798. 21 R. Passing and D. Schubert, The binding of Ca r' to solubilized band 3 protein of the human erythrocyte membrane. Hoppe.Seyler's Z. Physiol. Chem., 364 (1983) 873-878. 22 V. Castranova, M.J. Weise and J.F. Hoffman, Anion transport in dog, cat and human red cells. Effects of varying ceil volume and Donnan ratio. J. Gen. PhysioL, 74 (1979) 319-334. 23 G. Bartosz, E. Grzelir~ska and A. Bartkowiak, Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes. Mech. Ageing Dev., 24 (1984) 1-7.
245
Elsevier ScientificPublishersIreland Ltd.
AGED ERYTHROCYTES EXHIBIT DECREASED ANION EXCHANGE
GRZEGORZ BARTOSZ, MARIA GACZY~SKA, EWA GRZELII~ISKA, MIROS[AW SOSZYI~ISKI,W]~ODZIMIERZ MICHALAKa and ROMAN GONDKOa Laboratory of Biophysics of Development and Aging, Chair of Biophysics and aLaboratory of Bio. logical Sciences, University of l~6d[, 90-237 l]6d~ fPoland}
(Received October 15th, 1986) (RevisionreceivedFebruary 26th, 1987) SUMMARY
The rate of transport of [3~p] phosphate into human and bovine erythrocytes and of a spin-label analogue of phosphate (Tempo-phosphate) into human erythrocytes was found to decrease with increasing erythrocyte age by 15-20% when comparing 20% most dense celis with 20% of lightest cells. The activation energy of Tempo-phosphate transport did not show significant changes upon erythrocyte aging. Key words: Erythrocyte aging; Phosphate transport; Spin label; Electron spin resonance
INTRODUCTION Aging of a mammalian erythrocyte has been demonstrated to involve structural and functional alterations in red cell membrane proteins. They include minor changes in the polypeptide composition [1], progressive cross-linking [2] and aggregation [3], racemization [4], alterations in the physical state of the proteins as evidenced by spin-labeling [5] and fluorescence quenching (submitted), decrease in the number of receptors [6] and enzymatic activities [7] and exposure of a "senescent cell antigen" [8]. It has been proposed [9] that the "senescent cell antigen" is formed by proteolytic degradation in situ of the Band 3 protein of the erythrocyte membrane (the anion exchange protein). Progressive proteolysis of the Band 3 protein was demonstrated to .occur during erythrocyte aging [10]. It seemed therefore worthwhile to examine functional properties of the Band 3 protein in red blood cells of various ages. We have studied the transport of an anionic spin label 2,2,5,5-tetramethyl-3-carboxypyrrolidine-l-oxyl into bovine erythrocytes finding a hampering of its transport in aged cells [11]. This Address all correspondence and reprint requests to: Dr. Grzegorz Bartosz, Laboratory of Biophysics
of Development and Aging, Chair of Biophysics, University of L6dl, Banacha 12/16, PL 90-237 L6d~, Poland. 0047-6374/87/$03.50 Printed and Publishedin Ireland
© 1987 ElsevierScientificPublishers Ireland Ltd.
