- Email: [email protected]
Crosslinking of membrane proteins in red blood cells from vitamin E-deficient lead-poisoned rats
Life Sciences, Vol. 28, pp. 147-154 Printed in the U.S.A.
Pergamon Press
CROSSLINKING OF MEMBRANE PROTEINS IN RED BLOOD CELLS FROM VITAMIN E-DEFICIENT LEAD-POISONED RATS
Orville A. Levander and Susan O. Welsh
Nutritlon Institute Agricultural Research Center Beltsville, Maryland 20705 (Received in final form October 24, 1980) Summary Red blood cells from vitamin E-deficient rats lose their filterability after ineubatlon in vitro and concurrent lead poisoning of the rats accelerates this decline. Thls decreased red cell filterability is associated with an increased crosslinking of red cell membrane protelns. Previous reports by others suggested an association between red cell glutathione levels and fllterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a roleein the discocyte/ spherocyte shape change that accounts for the impalred filterability of red blood cells from vitamin E-deflclent lead-poisoned rats. The useful physiological lifetime partially determined by their abillty narrow as 3 microns in diameter. The deformable since the dlameter of, for morons.
of red blood cells (RBC) is at least to pass through mlcrocapillarles as latter requlres that RBC be highly example, the rat RBC is about 7.5
RBC from vitamin E-deflcient rats (-E RBC) lose thelr ability to pass through 3 micron polycarbonate membrane filters after incubation in vitro and lead poisonlng of the anlmals exacerbates thls effect (I). Thls decreased RBC filterabillty is accentuated by incubation wlth pro-oxidants (2), is correlated with lipld peroxidation (I), and can be prevented by feeding a synthetzc antioxidant (I). Old (denser) -E RBC show thls defect in filterability more strongly than young -E RBC and lead poisoning exaggerates this difference related to cell age (3). The decreased filterab111ty of -E RBC is accompanled by a morphological transition from the discocytlc to the spherocytlc shape (4). Others have associated changes in RBC shape and deformability wlth crosslinking of membrane proteins (5). Our present purpose was to determine whether membrane proteins are crossllnked in nonfilterable RBC from vitamln E-deficlent rats with and without concomitant lead polsoning. RBC filterability is used as an index of RBC deformab111ty. Since RBC f~lterabllity is thought to depend partly on intracellular GSH levels (6), the role of GSH in the f11terabllity of -E RBC was also studled. 0024-3205/81/020147-08502.00/0
148
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
Methods Weanling male Fischer 344 rats (Microblologlcal Associates, Walkersville, MD) were housed individually in hanging stainless steel wire cages and were fed elther a vitamin E-deficient Torula yeast diet (I) supplemented with 0.5 ppm selenium as sodium selenite or the same diet supplemented with both selenium and I00 ppm vltamin E as dl-~-tocopheryl acetate (Powder, General Biochemicals, Inc., Chagrin Falls, OH). Lead-poisoned rats received 500 ppm lead as lead acetate in the drlnking water. All rats had free access to food and water. In experiments relating RBC membrane protein crosslinking to RBC filterability, heparinized arterial blood from ether anesthetized rats was centrlfuged at 1000 x g for 10 min. at 4 ° . The plasma and bully coat were removed by aspiration and the packed RBC were washed 3x by resuspension in 3-4 volumes of 0.15 M NaCI-0.5 mM glucose-O.01M trls-Cl buffer (pH 7.4) and centrlfuged as above. For filterability measurements (see below), 0.9% RBC suspensions were prepared in the buffer. Either at zero time or after 4-6 hr. of Incubation at 30 °, RBC were pelleted by centrifugation as above. The packed RBC were lysed in 25-35 volumes of cold i0 mM tris-Cl (pH 7.4) followed by centrifugation at 29,000 x g for 10 mln. at 4° . The pelleted membranes were washed 2x with the hypotonic buffer and then were solubllized in 0.25 volumes of 5% SDS, 250 mM tris-Cl (pH 7.4), 10 mM EDTA, 1.25 M sucrose and 0.1 mE/ml pyronin Y and stored at -20 °. Protein concentration was determined by a modified Lowry procedure (7). Aliquots of solubilized membrane solutlon containlng 0.05 mE of proteln were applied to 5.5xi00 mm 4% acrylamide gels (Bio-Rad Labs., Richmond, CA 94804) whlch had been electrophoresed previously with the running buffer. The samples were allowed to enter the gels at a current of 2 mA/tube for 30 min., followed by a current of 3.5 mA/tube for 5 hr. The buffers and stainlng solutlons were prepared according to Falrbanks et al. (8). In experiments relating RBC GSH levels to RBC filterability, filterability of suspensions