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Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes
Comparative Biochemistry and Physiology Part A 129 Ž2001. 355᎐362
Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes Mohammad K. Ali, Saad TayyabU Interdisciplinary Biotechnology Unit, Aligarh Muslim Uni¨ ersity, Aligarh, U.P. 202002, India Received 28 July 2000; received in revised form 30 November 2000; accepted 4 December 2000
Abstract Binding of bilirubin to erythrocyte membranes of human, buffalo, sheep and goat was studied after phospholipase C, trypsin and neuraminidase treatment. Phospholipase C and trypsin treatment of membranes greatly enhanced the bilirubin binding in all mammalian species, whereas, neuraminidase treatment resulted into a small increase in the membrane-bound bilirubin. Human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human ) sheep ) buffalo ) goat; the order was: human ) buffalo ) sheep ) goat; in phospholipase C- and trypsin-treated erythrocyte membranes. These binding results indicate that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes as the differences observed in the membrane-bound bilirubin among mammalian species were directly correlated with the sum of choline phospholipids, especially phosphatidylcholine and sphingomyelin content of the erythrocyte membranes. The negatively charged phosphate moiety of phospholipids of the membranes appears to inhibit a large amount of bilirubin binding to the membrane as its removal by phospholipase C greatly enhanced the binding. Furthermore, membrane proteins and carbohydrate also seem to play a significant regulatory function on the binding as their degradation andror removal in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Bilirubin; Buffalo; Erythrocyte membranes; Goat; Human; Neuraminidase; Phospholipase C; Sheep
1. Introduction Interaction of bilirubin with erythrocyte membranes has been studied ŽSato and Kashiwamata, 1983; Sato et al., 1987; Hayer et al., 1989; Rashid et al., 2000. to understand the mechanism of entry of bilirubin inside the cells which may be U
Corresponding author. Tel.: q91-571-701718; fax: q91571-701081. E-mail address: [email protected] ŽS. Tayyab..
helpful in developing preventive measures against bilirubin encephalopathy in new born infants. It is generally believed that bilirubin mainly interacts with the lipid bilayer of erythrocyte membranes ŽSato and Kashiwamata, 1983; Sato et al., 1987., however, the mechanism of such interaction is poorly understood. It has been suggested that bilirubin is bound to the lipid bilayer through strong ionic interactions between a cationic head group of the lipid and anionic bilirubin ŽNagaoka and Cowger, 1978.. The dianion form of bilirubin
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 0 . 0 0 3 5 3 - 6
356
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
binds weakly to the polar heads of the phospholipids, whereas, acid bilirubin binds strongly and is hydrophobically inserted into the lipophilic region of the bilayer ŽCestaro et al., 1983.. A decrease in binding constant for bilirubin-membrane interaction has been reported after phospholipase C treatment of synaptosomal membranes whereas phospholipase D-treated membranes showed a higher binding constant suggesting that the polar protruding ends of lipids are involved in the initial interaction with bilirubin ŽVazquez et al., 1988.. Involvement of electrostatic interactions between bilirubin and polar head groups of lipids was challenged by Sato and his group ŽSato et al., 1987. who showed no change in bilirubin binding after phospholipase D treatment of the membranes, whereas, phospholipase C-treated membranes showed greatly enhanced bilirubin binding. They are of the view that the exposure of non-polar diacylglycerols at the surface of the membrane after removal of polar head groups of phosphatidylcholine ŽPC., phosphatidylethanolamine ŽPE. as well as sphingomyelin ŽSph. of membrane, may potentiate a large amount of non-specific binding of bilirubin to the fatty acid moiety of diacylglycerols ŽSato et al., 1987.. The role of membrane proteins in bilirubin binding to membranes is also not very clear. Sato and Kashiwamata Ž1983. ruled out the involvement of membrane proteins as the bilirubin binding sites, based on the increased bilirubin binding after trypsin treatment of human erythrocyte membranes but suggested that membrane proteins may act as a barrier in the binding process. These findings are questionable in view of enhanced bilirubin binding to model membranes containing an integral protein compared to protein lacking model membranes ŽLeonard et al., 1989.. Furthermore, the role of negatively charged sialic acid residues present on the membrane surface in bilirubin binding phenomenon is not certain. Whereas, Vazquez et al. Ž1988. have reported that the negative charges contributed by sialic acid residues exert an inhibitory effect on the bilirubin binding to membrane, Sato et al. Ž1987. have found no significant change in the binding of bilirubin after neuraminidase treatment of membranes. In order to clarify the conflicting observations of the role of polar head groups of phospholipids, membrane proteins and sialic acid residues, we
studied the binding of bilirubin to the human erythrocyte membranes in detail before and after treating them with phospholipase C, trypsin and neuraminidase. As protein and lipid make-up of erythrocyte membranes varies in different mammalian species ŽO’Kelly, 1979; Barenholz and Thompson, 1980; Inaba and Maede, 1988., bilirubin binding results of treated human erythrocyte membranes were also compared with the membranes obtained from buffalo, sheep and goat under similar conditions which will be helpful in understanding the specific roles of various membrane components in bilirubin binding.
