Effects of Temperature and ph on Hemoglobin Release from Hydrostatic Pressure-Treated Erythrocytes 1

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1 Effects of Temperature and ph on Hemoglobin Release from Hydrostatic Pressure-Treated Erythrocytes 1 Takeo Yamaguchi, Hideo Kawamura, Eiji Kimoto, and Mitsuru Tanaka Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka, Fukuoka Received for publication, Junuary 25, 1989 J. Biochem. 106, (1989) The release of hemoglobin from human erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thaw methods was examined as a function of temperature (0-45 C) and ph ( ) at atmospheric pressure. Only in the case of high pressure (2,000 bar) did the release of hemoglobin increase significantly with decreasing temperature and ph. Maleimide spin label studies showed that the temperature and ph dependences of hemoglobin release were qualitatively explicable in terms of those of the conformational changes of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins showed the diminution of band intensities corresponding to spectrin, ankyrin, and actin in the erythrocytes hemolyzed by high pressure. Cross-linking of cytoskeletal proteins by diamide stabilized the membrane structure against high pressure and suppressed hemoglobin release. These results indicate that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky. The structures of biomembranes, composed mainly of phospholipids and proteins, are maintained by hydrophobic and ionic interactions (1). Hydrostatic pressure significantly affects these interactions (2). The application of high pressure to biomembranes is therefore useful to analyze the interactions between membrane components. The membrane structure of human erythrocytes is known in detail and so erythrocytes are suitable as test samples to observe the pressure effect. The biconcave shape and the deformability of mammalian erythrocytes are controlled by cytoskeletal apparatus which has a cross-linked meshwork consisting of spectrin, actin, band 4.1, and band 4.9 (3, 4). Current interest in membrane structures of erythrocytes is centered on the properties of complexes of cytoskeletal proteins and the interaction of the cytoskeletal components with the membrane (5-7). The affinity of spectrin to actin filaments is greatly enhanced by the addition of band 4.1 (6, 8). The association of the spectrin - actin complex with the membrane is mediated by the two linking proteins, band 4.1 and ankyrin (9). Band 4.1 is known to bind to integral membrane proteins such as band 3 (10), the membrane anionchannel, and glycophorin (11). Further, ankyrin (12) and band 4.2 (13) are also bound to band 3. Thus, a study on the hemolysis of erythrocytes by the application of hydrostatic pressure is expected to provide information on the interactions among cytoskeletal proteins and on those of the proteins with the membrane. The first application of hydrostatic pressure to eryth- 1 This work was supported by grants from the Ministry of Education, Science and Culture of Japan, and the Central Research Institute of Fukuoka University. Abbreviations: PMSF, phenylmethanesulfonyl fluoride; DFP, diisopropylfiuorophosphate; diamide, diazinedicarboxylic acid bis{n,n'- dimethylamide); DTT, dithiothreitol; 5P8, 5 mm sodium phosphate, ph 8; SDS-PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis. rocytes was carried out by Ebbecke (14), who reported that no hemolysis occurred until 2,000 bar. Haubrich (15) found that erythrocytes subjected to hydrostatic pressure were more fragile. Since then, there have been few studies on the pressure effect on hemolysis of human erythrocytes. In the present study, we examined the effects of temperature and ph on the release of hemoglobin from red ghosts prepared by high pressure. These results are compared with those obtained by the osmotic pressure and freeze-thaw methods. Furthermore, it is shown that the disruption of cytoskeletal apparatus by high pressure makes the membrane more leaky. MATERIALS AND METHODS Materials Compounds were obtained from the following sources: phenylmethanesulfonyl fluoride (PMSF), Nakarai Chemicals; Dextran (MW= 100, ,000) and dithiothreitol (DTT), Wako Chemicals; diisopropylfluorophosphate (DFP) and diazinedicarboxylic acid bis- (N,N'-dimethylamide) (diamide), Sigma. All other chemicals were of reagent grade. Temperature and ph Dependences of Hemoglobin Release The heparinized blood from volunteer donors was centrifuged at 750 X g for 10 min at 4 C. The plasma and buffy coat were removed. The erythrocytes were washed three times in phosphate-buffered saline (PBS; 10 mm sodium phosphate, 150 mm NaCl, ph 7.3) and used within 24 h. The erythrocyte suspension (0.3 or 2% hematocrit) was put into a syringe-type cell with a piston. The sample cell was placed in a pressure bomb made of stainless steel. The pressure was generated by means of a hand-type pump (Hikari Kikai) and monitored with a Heise pressure gauge. Ligroin was used as the pressure transmitting fluid. The erythrocyte suspension was compressed and then kept for 1080 J. Biochem.