of RBC from rats at least 3 months on the experimental diets was determined by the method of Levander et al. (I). This technique involves measuring the amount of tlme required ("Filtration Time") for 2 ml of a 0.9% RBC suspension in buffer to pass through a pre-calibrated 25 mm polycarbonate filter membrane with a pore slze of 3 microns (Nuclepore Corp., Pleasanton, CA 94566) under a vacuum of i0 cm of water. The suspending buffer was 0.145 M NaCl-O.01M tris-Cl, pH 7.4, whlch contained 10 mg glucose/100 ml. The RBC suspensions were incubated at room temperature under alr. In the cell age experiment (Table II) old and young RBC were fractionated by differential centrlfugation (3). In the sulfhydryl blocking experiment (Table III), the N-ethylmaleimlde was dissolved where appropriate in the suspending buffer at the concentratlon indicated before the RBC were added to the buffer. Thus, the RBC were exposed to the blocking agent immediately before the first filtration measurement at 0 hr. RBC GSH was measured by colorimetry (9). Results Table I shows the decreased filterablllty of -E RBC incubated in vltro plus the exacerbating effect of concurrent in vlvo lead poisoning that we reported previously (I-4). In thls particular experiment, the lead impaired filterability of the -E RBC even without incubation. Lead poisoning had little or no effect on the filterability of RBC from vitamln E-supplemented rats (+E RBC). Electrophoretlc analysls of membrane protelns extracted from +E RBC of non-polsoned rats revealed typlcal normal stalning patterns before and after incubatlon of the RBC (Fig. I, A and B). Membrane protelns of -E RBC from non-poisoned rats also exhiblted a normal pattern before incubation but when
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinkin E
149
TABLE I Effects of vitamin E deflciency and lead poisoning on filterability of rat RBC
Diet
Lead in water ppm
Filtratlon t:me after incubation for 0 hr
2 hr
sec
4 hr
sec
sec
Control
0
17 +
1
19 ÷
Deficient
0
16 ÷
1
43 + 14
Control
500
21 ÷
2
26 +
Deficlent
500
263 + 91
1
5
457 ÷ 59
19 ÷
2
580 ÷ 20 37 +
7
>600
Mean values of 4 to 9 rats + S.E. the cells had lost thelr f11terability after incubat:on there was evidence of increased stalning at molecular weights greater than spectrin (Fig. I, C and D). Other changes included some increased generalized diffuse staining throughout the gel, possibly indicative of proteolysis, and a rather sharp decline in the sta:ning associated with bands 2.1 and 4.2. Membrane proteins of +E RBC st111 gave normal staining patterns even though the rats were poisoned w:th lead (F:g. 2, A and B). On the other hand, membrane proteins of -E RBC from lead-poisoned rats showed evidence of protein polymerization and the other changes noted above even wlthout :ncubation of the RBC (Fig. 2, C and D). Since the loss of filterability of -E RBC is due largely to non-filterable RBC of old cell age (3), GSH levels in young vs. old RBC were analyzed. As reported (3), -E RBC of old cell age lose thelr filterab:llty to a much greater extent after incubation in vitro than -E RBC of young cell age and the GSH levels of the non-filterable old -E RBC were about 22% less than those in any of the other incubated RBC (Table II). To investigate further the relationship between GSH and RBC filterability, RBC from def:clent and supplemented rats were incubated w:th and without the permeable sulfhydryl blocking agent, N-ethylmaleimide (NEM). Incubation with 10"4 M NEM accelerated the decl:ne in filterability of -E RBC but had no effect on the fllterabillty of +E RBC (Table III) even though the NEM caused an almost instantaneous loss of GSH in both -E RBC and +E RBC. Discussion The data presented here confirm earlier results that -E RBC suffer a decline in f11terabillty during incubatlon in vltro and that concurrent lead polsoning accelerates thls decline (I-4). The relevance of these in vitro f:ltration changes to decreased RBC survival in vivo is not certain but the stress of :ncubation may be needed to reveal subtle differences in RBC that are at increased risk with regard to splenic sequestration. Since the RBC samples taken from our rats are derived from per:pheral blood, any grossly abnormal RBC would presumably already have been culled by the spleen and one
150
RBC Filterability & Protein Crosslinking
A
B
C
Vol. 28, No. 2, 1981
D
I 2 2.1
3 4.1 4.2
5 6
7
FIG. 1 Effect of vitamin E deficiency on RBC membrane proteins separated by SDS gel electrophoresis. A: unincubated +E RBC, 8: ÷E RBC incubated 4 hr., C: unincubated -E RBC, D: -E RBC incubated 4 hr. The designation of the major membrane polypeptides is according to Steck (i0).