2. Materials and methods 2.1. Materials Bovine serum albumin, fraction V, trypsin, type III from bovine pancreas ŽLot 24H0070., phospholipase C, type I from Clostridium welchii ŽLot 10H6801., neuraminidase, type V from C. perfringens ŽLot 31H82302. and phenylmethylsulfonylfluoride ŽPMSF. were purchased from Sigma Chemical Company, USA. Bilirubin was the product of Sisco Research Laboratories, India. Other reagents used were of analytical grades. The blood of buffalo Ž Bos indicus., sheep Ž O¨ is aries. and goat Ž Cepra hircus. was collected fresh in 1.32% sodium citrate solution from slaughter houses. Human blood was obtained from the Blood Bank of J.N. Medical College, Aligarh Muslim University, Aligarh. 2.2. Methods Erythrocytes were collected by centrifugation of blood at 1000 = g for 20 min. After removing the upper buffy coat through gentle decantation, cells were washed with 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl followed by centrifugation at 1000 = g for 20 min. This process was repeated 3 times. The final packed cell volume was diluted with an equal volume of the same buffer to obtain a 50% hematocrit value. Erythrocyte membranes were prepared following the method of Palfrey and Waseem Ž1985.. Hemolysis of erythrocytes was carried out using 10 volumes of cold 10 mM Tris᎐HCl buffer, pH 7.4 containing 0.01 mM EDTA and 0.01 mM PMSF. The contents were mixed by gentle swirling
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
and then centrifuged at 16 000 = g at 4⬚C for 30 min. The dark red supernatant was removed carefully by gentle aspiration. The membrane pellet was resuspended by swirling in 5.0 ml of the same lysis buffer and then centrifuged at 16 000 = g at 4⬚C for 20 min. The pellet was washed 5᎐6 times with the same buffer until a clear supernatant was obtained. The membrane pellet was resuspended in 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl in a volume equivalent to the volume of erythrocytes used. It was stored at 10⬚C and used as such within 2 days. Phospholipid content of erythrocyte membranes was determined by the method of Chen et al. Ž1956. following the extraction of phospholipids ŽFolch et al., 1957.. Protein concentration was determined by the method of Lowry et al. Ž1951. after solubilizing the membranes in 1% SDS. Orcinol ŽSvennerholm, 1956. was used to determine the total carbohydrate content of the membranes. Phospholipase C digestion of erythrocyte membranes was carried out following the method of Sato et al. Ž1987.. To 1.0 ml of membrane suspension in 50 mM Tris᎐HCl buffer, pH 7.4 was added 3.75 ml of 0.1 M Tris᎐HCl buffer, pH 7.4, 50 l of 1.0 M CaCl 2 and 200 l of the enzyme solution Ž5 mgrml. and the mixture was incubated at 37⬚C. The reaction was stopped by adding 1.0 ml of 10 mM EDTA. All the contents were centrifuged at 16 000 = g for 20 min. The supernatant was assayed for released phosphate ŽChen et al., 1956.. Tryptic digestion of erythrocyte membranes was performed by the method of Steck et al. Ž1971.. Five milligrams of solid trypsin were added to 1.0 ml of erythrocyte membrane suspension in 2.5mM sodium phosphate buffer, pH 8.0 and the mixture was shaken until the enzyme was completely dissolved. The reaction was carried out at room temperature for varying periods. Then, the mixture was centrifuged at 16 000 = g for 5 min and the supernatant containing released glycopeptides was collected and subjected to carbohydrate measurement as above ŽSvennerholm, 1956.. Erythrocyte membranes Ž1.0 ml. were treated with neuraminidase following incubation with 0.5 units of the enzyme for 1 h at 37⬚C in 50 mM Tris᎐HCl buffer, pH 7.4. The mixture was centrifuged at 16 000 = g for 5 min and the supernatant containing released sialic acid was col-
357
lected and subjected to sialic acid estimation ŽWarren, 1959.. The pellets obtained in the above experiments were washed 2 times with cold 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl followed by centrifugation at 16 000 = g for 20 min. Then, membranes in each tube were resuspended in the same buffer making the total volume as 1.0 ml. These membranes were directly used for bilirubin binding studies. Bilirubin solution was prepared by dissolving a few crystals of bilirubin in 38 mM sodium carbonate solution containing 5.0 mM EDTA, pH 11.0. Its concentration was determined by Fog’s method ŽFog, 1958.. The bilirubin solution was protected from light and used within 1 h. All experiments were carried out under yellow light. Binding of bilirubin to erythrocyte membranes Žuntreatedrtreated . was studied by incubating 1.0 ml of stock bilirubin solution of desired concentration with 1.0 ml of erythrocyte membrane suspension Žequivalent to 1.0 ml of erythrocyte suspension of 50% hematocrit. and the final volume was made to 6.0 ml with 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl. After 30 min of incubation at 37⬚C, the mixture was centrifuged at 16 000 = g for 15 min at 4⬚C and the supernatant containing unbound bilirubin was discarded. Membranes were washed several times with the same buffer until the last supernatant was devoid of yellow color. After final washing, the membrane-bound bilirubin was determined by modified Fog’s method ŽFog, 1958. as described by Tayyab and Ali Ž1999.. Statistical analysis of the data included calculations of Pearson’s product moment correlation coefficients and dispersion. Correlation coefficients and difference of means were tested for significance using two-tailed ‘t’-tests.
3. Results and discussion 3.1. Interaction of bilirubin with untreated and phospholipase C-treated erythrocyte membranes In untreated mammalian erythrocyte membranes, the concentration of membrane-bound bilirubin increased with an increase in bilirubin concentration in the incubate Žsee Fig. 1.. Human erythrocyte membranes bound significantly higher amounts of bilirubin Ž P) 0.001. than other mam-
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M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
Fig. 1. Interaction of bilirubin with erythrocyte membranes of different mammalian species treated with phospholipase C Ž C. welchii . for different time periods, i.e. untreated Ž`., 10 min Ž䢇., 20 min Ž^. and 30 min Ž'.. Each point is the mean of two independent experiments.
