922
BIOCHEMICAL SOCIETY TRANSACTIONS
combined, pressure-dialysed to small volume and subjected to further analysis by sucrose-density centrifugation as follows: continuous sucrose density gradients (5-30 %, wlv, sucrose in HBM buffer, pH7.0) were prepared in 5ml centrifuge tubes. Two of these were each overlaid with 2 0 0 ~ 1of the sample containing ‘androstad,l6-dien-3~-ol synthetase’ activity and centrifuged alongside gradients containing reference compounds and controls. After centrifugation at 181OOOg for 24h at IO’C, the tubes were emptied by careful siphoning from the bottom, every eleven drops being collected. All samples were assayed for protein concentration and ‘androsta-5,16-dien-3~-ol synthetase’ activity. Incubations were performed for a period of 3h because of the low amount of activity expected. The results are expressed in Fig. 1, which shows that a sharp band of activity had migrated approximately the same distance as bovine serum albumin (mol.wt. 65000). However, in terms of specific activity this sample had a value of 14nmol of product/min per mg of protein compared with the original microsomal fraction (37.4nmol/min per mg of protein. This may be due to an alteration in protein structure, or the disaggregation of component proteins on solubilization. The financial support of G. M. C. by the A. R. C. (Grant no. AG 35/29) is gratefully acknowledged. Cooke, G. M. & Cower, D. B. (1977) Biochim. Biophys. Acta 498,265-271 Lowry, 0 .H., Rosebrough,N. J., Farr, A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Shimizu, K. & Nakada, F. (1976) Biochinr. Biophys. Acfa450,441449 Weber, K. & Kuter, D. J. (1971) J. Biol. Chem. 246,4504-4509
The Oleic Acid-Induced Shape Transformation of Human Erythrocytes MARTIN G . RUMSBY, SUSAN SPARROW and CLIVE LITTLE* Department of Biology, University of York, Heslington, York YO1 5 D D , U.K.
Oleic acid, like some detergents, drugs and alcohols (Deuticke, 1968), induces echinocyte formation when added to fresh human erythrocytes. It is not clear whether all these compounds cause enchinocytosis by a common mechanism (Sheetz & Singer, 1974). The mechanism will probably be different from that involved in the disc-spherocyte transformation induced in erythrocytes with ionophore A23187+Ca2+ (e.g. Allan & Michell, 1976) or during the storage of human blood in vitro for transfusion (Rumsby et al., 1977).In the present work the effect of oleic acid on erythrocyte shape was examined to test whether haemoglobin-filled vesicles are released during the echinocytosis and whether Ca2+is important in the fatty acid-induced bransformation. Human erythrocytes in acid citrateldextrose were obtained 24h after donation and were used within 2 days. Oleic acid (free; Sigma) in ethanol was added to washed erythrocytes suspended to 50 % haematocrit in 0.9 % NaCI. Control incubation mixtures containing 0.25 %ethanol were prepared similarly. Cell shape was monitored by stereoscan electron microscopy (C. Little & M . G. Rumsby, unpublished work). Haemoglobin release was measured spectrophotometrically at 412nm after sedimentation of the cells. Some incubation mixtures containing fatty acid were made 2 m with ~ respect to CaZ+. Phospholipase C (Bacillus cereus), prepared as described by Little et al. (1975), was allowed to react with cells pretreated with oleic acid ( 0 . 5 m ~ )or with oleate+Ca2+. Incubation of discoid erythrocytes with oleate (Table 1) causes an increasing echinocytosis with time. Extents of haemolysis also increase with time. Control erythrocytes incubated with 0.25 % ethanol showed 0.1 % haemolysis after 2h, and the cells were echinocytes 1 or discs. Oleate-treated erythrocytes gradually transformed from echinocytes to spheroechinocytes. Smooth spheres were not observed in the 2h incu-
* Permanent address: Institute of Medical €$ology,,University
of Tjomss, Tromsg, Norway
1979
583rd MEETING, CAMBRIDGE
923
Table 1. Action of oleic acid on erythrocyte shape and haemolysis Oleic acid in the free form was dissolved in ethanol and mixed with freshly washed discoid erythrocytes at the concentrations shown. Incubation media were mixed constantly at room temperature. Samples were removed at appropriate times for assay as described in the text. Cells were lysed with saponin for measurement of total haemoglobin. Concn. of oleic acid (mM) 0.5mM
Time of exposure (min) 5 15 30
60 2.0mM
120 5 15 30 60 120
Cell types* (% of total) Haemolysis , (%) D+El+E2 E3 0.18 0.21 47 53 0.29 0.34 38 55 58 0.38 33 0.65 0.79 22 71 1.04 1.33 20 57 2.37 24 39
*
SP1 -
7 9 -
7
-
23 37