246 might be due to a decrease in the anion-exchange function of the Band 3 protein since the bovine erythrocytes lack the monocarboxylate transporter [12] and the charged form of the label is expected to be transported by the Band 3 protein in these cells. However, that conclusion could not be definite since the spin-labeled carboxylate may as well penetrate the membrane in an uncharged form. Recently it has been reported [13] that the rate constant for 3sSO2- efflux is higher by 20% in old as compared with young human erythrocytes. It prompted us to reexamine the effect of erythrocyte aging on the anion exchange using [32p]phosphate and a spin-labeled phosphate analogue (Tempo-phosphate). MATERIALSAND METHODS Sodium [32p] phosphate was purchased from the Institute of Nuclear Research (gwierk, Poland) and Tempo-phosphate (4-phospho-2,2,6,6-tetramethylpiperidyl-oxyl) from Syva (Palo Alto, CA). Erythrocytes were obtained from ACD-anti.coagulated human blood provided by a local blood bank and from bovine blood anti-coagulated with citrate, taken from an abbatoir. Erythrocytes were separated by density (and age) by the method of Murphy [14]. From the stratified cell column, the upper 20% ("young" cells), middle 20% and bottom 20% ("old" cells) were withdrawn and used for the experiments. For experiments with [32p]phosphate, the cells were washed with 205 mM sucrose in 28 mM sodium citrate (pH 7.4) [15]. The solution used for the final wash and cell resuspension contained, moreover, 5 mM glucose and 2 mM adenosine. For experiments with Tempo-phosphate, the erythrocytes were washed with Eagle's minimal essential medium (Medium 1959; WSS Lublin, Poland, pH 7.35). The cells were brought to the hematocrit of 0.50, equilibrated at appropriate temperature (18°C for [32p]phosphate studies and usually 37°C, or 18-37°C for Tempo-phosphate studies) and added with an 0.01 vol. of [a2P]phosphate in 100 mM sodium phosphate (pH 7.4) or 10 mM Tempophosphate in phosphate-buffered saline (145 mM NaC1 in 10 mM phosphate, pH 7.4) and mixed thoroughly. The cell suspensions were incubated with gentle shaking. At appropriate time intervals (usually 10, 20, 40, 60, 90, 120, 180,240 and 300 min)aliquots were withdrawn, centrifuged, and radioactivity of 32p was measured in the supernatants by Cerenkov radiation in a Beckman LS 7800 scintillation counter. Alternatively, electron spin resonance (ESR) spectra of the suspensions containing Tempo-phosphate were taken [16]. RESULTSAND DISCUSSION Up to at least 180 min the semi-logarithmic plots of the fraction of [32p]phosphate remaining in the extracellular medium were linear. The absolute values of these slopes were taken as permeation rate constants Kp for the [32p]phosphate influx into the ceils under the experimental conditions employed. Similarly, the semi-logarithmic plots of the intensity of the middle peak of ESR spectra of Tempo-phosphate in human
247 erythrocyte suspensions were linear within the time-range of the experiments (Fig. 1). It has been demonstrated [16] that the spin label is transported into the erythrocyte by the Band 3 protein and that the transport of that spin label into the cells is the ratelimiting step in the decay of its ESR spectral intensity in erythrocyte suspensions. Therefore, absolute values of the slopes of lines from these plots were taken as permeation constants Ktp of Tempo-phosphate into erythrocytes under the conditions employed. Interestingly, no decrease in the ESR spectral intensity of Tempo-phosphate was observed in bovine erythrocyte suspensions (not shown). It might be due to the label either not penetrating into the cells or not being reduced inside. The latter possibility seems less likely as other spin labels are reduced by suspensions of bovine red blood cells [5,11,17]. It can be hypothesized that the anion transport site of the bovine erythrocyte Band 3 protein differs from that of the human in being unable to bind and transport the tempo analogue of the phosphate anion. Comparison of the permeation constants revealed a consistent decrease with advancing erythrocyte age of the phosphate transport rate into human and bovine erythrocytes (Table I) and of the transport rate of Tempo-phosphate into human red blood cells (Table II). The obtained values of activation energy for the Tempo-phosphate transport are in good agreement with those found by Ross and McConnell [16] and were not subject to significant changes during erythrocyte aging (Table II).
1.0 0.9 0.8 _
0.7
~-
0.6
? "7,
"6 0.5
!
~6 0.4
"
Ia-
0
2b
4'0
60
8'0
100
12b t (~i..)
"
Fig. 1. Time-course of the decrease in the height of the middle peak of ESR spectra of Tempo-
phosphate in suspensionsof variousage fractions of human erythrocytes.