malian erythrocyte membranes. Significant difference in the membrane-bound bilirubin was noticed between sheep and buffalo erythrocyte membranes Ž Ps 0.01., whereas, buffalo and goat erythrocyte membranes were marginally different Ž Ps 0.03.. There was no correlation between the amount of membrane-bound bilirubin and membrane protein, phospholipid or carbohydrate content. However, using the reported data on membrane phospholipid composition of human, buffalo, sheep and goat erythrocyte membranes ŽO’Kelly, 1979; Barenholz and Thompson, 1980., a strong positive correlation Ž r s 0.98, P) 0.0001. was found between the membrane-bound bilirubin and the sum of choline phospholipids ŽPC q Sph.. Differences in the membrane-bound bilirubin among different mammals, as observed in this study, were probably due to the differences in their choline phospholipid content, which was reported to be highest in human erythrocyte membranes Ž; 60.4% of total phospholipids., followed by sheep Ž55.3%., buffalo and goat Ž; 50%. erythrocyte membranes ŽO’Kelly, 1979; Barenholz and Thompson, 1980.. The calculated value of membrane-bound bilirubinrmg choline phospholipid Žusing data from Table 1 and reported percentage content of choline phospholipids. was found to be 47.0" 0.9 nmoles in erythrocyte membranes of all four mammals when the concentration of bilirubin in the incubate was 68.3 nmolesrml. Phospholipase C treatment of human erythro-
cyte membranes resulted in a marked increase in the membrane-bound bilirubin compared to untreated membranes at any given bilirubin concentration ŽFig. 1.. Furthermore, increase in bilirubin binding was directly correlated with the time of incubation with phospholipase C. This increase in the amount of membrane-bound bilirubin was directly correlated with the amount of membrane phosphorus released ŽTable 1.. Phospholipase Ctreated membranes also showed an increase in the membrane-bound bilirubin with the increase in bilirubin concentration in the incubate in all four species ŽFig. 1.. However, they differed quantitatively in the amount of membrane-bound bilirubin at any bilirubin concentration in the same way as reported for untreated membranes. The difference in bound bilirubin between treated and untreated membranes was plotted against bilirubin concentration in the incubate ŽFig. 2.. Increase in bound bilirubin was found highest in human erythrocyte membranes after phospholipase C treatment for 10, 20 or 30 min followed by buffalo, sheep and goat membranes. Since phospholipase C from C. welchii removes membrane phosphorus predominantly from PC, PE and Sph, bilirubin binding data were also subjected to statistical analysis to find the correlation between the composition of membrane phospholipids and increase in bilirubin binding to enzyme-treated membranes. A positive correlation Ž r s 0.95. was found between the increase in membrane-bound bilirubin and a sum of PC, PE and Sph content
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
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Table 1 Effect of phospholipase C Ž C. welchii . treatment on the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Total membrane phosphorus ŽPi. Žg.
Phospholipid Žmg.
119.6" 14.9 97.0" 8.8 86.2" 9.4 102.9" 11.5
1.33" 0.03 1.26" 0.04 1.20" 0.03 1.18" 0.05
Membrane phosphorus ŽPi. removed after enzyme treatment for 10 min
20 min
30 min
amount Žg.
amount Žg.
amount Žg.
42.8" 1.5 30.8" 1.7 30.1" 4.2 20.7" 2.6
63.4" 4.6 54.8" 2.7 45.7" 4.8 41.4" 3.3
74.5" 5.7 61.9" 4.1 55.4" 3.1 48.8" 4.1
Each value represents the mean " S.D. of eight observations.
which was highest in human erythrocyte membranes Ž1.13 mg., followed by buffalo Ž1.08 mg., sheep Ž0.97 mg. and goat Ž0.91 mg. erythrocyte membranes. These results suggest that removal of polar head groups from PC, PE and Sph greatly enhanced the bilirubin binding implying that exposure of non-polar fatty acid diacylglycerols and sphingosine was responsible for increased binding of bilirubin to these membranes. This was in accordance with an earlier observation ŽSato et al., 1987. and contradicted the results reported by Vazquez et al. Ž1988.. 3.2. Interaction of bilirubin with trypsin-treated erythrocyte membranes It can be seen from the figure that the amount of membrane-bound bilirubin increased after trypsin treatment in all the four species at any
concentration of bilirubin in the incubate ŽFig. 3.. However, in terms of membrane-bound bilirubin, significant differences were found. The increase in membrane-bound bilirubin was highest in human erythrocyte membranes followed by buffalo and sheep erythrocyte membranes, while goat erythrocyte membranes showed a small increase. As erythrocyte membranes treated with trypsin resulted in the loss of membrane proteins and glycopeptides, the amount of released carbohydrate was estimated ŽTable 2.. A positive correlation was found between the increase in membrane-bound bilirubin and the percentage of carbohydrate released from these membranes. It appears that trypsin treatment of the membranes resulted in the opening of a large number of low affinity sites for bilirubin. Hence, it can be concluded that bilirubin binding sites are not composed of carbohydrate, instead, a shielding effect
Fig. 2. Plot of the difference in the amount of bilirubin bound to phospholipase C-treated and untreated erythrocyte membranes of different mammalian species. The treatment time was: 10 min Ž`.; 20 min Ž䢇. and 30 min Ž^..
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
360
Fig. 3. Interaction of bilirubin with erythrocyte membranes of different mammalian species treated with trypsin for different time periods, i.e. untreated Ž`., 1 h Ž䢇., 2 h Ž^. and 3 h Ž'.. Each point is the mean of two independent experiments.
of carbohydrate on bilirubin-membrane interaction was noted. Extensive degradation of membrane proteins upon trypsin treatment has been reported earlier ŽSteck et al., 1971; Inaba and Maede, 1988.. It appears that differential degradation of membrane proteins causing the release of glycopeptides from different erythrocyte membranes may account for the different increase in bilirubin binding after trypsin treatment.
Although the pattern of bilirubin binding was similar in both untreated as well as treated erythrocyte membranes, i.e. human ) sheep G buffalo ) goat, the amount of membrane-bound bilirubin was higher Ž; 14%. in treated than untreated erythrocyte membranes in all species. Sato and his group ŽSato et al., 1987. also reported little increase in bilirubin binding to human erythrocyte membranes after neuraminidase treatment. A slight increase in bilirubin binding to neuraminidase-treated membranes can be ascribed to the loss of negatively charged sialic acid residues which otherwise may repel negatively charged bilirubin monoanions. In the absence of any correlation between the release in sialic acid and increase in bilirubin binding to neuraminidase-treated membranes, it appears that some other factors are responsible for the enhanced binding in these membranes in addition
3.3. Interaction of bilirubin with neuraminidasetreated erythrocyte membranes Neuraminidase treatment resulted in the loss of sialic acid. The amount of sialic acid removed from these membranes was higher in human erythrocyte membranes followed by buffalo erythrocyte membranes whereas, sheep and goat erythrocyte membranes showed similar loss ŽTable 3..