* For cell types D = discocyte, El = echinocytes 1 , E2 = echinocytes 2, E3 = echinocytes 3 and SP1= spheroechinocytes 1 (after Bessis, 1972). bations with either oleate concentration. With longer incubation up to 4h with 2 m ~ oleate smooth spheres become apparent. Extents of haemolysis were over 5 % by this stage, and erythrocyte ‘ghosts’ were present. CaZ+added at the start of incubations diminished the echinocytogenic potency of the oleate. In the presence of 2m~-Ca’+ erythrocyte transformation to echinocytes did not proceed to the same extent. This may be due to the fact that calcium oleate does not partition into the erythrocyte membrane as effectively as in the free form or that the Ca2+somehow modifies the surface of the erythrocyte membrane to diminish the action of the oleate. In contrast with the situation with ionophore A231 87 therefore, the echinocytosis induced by oleate is Ca2+-independent. Oleate presumably induces echinocyte formation by partitioning info the erythrocyte membrane, where it may disorder bilayer organization, as diacylglycerol has been suggested to do (Allen et al., 1978). Cullis & Hope (1978), however, note that Ca2+is essential for oleate-induced membrane fusion processes. The shapes of echinocytes and spheroechinocytes generated by oleate differ from those of echinocytes obtained by treatment with ionophore A23187+Ca2+ or by aging in uitro; oleatetreated cells had many more projections in the echinocyte 3 form that were more stubby. Spheroechinocytes that form during oleate treatment are smaller than the original discs. Thus it is likely that membrane is lost from cells during the transformation of the discocyte to the spherocyte form. Attempts to recover microvesicles, however, have been complicated by the erythrocyte ‘ghosts’ generated during haemolysis. Densitygradient centrifugation may allow us to resolve this problem to check whether microvesicles are produced and whether they are depleted in spectrin, as is the case for microvesicles that form with ionophore A23187 and during aging in vitro. Erythrocytes pretreated with oleate were much more susceptible to attack by phospholipase C (Fig. 1 ) relative to control cells. This increased susceptibility to lysis is presumably due to the presence of the fatty acid in the membrane, which may influence the packing pressure between lipids. Such changes could allow phospholipase C better access to its substrates in the membrane. Ca2+ somewhat decreased this susceptibility to phospholipase C (Fig. l), but this can be explained on the basis of less fatty acid partitioning into the membrane. The enhanced susceptibility of oleate-treated erythro-
Vol. 7
924
BIOCHEMICAL SOCIETY TRANSACTIONS
Time (rnin) Fig. 1 . Haemolysis of oleic acid-pretreated human erythrocytes by phospholipase C (B. cereus) Erythrocytes, pretreated with 0.5 mwoleic acid.(.) or with0.5m~-oleicacid+2m~-Ca’+ (a), were suspended in 0.9% NaCl buffered with SOmM-Tris/HCl, pH7.4, to 50% haematocrit. Phospholipase C was added at a concentration of 55 units of enzyme/ml of cell suspension. Incubations were at 20°C. Control cells treated with ethanol but no fatty acid (A) were incubated similarly. Haemolysis was assayed by release of haemoglobin to the supernatant after sedimentation of cells. Further details are in the text.
cytes to lysis by the enzyme could be reversed by further treating them with fat-free albumin. This work is supported by funds from The Medical Research Council and the Norwegian Research Council for Science and the Humanities. Allan, D. & Michell, R. H. (1976) Biochem. J. 156,225-232 Allan, D., Thomas, P. & Michell, R. H. (1978) Nature (London) 276,289-290 Bessis, M. (1972) Nouo. Rev. Fr. HPmatol. 12, 1-25 Cullis, P. R. & Hope, M. J. (1978) Nature (London) 271,672-674 Deuticke, B. (1968) Biochim. Biophys. Acta 163,494-500 Little, C., Aurebekk, B. & Otnaess, A,-B. (1975) FEBSLett. 52,175-179 Rumsby, M. G., Trotter, J., Allan, D. & Michell, R. H. (1977) Biochem. SOC.Trans. 5 , 126-128 Sheetz, M. P. & Singer, S. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,4457-4461
Mutability of the Kinetic Properties of the Acetylcholine Hydrolase Activity of Human Erythrocytes S. FAZLI MABOOD,* PETER F. J. NEWMAN? and IAN A. NIMMO* *Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, and ?Department of Haematology, The Royal Infirmary, Edinburgh EH3 9 YW, Scotland, U.K. Human erythrocyte acetylcholine hydrolase (EC 3.1 .I .7) is a membrane-bound enzyme whose kinetic properties seem to be influenced by the integrity and composition of the
1979