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinking
A
B
C.
D
! 2 2.1
3 4.1 4.2
5 6
7
FIG. 2. Effect of vitamin E deflciency and lead toxicity on RBC membrane proteins separated by SDS gel electrophoresls. A: unincubated ÷E RBC from leadpoisoned rats, B: same as A after 4 hrs. incubation, C: unincubated -E RBC from lead-poisoned rats, D: same as C after 4 hrs. incubation. Membrane polypeptides labelled as in Fig. i.
151
152
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
TABLE II Effect of cell age on filterability and GSH levels of RBC from vitamin E-deflcient and supplemented rats
Diet
Cell age
Incubation period 0 hr
3 hr
Filtration time (seconds) Control Control Deficient Deficient
young old young old
18 + 2 20+3 15-+1 17_+i
25 46 26 >
+ 7 + 16 + 6 ~00
GSH ( q / l O 0 ml packed RBC) Control Control Deficient Deficient
young old young old
129 131 108 98
~ ,+ ,+ ,+
14 14 3 3
105 105 99 77
,+ ,+ ± ,+
3 6 4 3
Mean values of 3 rats + S.E. TABLE III Effect of N-ethylmaleimide on filterability and GSH levels of RBC from vltamin E-deficient and supplemented rats
Diet
N-ethylmaleimide
Incubation period 0 hr
3 hr
5 hr
Filtration time (seconds) Control Deficient Control Deficient
0 0 10 -4 10 -4
15 16 13 14
+ + ~ ~
1 1 2 1
21 + 2 68 + 32 16 ~ 1 >600
22+2 > EO0
GSH (m~/lOO ml packed RBC) Control Deficient Control Deficient
0 0 10 -4 10 -4
Mean values of 3 rats + S.E.
109 i00 14 13
,+ 3 + 2 ,+ 3 ,+ 3
12 + 5 16 _~ 1
104 + 5 94+4
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinking
153
would not necessarily expect any differences in the filterability of fresh RBC in the various treatment groups. Hypothetical mechanisms of splenic trapping of RBC from vitamin E-deflcient lead-poisoned rats have been discussed elsewhere (11). The biochemical mechanism by which vitamin E deficiency and lead toxicity decrease RBC filterability is not understood in detail although a discocyte/ spherocyte shape change appears involved (4). Vitamin E deficiency is known to impair the active transport of cations in rat liver slices (12), but cation shifts do not seem to play a role in the decreased filterability reported here. Incubation of -E RBC at 0 °, which would cause massive loss of intracellular potassium and influx of sodium, actually protected such cells against the typical filtration changes observed in -E RBC incubated at room temperature
(13). Intracellular GSH concentration also does not appear to play a primary role in determining RBC filterability. Under different conditions, it was possible to demonstrate elther complete loss of RBC filterability with only mild loss of RBC GSH or, conversely, an almost total loss of RBC GSH wlth no loss of RBC filterability. Others have concluded that intracellular GSH levels have little to do with various RBC hemolytic phenomena and have suggested that membrane protein sulfhydryl groups are more important in thls regard (14). The decreased filterability of incubated -E RBC observed in our studies was associated with increased crosslinking of RBC membrane proteins and concurrent lead poisonlnE of the rats accelerated both of these changes. We were concerned that the proteins prepared from membranes of -E RBC might be susceptible to artifactual oxidative crosslinking during extraction because of the lack of antioxidant protection in such membranes. However, since no crosslinkinE was seen In membrane proteins from unincubated (i.e., zero time) -E RBC, the proteins seemed to be stable to the extraction process per se even in the absence of antloxldant and the polymerization apparently occurred durlng incubation and not during the isolation of stroma from the RBC lysate. Therefore, the morphological transltion from discocyte to spheroeyte thought responsible