2 Hemolytic Properties of High Pressure- Treated Erythrocytes min at 37 C and 2,000 bar. After decompression, the hemolysates were incubated at various temperatures (0-45 C) and incubation times (0-30 min), and then centrifuged for 10 min at 750 X g. Hemoglobin release into the supernatant was measured at 542 ran. Then, the supernatant was mixed with the pellet. The suspension was frozen in chilled methanol ( 40 C), thawed at 37 C, and centrifuged for 10 min at 750 x g. One hundred percent hemolysis (or hemoglobin release) was taken as the absorbance of the supernatant thus obtained (Figs. 1-3 and Table I). For the ph dependence of hemoglobin release, the hemolysates after decompression were centrifuged for 10 min at 750 x g. The pellets (red ghosts) were washed three times with warm PBS (ph 7.3) and then suspended in 150 mm NaCl adjusted to ph with 10 mm sodium phosphate. The suspensions were incubated for 30 min at various temperatures (0-37 C) and centrifuged (10 min at 750 X g). Hemoglobin release was measured as mentioned above. In this case, the full release of hemoglobin from isolated red ghosts was regarded as 100% hemoglobin release (Fig. 4). For osmotic hemolysis, one volume of erythrocytes was added to 300 volumes of 10 mm sodium phosphate (ph 7.3) containing 55 mm NaCl at 37 C. The hemolysates were made isotonic using NaCl and used to examine the temperature and ph dependences of hemoglobin release, as mentioned above. For hemolysis by the freeze-thaw method, one volume of erythrocytes was suspended in 300 volumes of PBS (ph 7.3) containing 5% (v/v) dimethylsulfoxide, frozen (at 40 C), and thawed. Using this hemolysate, hemoglobin release was similarly examined. In this case, 100% hemoglobin release was obtained by the freeze-thaw method in the absence of dimethylsulf oxide. To examine whether all erythrocytes are partially hemolyzed, the hemolysates were centrifuged for 5 min at 750 x g. The pellets were layered on a discontinuous Dextran density gradient solution, which was prepared by dissolving 0.2 or 0.3 g of Dextran in 1 ml of PBS (ph 7.3), and spun at 370 Xg for 10 min. Spin Labeling of Membrane Proteins The membranes prepared using 5 mm sodium phosphate, ph 8 (5P8) buffer [16) were suspended in PBS (ph7.3), subjected to a pressure of 2,000 bar for 30 min at 37 C, and then spinlabeled with a maleimide spin label as previously described (27). To examine the ph dependence of the ESR spectra, the spin-labeled membranes were incubated for 30 min at 25 C in various ph ( ) buffers as mentioned above. The ESR spectra were recorded on a JEOL JES FE-1X spectrometer. Erythrocyte Membrane Preparation and Gel Electrophoresis The methods of membrane preparation described here were essentially the same as those used in the case of hemoglobin release (Figs. 2-4), except that different hematocrits were used. Erythrocyte membranes were prepared in 5P8 and suspended in PBS (ph7.3). To prepare the membranes by the high pressure method, the erythrocyte suspension (2% hematocrit) in PBS (ph 7.3) containing 0.4 mm DFP and 0.1 mm PMSF was subjected to a pressure of 2,000 bar for 30 min at 37 C and then decompressed. To obtain the membranes by the freezethaw method, the erythrocyte suspension (0.05% hematocrit) in PBS (ph 7.3) containing 5% (v/v) dimethylsulf oxide was frozen at 40 C and then thawed. The membrane suspensions obtained by the three methods were incubated for 30 min at 0 C and then centrifuged at 36,000 x g for 20 min at 4 C. The supernatants were concentrated about 50-fold using Amicon ultrafiltration (PM 10 membrane). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of membrane proteins was performed according to the method of Fairbanks et al. (18). Protein bands were stained with Coomassie Blue. Treatment of Erythrocytes with Diamide In order to cross-link membrane proteins, the erythrocyte suspension (0.3% hematocrit) in PBS (ph 7.3) was incubated with 0.1 mm diamide for 30 min at 37 C and 800 bar. After decompression, the diamide-treated erythrocytes were washed twice with 150 volumes of PBS (ph 7.3). The reduction of cross-linking of membrane proteins was performed by incubating diamide-treated erythrocytes with 10 mm DTT for 30 min at 37 C and atmospheric pressure. No hemolysis was observed by membrane protein cross-linking and its reduction. The erythrocytes thus prepared were suspended in PBS (ph 7.3) and then subjected to a pressure of 2,000 bar for 30 min at 37 C. After decompression, hemoglobin release at 0 C was examined (Table I) and the membranes were prepared. For membrane or supernatant preparation, the same methods as mentioned above were used, except that the erythrocytes were washed once