BIOCHEMICAL SOCIETY TRANSACTIONS
combined, pressure-dialysed to small volume and subjected to further analysis by sucrose-density centrifugation as follows: continuous sucrose density gradients (5-30 %, wlv, sucrose in HBM buffer, pH7.0) were prepared in 5ml centrifuge tubes. Two of these were each overlaid with 2 0 0 ~ 1of the sample containing ‘androstad,l6-dien-3~-ol synthetase’ activity and centrifuged alongside gradients containing reference compounds and controls. After centrifugation at 181OOOg for 24h at IO’C, the tubes were emptied by careful siphoning from the bottom, every eleven drops being collected. All samples were assayed for protein concentration and ‘androsta-5,16-dien-3~-ol synthetase’ activity. Incubations were performed for a period of 3h because of the low amount of activity expected. The results are expressed in Fig. 1, which shows that a sharp band of activity had migrated approximately the same distance as bovine serum albumin (mol.wt. 65000). However, in terms of specific activity this sample had a value of 14nmol of product/min per mg of protein compared with the original microsomal fraction (37.4nmol/min per mg of protein. This may be due to an alteration in protein structure, or the disaggregation of component proteins on solubilization. The financial support of G. M. C. by the A. R. C. (Grant no. AG 35/29) is gratefully acknowledged. Cooke, G. M. & Cower, D. B. (1977) Biochim. Biophys. Acta 498,265-271 Lowry, 0 .H., Rosebrough,N. J., Farr, A. L. &Randall, R. J. (1951)J. Biol. Chem. 193,265-275 Shimizu, K. & Nakada, F. (1976) Biochinr. Biophys. Acfa450,441449 Weber, K. & Kuter, D. J. (1971) J. Biol. Chem. 246,4504-4509
The Oleic Acid-Induced Shape Transformation of Human Erythrocytes MARTIN G . RUMSBY, SUSAN SPARROW and CLIVE LITTLE* Department of Biology, University of York, Heslington, York YO1 5 D D , U.K.
Oleic acid, like some detergents, drugs and alcohols (Deuticke, 1968), induces echinocyte formation when added to fresh human erythrocytes. It is not clear whether all these compounds cause enchinocytosis by a common mechanism (Sheetz & Singer, 1974). The mechanism will probably be different from that involved in the disc-spherocyte transformation induced in erythrocytes with ionophore A23187+Ca2+ (e.g. Allan & Michell, 1976) or during the storage of human blood in vitro for transfusion (Rumsby et al., 1977).In the present work the effect of oleic acid on erythrocyte shape was examined to test whether haemoglobin-filled vesicles are released during the echinocytosis and whether Ca2+is important in the fatty acid-induced bransformation. Human erythrocytes in acid citrateldextrose were obtained 24h after donation and were used within 2 days. Oleic acid (free; Sigma) in ethanol was added to washed erythrocytes suspended to 50 % haematocrit in 0.9 % NaCI. Control incubation mixtures containing 0.25 %ethanol were prepared similarly. Cell shape was monitored by stereoscan electron microscopy (C. Little & M . G. Rumsby, unpublished work). Haemoglobin release was measured spectrophotometrically at 412nm after sedimentation of the cells. Some incubation mixtures containing fatty acid were made 2 m with ~ respect to CaZ+. Phospholipase C (Bacillus cereus), prepared as described by Little et al. (1975), was allowed to react with cells pretreated with oleic acid ( 0 . 5 m ~ )or with oleate+Ca2+. Incubation of discoid erythrocytes with oleate (Table 1) causes an increasing echinocytosis with time. Extents of haemolysis also increase with time. Control erythrocytes incubated with 0.25 % ethanol showed 0.1 % haemolysis after 2h, and the cells were echinocytes 1 or discs. Oleate-treated erythrocytes gradually transformed from echinocytes to spheroechinocytes. Smooth spheres were not observed in the 2h incu-
* Permanent address: Institute of Medical €$ology,,University
of Tjomss, Tromsg, Norway
1979
583rd MEETING, CAMBRIDGE
923
Table 1. Action of oleic acid on erythrocyte shape and haemolysis Oleic acid in the free form was dissolved in ethanol and mixed with freshly washed discoid erythrocytes at the concentrations shown. Incubation media were mixed constantly at room temperature. Samples were removed at appropriate times for assay as described in the text. Cells were lysed with saponin for measurement of total haemoglobin. Concn. of oleic acid (mM) 0.5mM
Time of exposure (min) 5 15 30
60 2.0mM
120 5 15 30 60 120
Cell types* (% of total) Haemolysis , (%) D+El+E2 E3 0.18 0.21 47 53 0.29 0.34 38 55 58 0.38 33 0.65 0.79 22 71 1.04 1.33 20 57 2.37 24 39
*
SP1 -
7 9 -
7
-
23 37