for the decreased filterability of -E RBC (4) may indeed be brought about by the aEEregatlon of RBC membrane proteins. On the basis of these data, however, we could not establish definitively whether crosslinking of RBC membrane proteins is the cause or an effect of the change in RBC shape. Several different biochemical mechanlsms have been impllcated in the polymerization of RBC membrane proteins. Lorand et al. (15) described a calciumactivated transglutamlnase which catalyzed the crossllnklng of RBC membrane proteins through the formation of 7-glutamyl-e-lysine bridges. That, however, probably was not the mechanism of the crosslinking observed by us since neither calcium nor ionophore was present in our incubation buffer. A second mechanism of RBC membrane protein crossllnklng is the oxidation of protein sulfhydryls to form intermolecular disulfide bridges. RBC membranes from patients wlth glucose-6-phosphate dehydrogenase deficiency and associated chronic hemolytic disease contain high molecular weight polypeptide aggregates that are formed by intermolecular disulfide bonds and RBC deformability is decreased in cells containing such aggregates (16). However, a good assoclatlon was noted between the quantlty of aggregates in the RBC of these patients and the RBC GSH concentration, whereas we found no association between RBC fllterabllity and GSH levels (see discussion above). Another explanation for the membrane protein crossllnklng observed in our incubated -E RBC is Schlff base formation by the reaction of amino groups with malonyldlaldehyde, an end product of polyunsaturated fatty acld peroxldatlon (17). Decreases in filterability of -E RBC are directly correlated wlth the production of malonyldlaldehyde (I) and the addition of malonyldlaldehyde to rat RBC in vitro causes an Increase in
154
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
high molecular weight protein polymers (18). Both of the latter 2 crosslinking mechanisms are compatible with the hypothesis that lipid peroxidation is the prlmary event causing shape change in -E RBC via protein polymerization either by "spilling over" of free radical chain reactions into the membrane proteins (oxidation to disulfide links) or by furnishing a chemically reactive crosslinking agent (mslonyldialdehyde). References i. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 107363-372 (1977). 2. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 1072135-2143 (1977). 3. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 108145-151 (1978). 4. O.A. LEVANDER, M. FISHER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 107 1828-1836 (1977). 5. J. PALEK, P.A. LIU and S.C. LIU, Nature 274505-507 (1978). 6. P. TEITEL, I. MARCU and A. XENAKIS, Fol. Haematologica 90281-295 (1968). 7. A. BENSADOUN and D. WEINSTEIN, Anal. Biochem. 70241-250 (1976). 8. G. FAIRBANKS, T.L. STECK and D.F.H. WALLACH, B~ohemlstry 102606-2617 (1971). 9. E. BEUTLER, O. DURON and B.M. KELLY, J. Lab. Clin. Med. 61882-888 (1963). 10. T.L. STECK, J. Mol. Biol. 66295-305 (1972). ii. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, in The Re d Cell (G.J. Brewer, Ed.) pp. 575-590, Alan R. Liss, Inc., New York (1978). 12. O.A. LEVANDER and V.C. MORRIS, J. Nutrition 1011013-1022 (1971). 13. V.C. MORRIS, Federation Proc. 3 8 8 7 5 (1979). 14. H.S. JACOB and J.H. JANDL, J. Clin. Invest. 411514-1523 (1962). 15. L. LORAND, L.B. WEISSMAN, D.L. EPEL and J. BRUNER-LORAND, Proc. Nat. Acad. Sci. 734479-4481 (1976). 16. G.J. JOHNSON, D.W. ALLEN, S. CADMAN, V.F. FAIRBANKS, J.G. WHITE, B.C. LAMPKIN and M.E. KAPLAN, New Eng. J. Med. 301522-527 (1979). 17. A.L. TAPPEL, Fed. Proo. 3_221870-1874 (1973). 18. S.K. JAIN and P. HOCBSTEIN, Biochem. Biophys. Res. Commun. 922247-254 (1980).