in 5P8 to remove a large amount of hemoglobin contained within membranes after incubation for 30 min at 0 C. In order to assess the state of cross-linking of membrane proteins, SDS-PAGE (28) was performed without reducing agent in the solubilization buffer (Fig. 7). RESULTS Effects of the Rates of Compression and Decompression on Hemolysis Human erythrocytes suspended in PBS (ph 7.3) were incubated for 30 min at 37 C and 2,000 bar. Figure 1 shows the effect of the rates ( bar/min) of compression and decompression on the degree of hemolysis. When the rate of compression was constant (200 bar/ min) and the rate of decompression was varied, the degree of hemolysis was independent of the rate of decompression from 200 to 500 bar/min. On the other hand, the degree of C RATE ( kbar/min ) Fig. 1. Effects of the rates of compression and decompression on the degree of hemolysis. Erythrocytes (0.3% hematocrit) suspended in PBS (ph 7.3) were incubated for 30 min at 37*C and 2,000 bar. While the rate of decompression was constant (400 bar/min), the rates of compression (o) were varied. Similarly, the rates of decompression (A) were varied when the rate of compression was held constant (200 bar/min). Vol. 106, No. 6, 1989

3 1082 T. Yamaguchi et al. hemolysis was unaffected by the rate of compression from 100 to 300 bar/min when the rate of decompression was held constant (400 bar/min). Therefore, the values of 200 and 400 bar/min were used as the rates of compression and decompression, respectively. Effects of Temperature and ph on Hemoglobin Release Figure 2 shows the time course of hemoglobin release during the incubation at 0 and 37 C from erythrocytes hemolyzed beforehand by hydrostatic pressure, osmotic pressure, and freeze-thawing. At 37 C, no release of hemoglobin occurred irrespective of the means of hemolysis. On the other hand, at 0 C, hemoglobin was released only in the case of high pressure. In this case, the amount of hemoglobin released from the membrane increased from 52 to 100% when the sample preincubated for 30 min at 37 C was incubated for 30 min at 0 C. To test whether some erythrocytes are completely hemolyzed while others remain intact or whether all are partially hemolyzed, each TIME(min) Fig. 2. Time course of hemoglobin release from hemolyzed erythrocytes. Hemolysates formed by the hydrostatic pressure (circles), osmotic pressure (triangles), and freeze-thaw (squares) methods were prepared as described under "MATERIALS AND METHODS." Each hemolysate was incubated at 0 (open symbols) or 37 C (closed symbols). Aliquots of the hemolysate were used to measure hemoglobin release. Values represent the mean±sd for at least two experiments , hemolysate was centrifuged and the pellets were layered on density barrier solution. Under conditions such that intact erythrocytes were sedimented to the bottom of the tube, the membrane pellets remained at the boundary region. This result indicates that all erythrocytes are hemolyzed partially. The hemolysates prepared by the high pressure method were incubated for 30 min at various temperatures (0- Fig. 4. ph dependence of hemoglobin release from isolated red ghosts. Isolated red ghosts prepared by the high pressure (o), osmotic pressure (A), or freeze-thaw method (o) were suspended in various ph buffers and incubated for 30 min at 37 C. The high pressure sample was also incubated similarly at 21 C ( ). Values represent the mean±sd for at least two experiments. TABLE I. Effects of cross-linking of membrane proteins on hemoglobin release at O'C. The erythrocytes pretreated with 0.1 mm diamide and 10 mh DTT as described under "MATERIALS AND METHODS" were subjected to a pressure of 2,000 bar for 30 min at 37'C. After decompression, hemoglobin release at O'C was measured. Values are the mean + SD for three experiments. Reagent None Diamide Diamide + DTT Hemoglobin release ± ± ±3.7 (%) at incubation times (min) ± ± i0 TEMPERATURE ( C ) Fig. 3. Temperature dependence of hemoglobin release from erythrocytes hemolyzed by high pressure. Erythrocytes (2% hematocrit) in PBS (ph 7.3) were subjected to high pressure. After decompression, aliquots of the hemolysate were diluted with 5 volumes of PBS (ph 7.3) and incubated for 30 min at various temperatures. Values represent the meail±sd for three experiments TEMPERATURE CO Fig. 5. Effects of temperature and ph on the A w /A s ratio of maleimidelabeled erythrocyte membranes. Maleimide spin labeling of membranes subjected to high pressure was performed as previously described (17). For the temperature effect (o), spin-labeled membranes were treated in PBS (ph 7.3). The ph dependence (A) of the ESR spectra were recorded at 25'C. J. Biochem.