248 TABLE I PERMEATION RATE CONSTANTS OF [a2P]PHOSPHATE INTO HUMAN AND BOVINE ERYTHROCYTES OF VARIOUS AGE FRACTIONS Temperature: 18.03C;P with respect to young cells Age fraction
Kp
Human cells (n = 5] Young Middle-age Old Bovine cells (n = 6) Young Middle-age Old
P
(6.77 -+0.32) X 10-3 min-1 = 100% 90.0 _+ 1.8% P < 0.001 83.9 _+ 4.4% P < 0.01 (2.45 -+0.12) × 10-3 min-~ = 100% 88.9 _+ 9.2% P < 0.05 85.3 _+10.6% P < 0.05
Our results evidence a decrease in the transport efficiency of the Band 3 protein with advancing erythrocyte age. The consistency o f data obtained under two different experimental conditions is an argument against an artifactual nature o f the changes observed. These results contradict those o f Zanner and Galey [13] who observed an increase in the rate constant for ~SO~- efflux when comparing the 10% most dense ( " o l d e s t " ) with 10% lightest ( " y o u n g e s t " ) human erythrocytes fractionated by the same method. The reasons for this discrepancy are unclear. They may be related to different experimental attitudes (measuring influx vs. efflux) and studying various anions but in principle this should not be a probable source o f qualitative differences. The temperature of transport studies was different in experiments with sulfate [13] and phosphate (this paper) but the transport of Tempo-phosphate was monitored at 37°C and no significant celt-age related alterations in the value of activation energy for the transport of this anion were found (Table II). Lately a paper appeared [18] stating a considerable impairment o f sulfate exchange in aged rat erythrocytes measured under Donnan equilibrium conditions (a more than two-fold decrease of maximal flux and a three-fold increase of the apparent Michaelis-
TABLE I1 RATE CONSTANTS AND ACTIVATION ENERGY E FOR TEMPO-PHOSPHATE TRANSPORT INTO HUMAN ERYTHROCYTES OF VARIOUS AGE FRACTIONS Ktp values for 37.0°C; E for the range of 18.0-37.0°C; n = 5; P with respect to young cells (Student's t-test) Age fraction
Ktp
E
Young Middle-age Old
(16.2 _+0.9) X 10-3 min -1 = 100% 87.2 -+8.2% P < 0.05 82.6 -+7.3% P < 0.01
102 _+9 kJ/mol = 100% 91.1 _+12.2% NS 96.1 _+10.6% NS
249 Menten constant in old vs. middle-aged ceils). Our data confirm this conclusion qualitatively, though a direct comparison is hardly possible because of employment of various quantities (Krp vs. Vmax), apparently the magnitude of cell-age related changes in anion transport was much higher in the study of Kay etal. [18] than in ours. This may be due to a lower efficiency of the method of Murphy [14] of cell separation according to age as compared with centrifugation on Percoll density gradient, [18], to comparison of large fractions of cells which may attenuate changes occurring in the few per cent of very oldest cells and/or to species differences in the extent of alterations related to erythrocyte aging. A decrease in the anion transport rate in aging erythrocytes is more comprehensible than its increase since: (i) Progressive proteolysis of the Band 3 protein occurs during erythrocyte aging [10] and proteolysis of this protein is known to decrease, if any, the anion exchange [19]. (ii) Calcium which accumulates in aged erythrocytes is known to inhibit the anion exchange [20,21]. (iii) Moreover, anion exchange through the erythrocyte membrane depends on membrane potential [16,22]. A decrease in membrane potential diminishes the rate of Tempo-phosphate uptake by erythrocytes [16]. Our preliminary studies indicate a decrease in the value of membrane potential in aged erythrocytes (in preparation). During erythrocyte aging, cell volume decreases without apparent changes in the cellular shape. This indicates an increase in the cell surface to volume ratio (by over 2% for bovine red blood cells separated according to age under the same conditions, as found by recalculation of our previous data [22] ). This effect would compensate for the phenomenon of decrease in the transport rate, so if to express the anion flux/cell, the actual magnitude of the diminution in the transport rate would be even higher than that given here in relation to unit cell volume (Tables I and II). ACKNOWLEDGEMENTS We are indebted to Prof. W. Leyko for stimulating discussions. REFERENCES 1 M. Kadlubowskiand J.R. Harris, The appearance of a protein in the human erythrocyte membrane during agein_g.FEBS Lett., 47 (1974) 252-254. 2 S.K. Jain and P. Hochstein, Polymerization of membrane components in aging red blood cells. Bioehem. Biophys. Res. Commun., 92 (1980) 247-254. 3 M. Gaczyflska and G. Bartosz, CrossUnking of membrane proteins during erythrocyte aging. Int. J. Bioehem., 18 (1986) 377-382. 4 J.R. Barber and S. Clarke, Membraneprotein carboxyl methylation increases with human erythrocyte age. EvidenCefor an increase in the number of methylable sites, d. Biol. Chem., 258 (1983) 1189-1196. 5 G. Bartosz, Spin-label studies of membrane lipids, proteins and permeability. Bioehim. Biophys. Aeta, 664 (1981) 69-73. 6 R.F. Dons, L.M. Corash and P. Gorden, The insulin receptor is an age-dependent integral component of the human erythrocyte membrane.J. Biol. Chem., 256 (1981) 2982-2987. 7 N.S. Cohen, J.E. Ekholm, M.G. Luithra and D.L Hanahan, Biochemical characterization of density-separatedhuman erythrocytes. Biochim. Biophys. Acra, 419 (1976) 229-242.