Pergamon Press
CROSSLINKING OF MEMBRANE PROTEINS IN RED BLOOD CELLS FROM VITAMIN E-DEFICIENT LEAD-POISONED RATS
Orville A. Levander and Susan O. Welsh
Nutritlon Institute Agricultural Research Center Beltsville, Maryland 20705 (Received in final form October 24, 1980) Summary Red blood cells from vitamin E-deficient rats lose their filterability after ineubatlon in vitro and concurrent lead poisoning of the rats accelerates this decline. Thls decreased red cell filterability is associated with an increased crosslinking of red cell membrane protelns. Previous reports by others suggested an association between red cell glutathione levels and fllterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a roleein the discocyte/ spherocyte shape change that accounts for the impalred filterability of red blood cells from vitamin E-deflclent lead-poisoned rats. The useful physiological lifetime partially determined by their abillty narrow as 3 microns in diameter. The deformable since the dlameter of, for morons.
of red blood cells (RBC) is at least to pass through mlcrocapillarles as latter requlres that RBC be highly example, the rat RBC is about 7.5
RBC from vitamin E-deflcient rats (-E RBC) lose thelr ability to pass through 3 micron polycarbonate membrane filters after incubation in vitro and lead poisonlng of the anlmals exacerbates thls effect (I). Thls decreased RBC filterabillty is accentuated by incubation wlth pro-oxidants (2), is correlated with lipld peroxidation (I), and can be prevented by feeding a synthetzc antioxidant (I). Old (denser) -E RBC show thls defect in filterability more strongly than young -E RBC and lead poisoning exaggerates this difference related to cell age (3). The decreased filterab111ty of -E RBC is accompanled by a morphological transition from the discocytlc to the spherocytlc shape (4). Others have associated changes in RBC shape and deformability wlth crosslinking of membrane proteins (5). Our present purpose was to determine whether membrane proteins are crossllnked in nonfilterable RBC from vitamln E-deficlent rats with and without concomitant lead polsoning. RBC filterability is used as an index of RBC deformab111ty. Since RBC f~lterabllity is thought to depend partly on intracellular GSH levels (6), the role of GSH in the f11terabllity of -E RBC was also studled. 0024-3205/81/020147-08502.00/0
148
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
Methods Weanling male Fischer 344 rats (Microblologlcal Associates, Walkersville, MD) were housed individually in hanging stainless steel wire cages and were fed elther a vitamin E-deficient Torula yeast diet (I) supplemented with 0.5 ppm selenium as sodium selenite or the same diet supplemented with both selenium and I00 ppm vltamin E as dl-~-tocopheryl acetate (Powder, General Biochemicals, Inc., Chagrin Falls, OH). Lead-poisoned rats received 500 ppm lead as lead acetate in the drlnking water. All rats had free access to food and water. In experiments relating RBC membrane protein crosslinking to RBC filterability, heparinized arterial blood from ether anesthetized rats was centrlfuged at 1000 x g for 10 min. at 4 ° . The plasma and bully coat were removed by aspiration and the packed RBC were washed 3x by resuspension in 3-4 volumes of 0.15 M NaCI-0.5 mM glucose-O.01M trls-Cl buffer (pH 7.4) and centrlfuged as above. For filterability measurements (see below), 0.9% RBC suspensions were prepared in the buffer. Either at zero time or after 4-6 hr. of Incubation at 30 °, RBC were pelleted by centrifugation as above. The packed RBC were lysed in 25-35 volumes of cold i0 mM tris-Cl (pH 7.4) followed by centrifugation at 29,000 x g for 10 mln. at 4° . The pelleted membranes were washed 2x with the hypotonic buffer and then were solubllized in 0.25 volumes of 5% SDS, 250 mM tris-Cl (pH 7.4), 10 mM EDTA, 1.25 M sucrose and 0.1 mE/ml pyronin Y and stored at -20 °. Protein concentration was determined by a modified Lowry procedure (7). Aliquots of solubilized membrane solutlon containlng 0.05 mE of proteln were applied to 5.5xi00 mm 4% acrylamide gels (Bio-Rad Labs., Richmond, CA 94804) whlch had been electrophoresed previously with the running buffer. The samples were allowed to enter the gels at a current of 2 mA/tube for 30 min., followed by a current of 3.5 mA/tube for 5 hr. The buffers and stainlng solutlons were prepared according to Falrbanks et al. (8). In experiments relating RBC GSH levels to RBC filterability, filterability of suspensions of RBC from rats at least 3 months on the experimental diets was determined by the