4 1083 Hemolytic Properties of High Pressure- Treated Erythrocytes kda ! 7- Hb- Fig. 7. SDS-PAGE of cross-linked membrane proteins. The erythrocytes were pretreated with 0.1 mm diamide and 10 mm DTT. All erythrocytes (Lanes a-c) were subjected to high pressure (2,000 bar) and then washed once in 5P8. SDS-PAGE (18) was carried out without 2-mercaptoethanol in the solubilization buffer. Lane a, untreated membrane; lane b, membrane treated with diamide; lane c, membrane treated with diamide and then DTT; lane d, supernatant concentrated 50fold after pressure treatment of diamide-treated erythrocytes. Hb, hemoglobin. * 2- I C). Figure 3 shows the relation between hemoglobin release and incubation temperature. At 0-15 C, hemoglobin was fully released from the membrane. The amount of released hemoglobin decreased gradually with increasing temperature up to about 30 C, kept an almost constant value between 30 and 37 C, and then increased. To examine the effect of ph on hemoglobin release, the isolated red ghosts prepared by each method were suspended in various ph buffers and incubated for 30 min at 037 C. Figure 4 shows typical results at 21 and 37 C. The ph dependence of hemoglobin release was remarkable only in the case of high pressure. At 37 C, hemoglobin was fully released below 5.9. Then, with increasing ph up to about 7.3, the amount of released hemoglobin steeply decreased to an almost constant small value. In the case of 21 C, the hemoglobin release-ph profile shifted toward higher ph. The relationship between the incubation temperature and the ph at 50% hemoglobin release, as in Fig. 4, was examined. A plot of ph ( ) us. temperature (0-37 C) gave a straight line with a negative slope (0.03 ph unit/ C; correlation coefficient of 0.999). In order to suppress hemoglobin release, the ph should be increased as the incubation temperature is decreased and vice versa. Effect of Cross-Linking of Membrane Proteins on Hemoglobin Release We investigated the effect of cross-linking of membrane proteins on hemoglobin release at 0 C (Table I). The cross-linking was performed by using 0.1 mm diamide at 800 bar. The degree of hemolysis under 2,000 bar of diamide-treated erythrocytes was significantly smaller (4.3%) than that (58.5%) of untreated erythrocytes. During incubation at 0 C for 10 min, hemoglobin was slightly released from diamide-treated erythrocytes, but fully released from untreated ones. In the case of diamide-treated erythrocytes which were further incubated with 10 mm DTT to reduce oxidative crosslinking, hemoglobin release was greatly increased. On the other hand, such a cross-linking effect on hemoglobin Hb-l a b c release was not observed in erythrocytes treated with 0.1 mm diamide at atmospheric pressure (data not shown). Effects of Temperature and ph on the ESR Spectrum of Membranes Labeled with Maleimide Spin Label The ESR spectra of a maleimide spin label bound to the sulfhydryl group of membrane proteins are characterized by a weakly immobilized component (w) and a strongly immobilized one (s) (17, 19). The ratio, Aw/As, of the amplitude from the horizontal base line to each peak at the low field of both components has been used as an index of protein conformational changes, i.e., a decrease in the ratio may reflect folding or aggregation of membrane proteins (17, 19). The effects of temperature (3-40 C) and ph ( ) on Aw/As in membranes subjected to high pressure were examined. The values of Aw/As increased with increasing temperature and ph (Fig. 5). SDS-PAGE Analysis of Membrane Proteins The changes in membrane proteins of hemolyzed erythrocytes were studied by SDS-PAGE (Fig. 6). The same electrophoretic patterns as seen in Fig. 6 were obtained for each sample in Figs. 2-4, except that the band intensities of hemoglobin were different (data not shown). Some significant changes were seen in red ghosts prepared by the high pressure method. The membrane proteins affected most under high pressure seem to be spectrin (bands 1 and 2), ankyrin, actin (band 5), and glyceraldehyde 3-phosphate dehydrogenase (band 6) (Fig. 6b). This characteristic pattern was observed in the presence and absence of protease inhibitors. Among