250 8 M.M.B. Kay, Role of physiologic autoantibody in the removal fo senescent human red cells. J. Supramol. Struct., 9 (1978) 329-341. 9 M.M.B. Kay, Localization of senescent cell antigen on Band 3. Prec. Natl. Acad. ScL U.S.A., 81 (1984) 5753-5757. 10 M.M.B. Kay, Band 3, the predominant transmembrane polypeptide, undergoes proteolytic degradation as cell age. Monogr. Dev. Biol., 17 (1984) 245-253. 11 G. Bartosz and K. Gwo~dzifiski, Aging of the erythrocyte. 23. Changes in the permeation of spinlabeled electrolytes. Am. J. Hematol., 14 (1983) 377-379. 12 B. Deuticke, I. Rickert and E. Beyer, Stereoseleetive, SH-dependent transfer of lactate in mammarian erythrocytes. Biochim. Biophys. Acta, 507 (1978) 137-155. 13 M.A. Zanner and W.R. Galey, Aged erythrocytes exhibit increased anion exchange. Biochim. Biophys. Acta, 818 (1985) 310-315. 14 J.R. Murphy, Influence of temperature and method of centrifugation on the separation of erythrocytes. J. Lab. Clin. Med., 82 (1973) 334-341. 15 J.D. Craik and R.A.F. Reithmeier, Inhibition of phosphate transport in human erythrocytes by water-soluble carbodimides. Biochim. Biophys. Acta, 778 (1984) 429-434. 16 A.H. Ross and H.M. McCormell, Permeation of a spin-label phosphate into the human erythrocyte. Biochemistry, 14 (1975) 2793-2798. 17 K. Gwo~dzifiski, G. Bartosz and W. Leyko, Effect of gamma radiation on the transport of spinlabeled compounds across the erytkrocyte membrane. Radiat. Environ. Biophys., 19 (1981) 275-283. 18 M2~.B. Kay, G.J.C.G.M. Bosman, S.S. Shapko, A. Bendich and P.S. Bassel, Oxidation as a possible mechanism of cellular aging: vitamin E deficiency causes premature aging and IgG binding to erythrocytes. Prec. Natl. Acad. ScL U.S.A., 83 (1986) 2463-2467. 19 M.L. Jennings and M.F. Adams, Modification by papain of the structure and function of Band 3, the erythrocyte anion transport protein. Biochemistry, 20 (1981) 7118-7123. 20 P.L. LaCeUe, F.H. Kirkpatrick, M.P. Utkow and B. Arkin, Membrane fragmentation and Ca ~membrane interaction: potential mechanism of shape change in senescent red cells. Nouv. Rev. Fr. Hematol., 12 (1972) 789-798. 21 R. Passing and D. Schubert, The binding of Ca r' to solubilized band 3 protein of the human erythrocyte membrane. Hoppe.Seyler's Z. Physiol. Chem., 364 (1983) 873-878. 22 V. Castranova, M.J. Weise and J.F. Hoffman, Anion transport in dog, cat and human red cells. Effects of varying ceil volume and Donnan ratio. J. Gen. PhysioL, 74 (1979) 319-334. 23 G. Bartosz, E. Grzelir~ska and A. Bartkowiak, Aging of the erythrocyte. XIX. Decrease in surface charge density of bovine erythrocytes. Mech. Ageing Dev., 24 (1984) 1-7.