method of Levander et al. (I). This technique involves measuring the amount of tlme required ("Filtration Time") for 2 ml of a 0.9% RBC suspension in buffer to pass through a pre-calibrated 25 mm polycarbonate filter membrane with a pore slze of 3 microns (Nuclepore Corp., Pleasanton, CA 94566) under a vacuum of i0 cm of water. The suspending buffer was 0.145 M NaCl-O.01M tris-Cl, pH 7.4, whlch contained 10 mg glucose/100 ml. The RBC suspensions were incubated at room temperature under alr. In the cell age experiment (Table II) old and young RBC were fractionated by differential centrlfugation (3). In the sulfhydryl blocking experiment (Table III), the N-ethylmaleimlde was dissolved where appropriate in the suspending buffer at the concentratlon indicated before the RBC were added to the buffer. Thus, the RBC were exposed to the blocking agent immediately before the first filtration measurement at 0 hr. RBC GSH was measured by colorimetry (9). Results Table I shows the decreased filterablllty of -E RBC incubated in vltro plus the exacerbating effect of concurrent in vlvo lead poisoning that we reported previously (I-4). In thls particular experiment, the lead impaired filterability of the -E RBC even without incubation. Lead poisoning had little or no effect on the filterability of RBC from vitamln E-supplemented rats (+E RBC). Electrophoretlc analysls of membrane protelns extracted from +E RBC of non-polsoned rats revealed typlcal normal stalning patterns before and after incubatlon of the RBC (Fig. I, A and B). Membrane protelns of -E RBC from non-poisoned rats also exhiblted a normal pattern before incubation but when
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinkin E
149
TABLE I Effects of vitamin E deflciency and lead poisoning on filterability of rat RBC
Diet
Lead in water ppm
Filtratlon t:me after incubation for 0 hr
2 hr
sec
4 hr
sec
sec
Control
0
17 +
1
19 ÷
Deficient
0
16 ÷
1
43 + 14
Control
500
21 ÷
2
26 +
Deficlent
500
263 + 91
1
5
457 ÷ 59
19 ÷
2
580 ÷ 20 37 +
7
>600
Mean values of 4 to 9 rats + S.E. the cells had lost thelr f11terability after incubat:on there was evidence of increased stalning at molecular weights greater than spectrin (Fig. I, C and D). Other changes included some increased generalized diffuse staining throughout the gel, possibly indicative of proteolysis, and a rather sharp decline in the sta:ning associated with bands 2.1 and 4.2. Membrane proteins of +E RBC st111 gave normal staining patterns even though the rats were poisoned w:th lead (F:g. 2, A and B). On the other hand, membrane proteins of -E RBC from lead-poisoned rats showed evidence of protein polymerization and the other changes noted above even wlthout :ncubation of the RBC (Fig. 2, C and D). Since the loss of filterability of -E RBC is due largely to non-filterable RBC of old cell age (3), GSH levels in young vs. old RBC were analyzed. As reported (3), -E RBC of old cell age lose thelr filterab:llty to a much greater extent after incubation in vitro than -E RBC of young cell age and the GSH levels of the non-filterable old -E RBC were about 22% less than those in any of the other incubated RBC (Table II). To investigate further the relationship between GSH and RBC filterability, RBC from def:clent and supplemented rats were incubated w:th and without the permeable sulfhydryl blocking agent, N-ethylmaleimide (NEM). Incubation with 10"4 M NEM accelerated the decl:ne in filterability of -E RBC but had no effect on the fllterabillty of +E RBC (Table III) even though the NEM caused an almost instantaneous loss of GSH in both -E RBC and +E RBC. Discussion The data presented here confirm earlier results that -E RBC suffer a decline in f11terabillty during incubatlon in vltro and that concurrent lead polsoning accelerates thls decline (I-4). The relevance of these in vitro f:ltration changes to decreased RBC survival in vivo is not certain but the stress of :ncubation may be needed to reveal subtle differences in RBC that are at increased risk with regard to splenic sequestration. Since the RBC samples taken from our rats are derived from per:pheral blood, any grossly abnormal RBC would presumably already have been culled by the spleen and one
150
RBC Filterability & Protein Crosslinking
A
B
C
Vol. 28, No. 2, 1981
D
I 2 2.1
3 4.1 4.2
5 6
7
FIG. 1 Effect of vitamin E deficiency on RBC membrane proteins separated by SDS gel electrophoresis. A: unincubated +E RBC, 8: ÷E RBC incubated 4 hr., C: unincubated -E RBC, D: -E RBC incubated 4 hr. The designation of the major membrane polypeptides is according to Steck (i0).