cytoskeletal proteins, only spectrin appeared in the supernatant (Fig. 6e). In addition to a new band (184 kda), more diffuse bands were observed between actin and 62 kda peptide (Fig. 6b). It is, however, unclear whether the appearance of these bands is due in part to ankyrin and actin. Furthermore, other new bands (24, 28, and 62 kda) appeared (Fig. 6b). These bands were also observed in the supernatant (Fig. 6d) prepared by the 5P8 method (16). These results indicate that the diminution in the band intensities of cytoskeletal proteins is not due to the digestion of these proteins with proteases but is an effect of the application of high pressure. To study the influence of high pressure on cross-linked membrane proteins, erythrocytes pretreated with 0.1 mm diamide or 0.1 mm diamide followed by 10 mm DTT were subjected to a pressure of 2,000 bar (Fig. 7). The electrophoretic patterns shown here are not so clear compared II -28 Fig. 6. SDS-PAGE of erythrocyte membrane proteins. The preparations of erythrocyte membranes by the hypotonic pressure (5P8) (a), hydrostatic pressure (b), and freeze-thaw (c) methods were carried out as described under "MATERIALS AND METHODS." To examine the membrane proteins released from the membrane, the supernatants prepared by hypotonic pressure (d) and hydrostatic pressure (e) were concentrated by Amicon ultrafiltration (PM 10 membranes). SDS-PAGE was performed by the method of Fairbanks et al. (18). Hb, hemoglobin. Vol. 106, No. 6, 1989 *

5 1084 T. Yamaguchi et al. to those in Fig. 6 because of the deletion of 2-mercaptoethanol from the solubilization buffer. In the case of diamide-treated erythrocytes, the spectrin band disappeared in comparison with the electrophoretic pattern of untreated ones (Fig. 7, a and b). No band corresponding to spectrin appeared in the supernatant in the absence (Fig. 7d) or presence (data not shown) of 2-mercaptoethanol (1%). Upon incubation of diamide-treated erythrocytes with DTT, spectrin reappeared (Fig. 7c). These results indicate that the high-molecular-weight complex produced by cross-linking of spectrin is stable to high pressure and it can not enter the polyacrylamide gel (5.6%) in the absence of reducing agents. DISCUSSION The data presented here show that the red ghosts prepared by hydrostatic pressure significantly differ from those prepared by osmotic pressure and freeze-thaw methods in the temperature and ph dependences of hemoglobin release and the electrophoretic pattern of membrane proteins. In the present study, we have used the release of hemoglobin to monitor the membrane structure in hemolyzed erythrocytes. One problem with this approach is that the hemoglobin binding to the inner surface of the erythrocytes increases at low ph (20, 21). However, hemoglobin release increased with decreasing ph in red ghosts prepared by the high pressure method. This result indicates that the ph dependence of hemoglobin release described here does not reflect the hemoglobin-membrane interaction. The plots of hemoglobin release (Fig. 3) and membrane fluidity (22, 23) against temperature show similar curves. With increasing temperature, the release of hemoglobin decreases, while the membrane fluidity increases. Both changes are particularly remarkable between 15 and 30 C. On the other hand, when we consider the ph dependence, both hemoglobin release and membrane fluidity decrease with increasing ph (24). This fact suggests that the temperature and ph dependences of hemoglobin release can not be interpreted in terms of the membrane fluidity only. The conformational changes of membrane proteins are significantly affected by temperature and ph. With decreasing temperature and ph, the membrane proteins subjected to high pressure fold and hemoglobin release increases. In addition, the spin label results demonstrate that the conformational changes of membrane proteins are irreversible above 40 C (17, 25). This suggests that the organization of membrane components is in part disturbed. The increment of hemoglobin release above 40 C in Fig. 3 may therefore be attributed to the disordering of membrane components caused by the irreversible conformational changes of membrane