Table 2 Effect of trypsin treatment on the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Total carbohydrate Žg.
242.8" 10.7 242.0" 4.5 274.8" 2.8 320.9" 8.8
Membrane carbohydrate released after enzyme treatment for 1h
2h
3h
amount Žg.
Ž%.
amount Žg.
Ž%.
amount Žg.
Ž%.
113.6" 11.2 86.5" 2.4 117.1" 6.4 130.5" 7.8
46.9 35.7 42.6 40.7
153.5" 8.8 117.6" 5.0 147.1" 2.1 146.4" 4.1
63.2 48.6 53.5 45.6
177.2" 7.1 161.1 " 11.8 179.0" 3.7 190.9" 2.6
73.0 66.6 65.1 59.5
Each value represents the mean " S.D. of eight observations.
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
361
Table 3 Effect of neuraminidase treatment on the binding of bilirubin to the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Sialic acid released in the supernatant Žnmoles.
Membrane-bound bilirubin Žnmoles. Control
Treated
Percent increase in bound bilirubin
168.0" 6.5 126.1" 4.3 94.8" 3.3 95.7" 3.3
198.0" 2.4 162.0" 2.4 174.0" 2.4 150.0" 4.8
229.2" 0.8 182.4" 0.4 189.0 " 0.6 162.2" 0.6
15.2 14.8 12.6 13.1
Each value represents the mean " S.D. of four observations.
to the loss of negatively charged sialic acid residues. Together, these results suggest that human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding under different conditions Žtrypsin, phospholipase C and neuraminidase treatments .. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human ) sheep ) buffalo ) goat; whereas the order was: human ) buffalo ) sheep ) goat; in phospholipase C and trypsin treated erythrocyte membranes. Thus, it appears that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes. The negatively charged phosphate moieties of phospholipids of the membrane appear to inhibit a large amount of bilirubin binding to the membrane as removal of negatively charged phosphate groups of phospholipids by phospholipase C greatly enhanced the binding. However, membrane proteins and carbohydrate play a significant regulatory function on the binding as degradation of membrane proteins or removal of carbohydrate in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites.
Acknowledgements This work was financially supported by a research grant ŽSPrSOrD-52r95. from the Department of Science and Technology, New Delhi, India. Facilities provided by Aligarh Muslim University and financial assistance to one of us ŽMKA. in the form of Research Associateship of the Department of Science and Technology, New Delhi, India are gratefully acknowledged.
References Barenholz, Y., Thompson, T.E., 1980. Sphingomyelins in bilayers and biological membranes. Biochim. Biophys. Acta 604, 129᎐158. Cestaro, B., Cervato, G., Ferrari, S., Di Silvestro, G., Monti, D., Manitto, P., 1983. Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine. Ital. J. Biochem. 32, 318᎐329. Chen, P.S., Toribara Jr., T.Y., Warner, H., 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756᎐1758. Fog, J., 1958. Determination of bilirubin in serum as alkaline ‘azobilirubin’. Scand. J. Clin. Lab. Invest. 10, 241᎐245. Folch, J., Less, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 479᎐509. Hayer, M., Piva, M-T., Sieso, V., de Bornier, B.M., 1989. Experimental studies on unconjugated bilirubin binding by human erythrocytes. Clin. Chim. Acta 186, 345᎐350. Inaba, M., Maede, Y., 1988. A new major transmembrane glycoprotein, gp 155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex. J. Biol. Chem. 263, 17763᎐17771. Leonard, M., Noy, N., Zakim, D., 1989. The interactions of bilirubin with model and biological membranes. J. Biol. Chem. 264, 5648᎐5652. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265᎐275. Nagaoka, S., Cowger, M.L., 1978. Interaction of bilirubin with lipids studied by fluorescence quenching method. J. Biol. Chem. 253, 2005᎐2011. O’Kelly, J.C., 1979. The lipid composition of erythrocytes in European cattle and buffalo steers. Lipids 14, 983᎐988. Palfrey, H.C., Waseem, A., 1985. Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9
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and other membrane proteins. J. Biol. Chem. 260, 16021᎐16029. Rashid, H., Ali, M.K., Tayyab, S., 2000. Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes. J. Biosci. 25, 157᎐161. Sato, H., Kashiwamata, S., 1983. Interaction of bilirubin with human erythrocyte membranes. Biochem. J. 210, 489᎐496. Sato, H., Aono, S., Semba, R., Kashiwamata, S., 1987. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes. Biochem. J. 248, 21᎐26. Steck, T.L., Fairbanks, G., Wallach, D.F.H., 1971. Disposition of the major protein in the isolated erythro-
cyte membrane. Proteolytic dissection. Biochemistry 10, 2617᎐2624. Svennerholm, L., 1956. The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem. 1, 42᎐45. Tayyab, S., Ali, M.K., 1999. A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods. J. Biochem. Biophys. Methods 39, 39᎐45. Vazquez, J., Garcia-Calvo, M., Valdivieso, F., Mayor, F., Mayor Jr., F., 1988. Interaction of bilirubin with the synaptosomal plasma membrane. J. Biol. Chem. 263, 1255᎐1265. Warren, L., 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234, 1971᎐1975.
Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes Mohammad K. Ali, Saad TayyabU Interdisciplinary Biotechnology Unit, Aligarh Muslim Uni¨ ersity, Aligarh, U.P. 202002, India Received 28 July 2000; received in revised form 30 November 2000; accepted 4 December 2000
Abstract Binding of bilirubin to erythrocyte membranes of human, buffalo, sheep and goat was studied after phospholipase C, trypsin and neuraminidase treatment. Phospholipase C and trypsin treatment of membranes greatly enhanced the bilirubin binding in all mammalian species, whereas, neuraminidase treatment resulted into a small increase in the membrane-bound bilirubin. Human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human ) sheep ) buffalo ) goat; the order was: human ) buffalo ) sheep ) goat; in phospholipase C- and trypsin-treated erythrocyte membranes. These binding results indicate that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes as the differences observed in the membrane-bound bilirubin among mammalian species were directly correlated with the sum of choline phospholipids, especially phosphatidylcholine and sphingomyelin content of the erythrocyte membranes. The negatively charged phosphate moiety of phospholipids of the membranes appears to inhibit a large amount of bilirubin binding to the membrane as its removal by phospholipase C greatly enhanced the binding. Furthermore, membrane proteins and carbohydrate also seem to play a significant regulatory function on the binding as their degradation andror removal in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Bilirubin; Buffalo; Erythrocyte membranes; Goat; Human; Neuraminidase; Phospholipase C; Sheep
1. Introduction Interaction of bilirubin with erythrocyte membranes has been studied ŽSato and Kashiwamata, 1983; Sato et al., 1987; Hayer et al., 1989; Rashid et al., 2000. to understand the mechanism of entry of bilirubin inside the cells which may be U
Corresponding author. Tel.: q91-571-701718; fax: q91571-701081. E-mail address: [email protected] ŽS. Tayyab..
helpful in developing preventive measures against bilirubin encephalopathy in new born infants. It is generally believed that bilirubin mainly interacts with the lipid bilayer of erythrocyte membranes ŽSato and Kashiwamata, 1983; Sato et al., 1987., however, the mechanism of such interaction is poorly understood. It has been suggested that bilirubin is bound to the lipid bilayer through strong ionic interactions between a cationic head group of the lipid and anionic bilirubin ŽNagaoka and Cowger, 1978.. The dianion form of bilirubin
1095-6433r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 0 . 0 0 3 5 3 - 6
356
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
binds weakly to the polar heads of the phospholipids, whereas, acid bilirubin binds strongly and is hydrophobically inserted into the lipophilic region of the bilayer ŽCestaro et al., 1983.. A decrease in binding constant for bilirubin-membrane interaction has been reported after phospholipase C treatment of synaptosomal membranes whereas phospholipase D-treated membranes showed a higher binding constant suggesting that the polar protruding ends of lipids are involved in the initial interaction with bilirubin ŽVazquez et al., 1988.. Involvement of electrostatic interactions between bilirubin and polar head groups of lipids was challenged by Sato and his group ŽSato et al., 1987. who showed no change in bilirubin binding after phospholipase D treatment of the membranes, whereas, phospholipase C-treated membranes showed greatly enhanced bilirubin binding. They are of the view that the exposure of non-polar diacylglycerols at the surface of the membrane after removal of polar head groups of phosphatidylcholine ŽPC., phosphatidylethanolamine ŽPE. as well as sphingomyelin ŽSph. of membrane, may potentiate a large amount of non-specific binding of bilirubin to the fatty acid moiety of diacylglycerols ŽSato et al., 1987.. The role of membrane proteins in bilirubin binding to membranes is also not very clear. Sato and Kashiwamata Ž1983. ruled out the involvement of membrane proteins as the bilirubin binding sites, based on the increased bilirubin binding after trypsin treatment of human erythrocyte membranes but suggested that membrane proteins may act as a barrier in the binding process. These findings are questionable in view of enhanced bilirubin binding to model membranes containing an integral protein compared to protein lacking model membranes ŽLeonard et al., 1989.. Furthermore, the role of negatively charged sialic acid residues present on the membrane surface in bilirubin binding phenomenon is not certain. Whereas, Vazquez et al. Ž1988. have reported that the negative charges contributed by sialic acid residues exert an inhibitory effect on the bilirubin binding to membrane, Sato et al. Ž1987. have found no significant change in the binding of bilirubin after neuraminidase treatment of membranes. In order to clarify the conflicting observations of the role of polar head groups of phospholipids, membrane proteins and sialic acid residues, we
studied the binding of bilirubin to the human erythrocyte membranes in detail before and after treating them with phospholipase C, trypsin and neuraminidase. As protein and lipid make-up of erythrocyte membranes varies in different mammalian species ŽO’Kelly, 1979; Barenholz and Thompson, 1980; Inaba and Maede, 1988., bilirubin binding results of treated human erythrocyte membranes were also compared with the membranes obtained from buffalo, sheep and goat under similar conditions which will be helpful in understanding the specific roles of various membrane components in bilirubin binding.