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinking
A
B
C.
D
! 2 2.1
3 4.1 4.2
5 6
7
FIG. 2. Effect of vitamin E deflciency and lead toxicity on RBC membrane proteins separated by SDS gel electrophoresls. A: unincubated ÷E RBC from leadpoisoned rats, B: same as A after 4 hrs. incubation, C: unincubated -E RBC from lead-poisoned rats, D: same as C after 4 hrs. incubation. Membrane polypeptides labelled as in Fig. i.
151
152
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
TABLE II Effect of cell age on filterability and GSH levels of RBC from vitamin E-deflcient and supplemented rats
Diet
Cell age
Incubation period 0 hr
3 hr
Filtration time (seconds) Control Control Deficient Deficient
young old young old
18 + 2 20+3 15-+1 17_+i
25 46 26 >
+ 7 + 16 + 6 ~00
GSH ( q / l O 0 ml packed RBC) Control Control Deficient Deficient
young old young old
129 131 108 98
~ ,+ ,+ ,+
14 14 3 3
105 105 99 77
,+ ,+ ± ,+
3 6 4 3
Mean values of 3 rats + S.E. TABLE III Effect of N-ethylmaleimide on filterability and GSH levels of RBC from vltamin E-deficient and supplemented rats
Diet
N-ethylmaleimide
Incubation period 0 hr
3 hr
5 hr
Filtration time (seconds) Control Deficient Control Deficient
0 0 10 -4 10 -4
15 16 13 14
+ + ~ ~
1 1 2 1
21 + 2 68 + 32 16 ~ 1 >600
22+2 > EO0
GSH (m~/lOO ml packed RBC) Control Deficient Control Deficient
0 0 10 -4 10 -4
Mean values of 3 rats + S.E.
109 i00 14 13
,+ 3 + 2 ,+ 3 ,+ 3
12 + 5 16 _~ 1
104 + 5 94+4
Vol. 28, No. 2, 1981
RBC Filterability & Protein Crosslinking
153
would not necessarily expect any differences in the filterability of fresh RBC in the various treatment groups. Hypothetical mechanisms of splenic trapping of RBC from vitamin E-deflcient lead-poisoned rats have been discussed elsewhere (11). The biochemical mechanism by which vitamin E deficiency and lead toxicity decrease RBC filterability is not understood in detail although a discocyte/ spherocyte shape change appears involved (4). Vitamin E deficiency is known to impair the active transport of cations in rat liver slices (12), but cation shifts do not seem to play a role in the decreased filterability reported here. Incubation of -E RBC at 0 °, which would cause massive loss of intracellular potassium and influx of sodium, actually protected such cells against the typical filtration changes observed in -E RBC incubated at room temperature
(13). Intracellular GSH concentration also does not appear to play a primary role in determining RBC filterability. Under different conditions, it was possible to demonstrate elther complete loss of RBC filterability with only mild loss of RBC GSH or, conversely, an almost total loss of RBC GSH wlth no loss of RBC filterability. Others have concluded that intracellular GSH levels have little to do with various RBC hemolytic phenomena and have suggested that membrane protein sulfhydryl groups are more important in thls regard (14). The decreased filterability of incubated -E RBC observed in our studies was associated with increased crosslinking of RBC membrane proteins and concurrent lead poisonlnE of the rats accelerated both of these changes. We were concerned that the proteins prepared from membranes of -E RBC might be susceptible to artifactual oxidative crosslinking during extraction because of the lack of antioxidant protection in such membranes. However, since no crosslinkinE was seen In membrane proteins from unincubated (i.e., zero time) -E RBC, the proteins seemed to be stable to the extraction process per se even in the absence of antloxldant and the polymerization apparently occurred durlng incubation and not during the isolation of stroma from the RBC lysate. Therefore, the morphological transltion from discocyte to spheroeyte thought responsible