proteins. Thus, the temperature and ph dependences of hemoglobin release from red ghost membranes prepared by high pressure are well correlated with those of the conformational changes of membrane proteins. In membranes prepared by the osmotic pressure method, however, no release of hemoglobin is observed despite the similar conformational changes of membrane proteins (17). Therefore, we can not say on the basis of spin labeling data only why hemoglobin is released at high pressure. Band 6 diminished in the membranes prepared by the high pressure (Fig. 6b) or freeze-thaw method (Fig. 6c). In unsealed ghosts, band 6 is released from the membrane with increasing ionic strength and ph (26). In the membrane ruptured by high pressure or freeze-thawing in PBS (ph 7.3), band 6 can easily be released from the membrane. Recent studies of the membrane skeleton demonstrate that the formation of a two-dimensional network is characterized by the head-to-head association of spectrin to form tetramers and the cross-linking of actin with the tail ends of the tetramers of spectrin (27). The membrane skeleton is linked to the lipid bilayer via a complex between the integral membrane proteins and the membrane linkage proteins (3, 4, 6-8). Thus, the bilayer structure of the membrane is greatly stabilized by cytoskeletal proteins (3, 5-7). In the present study, we have shown that the membrane skeleton is partially disrupted by the application of 2,000 bar. On the other hand, the membrane skeleton is relatively stable in 5P8 buffer (16, 28). Lieber and Steck have studied in detail the dynamics of the membrane holes produced by hypotonic hemolysis (5P mm MgSO 4 ) (29). So, it is of interest to compare the dynamics of the membrane holes produced by hydrostatic pressure with their results. The resealing of the holes produced by hypotonic hemolysis is favored by increasing membrane fluidity (30), i.e., higher temperature and lower ph (29). In addition, the hole dynamics are affected by lipid-intercalated compounds such as cholesterol and chlorpromazine (29, 30). The ghosts sealed at 37 C do not re-open at 0 C (29). In contrast, high pressure-induced holes are resealed under such conditions as the Av,/A s values increase, i.e., higher temperature and higher ph. The membrane sealed at 37 C re-opens at 0 C, i.e., the membrane which is impermeable to hemoglobin at 37 C becomes permeable at 0 C. Thus, the dynamics of the membrane holes produced by the two methods are clearly different. Recently, it has been reported that the lack of cytoskeletal proteins, e.g., spectrin (31) or band 4.2 (32), causes hemolytic anemia. We have established that crosslinking of membrane proteins (mainly spectrin) stabilizes the cytoskeleton against high pressure and resealed membranes do not readily re-open at 0 C. Therefore, the different behavior of the hole dynamics in both membranes is in part attributed to the differences in cytoskeletal structures. In general, a number of oligomeric proteins are dissociated by the application of high pressure (33, 34). For skeletal muscle actin, increased pressure (600 bar) shifts the filamentous state of actin toward the monomer state (35). In the erythrocyte membrane, cytoskeletal apparatus, which is composed of oligomeric proteins, is expected to be partially disrupted by high pressure. In fact, spectrin was observed in the supernatant (Fig. 6e). However, such a dissociation might be suppressed by cross-linking of oligomeric proteins so that the cytoskeletal apparatus is stabilized against high pressure. Diamide has been widely used to cross-link spectrin via intermolecular disulfide bonds in human erythrocytes (36, 37). When membrane proteins were cross-linked with diamide under pressure, hemoglobin release at 0 C was significantly decreased and no band corresponding to spectrin was observed in the supernatant (Fig. 7d). Upon the reduction of cross-linking of membrane proteins by DTT, hemoglobin release increased greatly. These results indicate that the disruption of cytoskeletal apparatus due to the dissociation of oligomeric proteins by high pressure makes the membrane more leaky. J. Biochem.

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