2. Materials and methods 2.1. Materials Bovine serum albumin, fraction V, trypsin, type III from bovine pancreas ŽLot 24H0070., phospholipase C, type I from Clostridium welchii ŽLot 10H6801., neuraminidase, type V from C. perfringens ŽLot 31H82302. and phenylmethylsulfonylfluoride ŽPMSF. were purchased from Sigma Chemical Company, USA. Bilirubin was the product of Sisco Research Laboratories, India. Other reagents used were of analytical grades. The blood of buffalo Ž Bos indicus., sheep Ž O¨ is aries. and goat Ž Cepra hircus. was collected fresh in 1.32% sodium citrate solution from slaughter houses. Human blood was obtained from the Blood Bank of J.N. Medical College, Aligarh Muslim University, Aligarh. 2.2. Methods Erythrocytes were collected by centrifugation of blood at 1000 = g for 20 min. After removing the upper buffy coat through gentle decantation, cells were washed with 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl followed by centrifugation at 1000 = g for 20 min. This process was repeated 3 times. The final packed cell volume was diluted with an equal volume of the same buffer to obtain a 50% hematocrit value. Erythrocyte membranes were prepared following the method of Palfrey and Waseem Ž1985.. Hemolysis of erythrocytes was carried out using 10 volumes of cold 10 mM Tris᎐HCl buffer, pH 7.4 containing 0.01 mM EDTA and 0.01 mM PMSF. The contents were mixed by gentle swirling
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
and then centrifuged at 16 000 = g at 4⬚C for 30 min. The dark red supernatant was removed carefully by gentle aspiration. The membrane pellet was resuspended by swirling in 5.0 ml of the same lysis buffer and then centrifuged at 16 000 = g at 4⬚C for 20 min. The pellet was washed 5᎐6 times with the same buffer until a clear supernatant was obtained. The membrane pellet was resuspended in 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl in a volume equivalent to the volume of erythrocytes used. It was stored at 10⬚C and used as such within 2 days. Phospholipid content of erythrocyte membranes was determined by the method of Chen et al. Ž1956. following the extraction of phospholipids ŽFolch et al., 1957.. Protein concentration was determined by the method of Lowry et al. Ž1951. after solubilizing the membranes in 1% SDS. Orcinol ŽSvennerholm, 1956. was used to determine the total carbohydrate content of the membranes. Phospholipase C digestion of erythrocyte membranes was carried out following the method of Sato et al. Ž1987.. To 1.0 ml of membrane suspension in 50 mM Tris᎐HCl buffer, pH 7.4 was added 3.75 ml of 0.1 M Tris᎐HCl buffer, pH 7.4, 50 l of 1.0 M CaCl 2 and 200 l of the enzyme solution Ž5 mgrml. and the mixture was incubated at 37⬚C. The reaction was stopped by adding 1.0 ml of 10 mM EDTA. All the contents were centrifuged at 16 000 = g for 20 min. The supernatant was assayed for released phosphate ŽChen et al., 1956.. Tryptic digestion of erythrocyte membranes was performed by the method of Steck et al. Ž1971.. Five milligrams of solid trypsin were added to 1.0 ml of erythrocyte membrane suspension in 2.5mM sodium phosphate buffer, pH 8.0 and the mixture was shaken until the enzyme was completely dissolved. The reaction was carried out at room temperature for varying periods. Then, the mixture was centrifuged at 16 000 = g for 5 min and the supernatant containing released glycopeptides was collected and subjected to carbohydrate measurement as above ŽSvennerholm, 1956.. Erythrocyte membranes Ž1.0 ml. were treated with neuraminidase following incubation with 0.5 units of the enzyme for 1 h at 37⬚C in 50 mM Tris᎐HCl buffer, pH 7.4. The mixture was centrifuged at 16 000 = g for 5 min and the supernatant containing released sialic acid was col-
357
lected and subjected to sialic acid estimation ŽWarren, 1959.. The pellets obtained in the above experiments were washed 2 times with cold 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl followed by centrifugation at 16 000 = g for 20 min. Then, membranes in each tube were resuspended in the same buffer making the total volume as 1.0 ml. These membranes were directly used for bilirubin binding studies. Bilirubin solution was prepared by dissolving a few crystals of bilirubin in 38 mM sodium carbonate solution containing 5.0 mM EDTA, pH 11.0. Its concentration was determined by Fog’s method ŽFog, 1958.. The bilirubin solution was protected from light and used within 1 h. All experiments were carried out under yellow light. Binding of bilirubin to erythrocyte membranes Žuntreatedrtreated . was studied by incubating 1.0 ml of stock bilirubin solution of desired concentration with 1.0 ml of erythrocyte membrane suspension Žequivalent to 1.0 ml of erythrocyte suspension of 50% hematocrit. and the final volume was made to 6.0 ml with 50 mM Tris᎐HCl buffer, pH 7.4 containing 100 mM NaCl. After 30 min of incubation at 37⬚C, the mixture was centrifuged at 16 000 = g for 15 min at 4⬚C and the supernatant containing unbound bilirubin was discarded. Membranes were washed several times with the same buffer until the last supernatant was devoid of yellow color. After final washing, the membrane-bound bilirubin was determined by modified Fog’s method ŽFog, 1958. as described by Tayyab and Ali Ž1999.. Statistical analysis of the data included calculations of Pearson’s product moment correlation coefficients and dispersion. Correlation coefficients and difference of means were tested for significance using two-tailed ‘t’-tests.
3. Results and discussion 3.1. Interaction of bilirubin with untreated and phospholipase C-treated erythrocyte membranes In untreated mammalian erythrocyte membranes, the concentration of membrane-bound bilirubin increased with an increase in bilirubin concentration in the incubate Žsee Fig. 1.. Human erythrocyte membranes bound significantly higher amounts of bilirubin Ž P) 0.001. than other mam-
358
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
Fig. 1. Interaction of bilirubin with erythrocyte membranes of different mammalian species treated with phospholipase C Ž C. welchii . for different time periods, i.e. untreated Ž`., 10 min Ž䢇., 20 min Ž^. and 30 min Ž'.. Each point is the mean of two independent experiments.