* For cell types D = discocyte, El = echinocytes 1 , E2 = echinocytes 2, E3 = echinocytes 3 and SP1= spheroechinocytes 1 (after Bessis, 1972). bations with either oleate concentration. With longer incubation up to 4h with 2 m ~ oleate smooth spheres become apparent. Extents of haemolysis were over 5 % by this stage, and erythrocyte ‘ghosts’ were present. CaZ+added at the start of incubations diminished the echinocytogenic potency of the oleate. In the presence of 2m~-Ca’+ erythrocyte transformation to echinocytes did not proceed to the same extent. This may be due to the fact that calcium oleate does not partition into the erythrocyte membrane as effectively as in the free form or that the Ca2+somehow modifies the surface of the erythrocyte membrane to diminish the action of the oleate. In contrast with the situation with ionophore A231 87 therefore, the echinocytosis induced by oleate is Ca2+-independent. Oleate presumably induces echinocyte formation by partitioning info the erythrocyte membrane, where it may disorder bilayer organization, as diacylglycerol has been suggested to do (Allen et al., 1978). Cullis & Hope (1978), however, note that Ca2+is essential for oleate-induced membrane fusion processes. The shapes of echinocytes and spheroechinocytes generated by oleate differ from those of echinocytes obtained by treatment with ionophore A23187+Ca2+ or by aging in uitro; oleatetreated cells had many more projections in the echinocyte 3 form that were more stubby. Spheroechinocytes that form during oleate treatment are smaller than the original discs. Thus it is likely that membrane is lost from cells during the transformation of the discocyte to the spherocyte form. Attempts to recover microvesicles, however, have been complicated by the erythrocyte ‘ghosts’ generated during haemolysis. Densitygradient centrifugation may allow us to resolve this problem to check whether microvesicles are produced and whether they are depleted in spectrin, as is the case for microvesicles that form with ionophore A23187 and during aging in vitro. Erythrocytes pretreated with oleate were much more susceptible to attack by phospholipase C (Fig. 1 ) relative to control cells. This increased susceptibility to lysis is presumably due to the presence of the fatty acid in the membrane, which may influence the packing pressure between lipids. Such changes could allow phospholipase C better access to its substrates in the membrane. Ca2+ somewhat decreased this susceptibility to phospholipase C (Fig. l), but this can be explained on the basis of less fatty acid partitioning into the membrane. The enhanced susceptibility of oleate-treated erythro-
Vol. 7
924
BIOCHEMICAL SOCIETY TRANSACTIONS
Time (rnin) Fig. 1 . Haemolysis of oleic acid-pretreated human erythrocytes by phospholipase C (B. cereus) Erythrocytes, pretreated with 0.5 mwoleic acid.(.) or with0.5m~-oleicacid+2m~-Ca’+ (a), were suspended in 0.9% NaCl buffered with SOmM-Tris/HCl, pH7.4, to 50% haematocrit. Phospholipase C was added at a concentration of 55 units of enzyme/ml of cell suspension. Incubations were at 20°C. Control cells treated with ethanol but no fatty acid (A) were incubated similarly. Haemolysis was assayed by release of haemoglobin to the supernatant after sedimentation of cells. Further details are in the text.
cytes to lysis by the enzyme could be reversed by further treating them with fat-free albumin. This work is supported by funds from The Medical Research Council and the Norwegian Research Council for Science and the Humanities. Allan, D. & Michell, R. H. (1976) Biochem. J. 156,225-232 Allan, D., Thomas, P. & Michell, R. H. (1978) Nature (London) 276,289-290 Bessis, M. (1972) Nouo. Rev. Fr. HPmatol. 12, 1-25 Cullis, P. R. & Hope, M. J. (1978) Nature (London) 271,672-674 Deuticke, B. (1968) Biochim. Biophys. Acta 163,494-500 Little, C., Aurebekk, B. & Otnaess, A,-B. (1975) FEBSLett. 52,175-179 Rumsby, M. G., Trotter, J., Allan, D. & Michell, R. H. (1977) Biochem. SOC.Trans. 5 , 126-128 Sheetz, M. P. & Singer, S. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,4457-4461
Mutability of the Kinetic Properties of the Acetylcholine Hydrolase Activity of Human Erythrocytes S. FAZLI MABOOD,* PETER F. J. NEWMAN? and IAN A. NIMMO* *Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, and ?Department of Haematology, The Royal Infirmary, Edinburgh EH3 9 YW, Scotland, U.K. Human erythrocyte acetylcholine hydrolase (EC 3.1 .I .7) is a membrane-bound enzyme whose kinetic properties seem to be influenced by the integrity and composition of the
1979