for the decreased filterability of -E RBC (4) may indeed be brought about by the aEEregatlon of RBC membrane proteins. On the basis of these data, however, we could not establish definitively whether crosslinking of RBC membrane proteins is the cause or an effect of the change in RBC shape. Several different biochemical mechanlsms have been impllcated in the polymerization of RBC membrane proteins. Lorand et al. (15) described a calciumactivated transglutamlnase which catalyzed the crossllnklng of RBC membrane proteins through the formation of 7-glutamyl-e-lysine bridges. That, however, probably was not the mechanism of the crosslinking observed by us since neither calcium nor ionophore was present in our incubation buffer. A second mechanism of RBC membrane protein crossllnklng is the oxidation of protein sulfhydryls to form intermolecular disulfide bridges. RBC membranes from patients wlth glucose-6-phosphate dehydrogenase deficiency and associated chronic hemolytic disease contain high molecular weight polypeptide aggregates that are formed by intermolecular disulfide bonds and RBC deformability is decreased in cells containing such aggregates (16). However, a good assoclatlon was noted between the quantlty of aggregates in the RBC of these patients and the RBC GSH concentration, whereas we found no association between RBC fllterabllity and GSH levels (see discussion above). Another explanation for the membrane protein crossllnklng observed in our incubated -E RBC is Schlff base formation by the reaction of amino groups with malonyldlaldehyde, an end product of polyunsaturated fatty acld peroxldatlon (17). Decreases in filterability of -E RBC are directly correlated wlth the production of malonyldlaldehyde (I) and the addition of malonyldlaldehyde to rat RBC in vitro causes an Increase in
154
RBC Filterability & Protein Crosslinking
Vol. 28, No. 2, 1981
high molecular weight protein polymers (18). Both of the latter 2 crosslinking mechanisms are compatible with the hypothesis that lipid peroxidation is the prlmary event causing shape change in -E RBC via protein polymerization either by "spilling over" of free radical chain reactions into the membrane proteins (oxidation to disulfide links) or by furnishing a chemically reactive crosslinking agent (mslonyldialdehyde). References i. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 107363-372 (1977). 2. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 1072135-2143 (1977). 3. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 108145-151 (1978). 4. O.A. LEVANDER, M. FISHER, V.C. MORRIS and R.J. FERRETTI, J. Nutrition 107 1828-1836 (1977). 5. J. PALEK, P.A. LIU and S.C. LIU, Nature 274505-507 (1978). 6. P. TEITEL, I. MARCU and A. XENAKIS, Fol. Haematologica 90281-295 (1968). 7. A. BENSADOUN and D. WEINSTEIN, Anal. Biochem. 70241-250 (1976). 8. G. FAIRBANKS, T.L. STECK and D.F.H. WALLACH, B~ohemlstry 102606-2617 (1971). 9. E. BEUTLER, O. DURON and B.M. KELLY, J. Lab. Clin. Med. 61882-888 (1963). 10. T.L. STECK, J. Mol. Biol. 66295-305 (1972). ii. O.A. LEVANDER, V.C. MORRIS and R.J. FERRETTI, in The Re d Cell (G.J. Brewer, Ed.) pp. 575-590, Alan R. Liss, Inc., New York (1978). 12. O.A. LEVANDER and V.C. MORRIS, J. Nutrition 1011013-1022 (1971). 13. V.C. MORRIS, Federation Proc. 3 8 8 7 5 (1979). 14. H.S. JACOB and J.H. JANDL, J. Clin. Invest. 411514-1523 (1962). 15. L. LORAND, L.B. WEISSMAN, D.L. EPEL and J. BRUNER-LORAND, Proc. Nat. Acad. Sci. 734479-4481 (1976). 16. G.J. JOHNSON, D.W. ALLEN, S. CADMAN, V.F. FAIRBANKS, J.G. WHITE, B.C. LAMPKIN and M.E. KAPLAN, New Eng. J. Med. 301522-527 (1979). 17. A.L. TAPPEL, Fed. Proo. 3_221870-1874 (1973). 18. S.K. JAIN and P. HOCBSTEIN, Biochem. Biophys. Res. Commun. 922247-254 (1980).