malian erythrocyte membranes. Significant difference in the membrane-bound bilirubin was noticed between sheep and buffalo erythrocyte membranes Ž Ps 0.01., whereas, buffalo and goat erythrocyte membranes were marginally different Ž Ps 0.03.. There was no correlation between the amount of membrane-bound bilirubin and membrane protein, phospholipid or carbohydrate content. However, using the reported data on membrane phospholipid composition of human, buffalo, sheep and goat erythrocyte membranes ŽO’Kelly, 1979; Barenholz and Thompson, 1980., a strong positive correlation Ž r s 0.98, P) 0.0001. was found between the membrane-bound bilirubin and the sum of choline phospholipids ŽPC q Sph.. Differences in the membrane-bound bilirubin among different mammals, as observed in this study, were probably due to the differences in their choline phospholipid content, which was reported to be highest in human erythrocyte membranes Ž; 60.4% of total phospholipids., followed by sheep Ž55.3%., buffalo and goat Ž; 50%. erythrocyte membranes ŽO’Kelly, 1979; Barenholz and Thompson, 1980.. The calculated value of membrane-bound bilirubinrmg choline phospholipid Žusing data from Table 1 and reported percentage content of choline phospholipids. was found to be 47.0" 0.9 nmoles in erythrocyte membranes of all four mammals when the concentration of bilirubin in the incubate was 68.3 nmolesrml. Phospholipase C treatment of human erythro-
cyte membranes resulted in a marked increase in the membrane-bound bilirubin compared to untreated membranes at any given bilirubin concentration ŽFig. 1.. Furthermore, increase in bilirubin binding was directly correlated with the time of incubation with phospholipase C. This increase in the amount of membrane-bound bilirubin was directly correlated with the amount of membrane phosphorus released ŽTable 1.. Phospholipase Ctreated membranes also showed an increase in the membrane-bound bilirubin with the increase in bilirubin concentration in the incubate in all four species ŽFig. 1.. However, they differed quantitatively in the amount of membrane-bound bilirubin at any bilirubin concentration in the same way as reported for untreated membranes. The difference in bound bilirubin between treated and untreated membranes was plotted against bilirubin concentration in the incubate ŽFig. 2.. Increase in bound bilirubin was found highest in human erythrocyte membranes after phospholipase C treatment for 10, 20 or 30 min followed by buffalo, sheep and goat membranes. Since phospholipase C from C. welchii removes membrane phosphorus predominantly from PC, PE and Sph, bilirubin binding data were also subjected to statistical analysis to find the correlation between the composition of membrane phospholipids and increase in bilirubin binding to enzyme-treated membranes. A positive correlation Ž r s 0.95. was found between the increase in membrane-bound bilirubin and a sum of PC, PE and Sph content
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
359
Table 1 Effect of phospholipase C Ž C. welchii . treatment on the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Total membrane phosphorus ŽPi. Žg.
Phospholipid Žmg.
119.6" 14.9 97.0" 8.8 86.2" 9.4 102.9" 11.5
1.33" 0.03 1.26" 0.04 1.20" 0.03 1.18" 0.05
Membrane phosphorus ŽPi. removed after enzyme treatment for 10 min
20 min
30 min
amount Žg.
amount Žg.
amount Žg.
42.8" 1.5 30.8" 1.7 30.1" 4.2 20.7" 2.6
63.4" 4.6 54.8" 2.7 45.7" 4.8 41.4" 3.3
74.5" 5.7 61.9" 4.1 55.4" 3.1 48.8" 4.1
Each value represents the mean " S.D. of eight observations.
which was highest in human erythrocyte membranes Ž1.13 mg., followed by buffalo Ž1.08 mg., sheep Ž0.97 mg. and goat Ž0.91 mg. erythrocyte membranes. These results suggest that removal of polar head groups from PC, PE and Sph greatly enhanced the bilirubin binding implying that exposure of non-polar fatty acid diacylglycerols and sphingosine was responsible for increased binding of bilirubin to these membranes. This was in accordance with an earlier observation ŽSato et al., 1987. and contradicted the results reported by Vazquez et al. Ž1988.. 3.2. Interaction of bilirubin with trypsin-treated erythrocyte membranes It can be seen from the figure that the amount of membrane-bound bilirubin increased after trypsin treatment in all the four species at any
concentration of bilirubin in the incubate ŽFig. 3.. However, in terms of membrane-bound bilirubin, significant differences were found. The increase in membrane-bound bilirubin was highest in human erythrocyte membranes followed by buffalo and sheep erythrocyte membranes, while goat erythrocyte membranes showed a small increase. As erythrocyte membranes treated with trypsin resulted in the loss of membrane proteins and glycopeptides, the amount of released carbohydrate was estimated ŽTable 2.. A positive correlation was found between the increase in membrane-bound bilirubin and the percentage of carbohydrate released from these membranes. It appears that trypsin treatment of the membranes resulted in the opening of a large number of low affinity sites for bilirubin. Hence, it can be concluded that bilirubin binding sites are not composed of carbohydrate, instead, a shielding effect
Fig. 2. Plot of the difference in the amount of bilirubin bound to phospholipase C-treated and untreated erythrocyte membranes of different mammalian species. The treatment time was: 10 min Ž`.; 20 min Ž䢇. and 30 min Ž^..
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
360
Fig. 3. Interaction of bilirubin with erythrocyte membranes of different mammalian species treated with trypsin for different time periods, i.e. untreated Ž`., 1 h Ž䢇., 2 h Ž^. and 3 h Ž'.. Each point is the mean of two independent experiments.
of carbohydrate on bilirubin-membrane interaction was noted. Extensive degradation of membrane proteins upon trypsin treatment has been reported earlier ŽSteck et al., 1971; Inaba and Maede, 1988.. It appears that differential degradation of membrane proteins causing the release of glycopeptides from different erythrocyte membranes may account for the different increase in bilirubin binding after trypsin treatment.
Although the pattern of bilirubin binding was similar in both untreated as well as treated erythrocyte membranes, i.e. human ) sheep G buffalo ) goat, the amount of membrane-bound bilirubin was higher Ž; 14%. in treated than untreated erythrocyte membranes in all species. Sato and his group ŽSato et al., 1987. also reported little increase in bilirubin binding to human erythrocyte membranes after neuraminidase treatment. A slight increase in bilirubin binding to neuraminidase-treated membranes can be ascribed to the loss of negatively charged sialic acid residues which otherwise may repel negatively charged bilirubin monoanions. In the absence of any correlation between the release in sialic acid and increase in bilirubin binding to neuraminidase-treated membranes, it appears that some other factors are responsible for the enhanced binding in these membranes in addition
3.3. Interaction of bilirubin with neuraminidasetreated erythrocyte membranes Neuraminidase treatment resulted in the loss of sialic acid. The amount of sialic acid removed from these membranes was higher in human erythrocyte membranes followed by buffalo erythrocyte membranes whereas, sheep and goat erythrocyte membranes showed similar loss ŽTable 3..
Table 2 Effect of trypsin treatment on the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Total carbohydrate Žg.
242.8" 10.7 242.0" 4.5 274.8" 2.8 320.9" 8.8
Membrane carbohydrate released after enzyme treatment for 1h
2h
3h
amount Žg.
Ž%.
amount Žg.
Ž%.
amount Žg.
Ž%.
113.6" 11.2 86.5" 2.4 117.1" 6.4 130.5" 7.8
46.9 35.7 42.6 40.7
153.5" 8.8 117.6" 5.0 147.1" 2.1 146.4" 4.1
63.2 48.6 53.5 45.6
177.2" 7.1 161.1 " 11.8 179.0" 3.7 190.9" 2.6
73.0 66.6 65.1 59.5
Each value represents the mean " S.D. of eight observations.
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
361
Table 3 Effect of neuraminidase treatment on the binding of bilirubin to the erythrocyte membranes Žequivalent to 1.0 ml of 50% hematocrit value. of different mammalian species a Species
Human Buffalo Sheep Goat a
Sialic acid released in the supernatant Žnmoles.
Membrane-bound bilirubin Žnmoles. Control
Treated
Percent increase in bound bilirubin
168.0" 6.5 126.1" 4.3 94.8" 3.3 95.7" 3.3
198.0" 2.4 162.0" 2.4 174.0" 2.4 150.0" 4.8
229.2" 0.8 182.4" 0.4 189.0 " 0.6 162.2" 0.6
15.2 14.8 12.6 13.1
Each value represents the mean " S.D. of four observations.
to the loss of negatively charged sialic acid residues. Together, these results suggest that human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding under different conditions Žtrypsin, phospholipase C and neuraminidase treatments .. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human ) sheep ) buffalo ) goat; whereas the order was: human ) buffalo ) sheep ) goat; in phospholipase C and trypsin treated erythrocyte membranes. Thus, it appears that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes. The negatively charged phosphate moieties of phospholipids of the membrane appear to inhibit a large amount of bilirubin binding to the membrane as removal of negatively charged phosphate groups of phospholipids by phospholipase C greatly enhanced the binding. However, membrane proteins and carbohydrate play a significant regulatory function on the binding as degradation of membrane proteins or removal of carbohydrate in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites.
Acknowledgements This work was financially supported by a research grant ŽSPrSOrD-52r95. from the Department of Science and Technology, New Delhi, India. Facilities provided by Aligarh Muslim University and financial assistance to one of us ŽMKA. in the form of Research Associateship of the Department of Science and Technology, New Delhi, India are gratefully acknowledged.
References Barenholz, Y., Thompson, T.E., 1980. Sphingomyelins in bilayers and biological membranes. Biochim. Biophys. Acta 604, 129᎐158. Cestaro, B., Cervato, G., Ferrari, S., Di Silvestro, G., Monti, D., Manitto, P., 1983. Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine. Ital. J. Biochem. 32, 318᎐329. Chen, P.S., Toribara Jr., T.Y., Warner, H., 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756᎐1758. Fog, J., 1958. Determination of bilirubin in serum as alkaline ‘azobilirubin’. Scand. J. Clin. Lab. Invest. 10, 241᎐245. Folch, J., Less, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 479᎐509. Hayer, M., Piva, M-T., Sieso, V., de Bornier, B.M., 1989. Experimental studies on unconjugated bilirubin binding by human erythrocytes. Clin. Chim. Acta 186, 345᎐350. Inaba, M., Maede, Y., 1988. A new major transmembrane glycoprotein, gp 155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex. J. Biol. Chem. 263, 17763᎐17771. Leonard, M., Noy, N., Zakim, D., 1989. The interactions of bilirubin with model and biological membranes. J. Biol. Chem. 264, 5648᎐5652. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265᎐275. Nagaoka, S., Cowger, M.L., 1978. Interaction of bilirubin with lipids studied by fluorescence quenching method. J. Biol. Chem. 253, 2005᎐2011. O’Kelly, J.C., 1979. The lipid composition of erythrocytes in European cattle and buffalo steers. Lipids 14, 983᎐988. Palfrey, H.C., Waseem, A., 1985. Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9
362
M.K. Ali, S. Tayyab r Comparati¨ e Biochemistry and Physiology Part A 129 (2001) 355᎐362
and other membrane proteins. J. Biol. Chem. 260, 16021᎐16029. Rashid, H., Ali, M.K., Tayyab, S., 2000. Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes. J. Biosci. 25, 157᎐161. Sato, H., Kashiwamata, S., 1983. Interaction of bilirubin with human erythrocyte membranes. Biochem. J. 210, 489᎐496. Sato, H., Aono, S., Semba, R., Kashiwamata, S., 1987. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes. Biochem. J. 248, 21᎐26. Steck, T.L., Fairbanks, G., Wallach, D.F.H., 1971. Disposition of the major protein in the isolated erythro-
cyte membrane. Proteolytic dissection. Biochemistry 10, 2617᎐2624. Svennerholm, L., 1956. The quantitative estimation of cerebrosides in nervous tissue. J. Neurochem. 1, 42᎐45. Tayyab, S., Ali, M.K., 1999. A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods. J. Biochem. Biophys. Methods 39, 39᎐45. Vazquez, J., Garcia-Calvo, M., Valdivieso, F., Mayor, F., Mayor Jr., F., 1988. Interaction of bilirubin with the synaptosomal plasma membrane. J. Biol. Chem. 263, 1255᎐1265. Warren, L., 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234, 1971᎐1975.