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Membranes undergoing phase transitions are preferentially hydrolyzed by beta-bungarotoxin
231
Biochimica et Biophysica Acta, 469 (1977) 231--235 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 71307
MEMBRANES UNDERGOING PHASE TRANSITIONS ARE PREFERENTIALLY HYDROLYZED BY BETA-BUNGAROTOXIN
PETER N. STRONG and REGIS B. KELLY
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, Calif. 94143 (U.S.A.) (Received April 13th, 1977 )
Summary ~-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited. Certain hydrolytic enzymes (e.g. proteases, nucleases) have long been powerful tools for the structural biochemist. In comparison, the potential of phospholipases as probes of membrane structure is only just being realized. Studies with phospholipase A2 have provided some of the most convincing evidence for the existence of an asymmetric distribution of phospholipid across the erythrocyte membrane [1--5]. In addition op den Kamp et al. [6, 7] have shown that pancreatic phospholipase A2 preferentially hydrolyzes phosphatidylcholine liposomes at the transition temperature (Tin) of the phospholipid. They have explained the p h e n o m e n o n using the concept of lateral compressibility [8] suggesting that the pancreatic enzyme requires widely spaced lipid molecules at the interface, a situation that is maximally realized at the transition temperature. ~-Bungarotoxin, the presynaptic neurotoxin isolated from the venom of the krait Bungarus multicinctus [9,10] has recently been shown to possess phospholipase activity [11--13]. Although phospholipases do not normally modify transmitter release [13,14], electrophysiological and anatomical evidence indicates that ~-bungarotoxin affects transmitter release by selective hydrolysis of nerve terminal phospholipids [13,14]. We have studied the substrate requirements of ~-bungarotoxin to gain some insight into the possible structure of presynaptic plasma membranes. We have found that ~-bungarotoxin
232 hydrolyzes phosphatidylcholine bilayers optimally at their transition temperature between liquid and gel phases. We have also demonstrated that the presence of cholesterol inhibits phospholipid hydrolysis, a finding which can explain some of the selectivity of toxin action. Table I demonstrates that fl-bungarotoxin preferentially hydrolyzed dilauroyl, dimyristoyl and dipalmitoyl phosphatidylcholines at their respective liquid crystalline transition temperatures. The lack of hydrolysis above the transition temperature of dipalmitoyl phosphatidylcholin e is not due to thermal inactivation of the enzyme. In the presence of the detergent sodium deoxycholate, (4 raM), which serves to disperse the lipid substrate [ 11], dipalmitoyl phosphatidylcholine was completely hydrolyzed by ~-bungarotoxin in 15 min at 58°C. In the absence of deoxycholate, the specific activity of the phospholipase was very low (1.9 gequiv./min/mg protein), compared with regular assay conditions in the presence of detergent (250 gequiv./min/mg protein) (Strong and Kelly, in preparation), using dimyristoyl phosphatidylcholine as a substrate. For reasons not yet understood, dimyristoyl phosphatidylcholine liposomes were the best substrate for the toxin when absolute rates of phospholipid hydrolysis were compared. Table I also demonstrates that fl-bungarotoxin preferentially hydrolyzed a binary mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, not at their individual transition temperatures but at an TABLE I HYDROL YSIS OF PHOSPHATIDYLCHOLINE LIPOSOMES BY fl-BUNGAROTOXIN AT D I F F E R E N T T E MPERAT URES Dilauroyl, d i m y r i s t o y l , and d i p a i m i t o y l p h o s p h a t i d y l c h o l i n e w e r e o b t a i n e d f r o m Sigma Co. [1-14C]D i m y r i s t o y l p h o s p h a t i d y l c h o l i n e w a s o b t a i n e d f r o m Applied Science. [ 1-14C] D i p a l m i t o y l phosp l i a t i d y l c h o l i n e w a s o b t a i n e d f r o m N e w England Nuclear. Hand-shaken p h o s p h a t i d y l c h o l i n e liposomes (2/~mol p h o s p h o l i p i d / m l ) w e r e dispersed in 100 mM NaC1, 5 mM CaCI2, 0.05 mM EDTA and 100 mM Tris .HCI, pH 7.6. A 200/J1 aliquot of Hposomes (400 n m o l ) w a s i n c u b a t e d w i t h f l - b u n g a r o t o x i n (8 nmol) at t h e t e m p e r a t u r e s i n d i c a t e d for e i t h e r 15 min ( d i m y r i s t o y l phos pha t i dyl c hol i ne ), 30 rain ( d i p a i m i t o y l p h o s p h a t i d y l c h o l i n e , d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e / d i p a i m i t o y lp h o s p b a t i d y l c h o l i n e (1:1)) or 2 h (dilauroyl p h o s p h a t i d y l c h o l i n e ) . I n c u b a t i o n s w e r e t e r m i n a t e d with 1 ml 0.5 M EDTA. If EDTA was added t o t h e i n c u b a t i o n m i x t u r e b e f o r e t o x i n , n o h y d r o l y s i s occurred. Phospholipids w e r e e x t r a c t e d and c h r o m a t o g r a p h e d as p r e v i o u s l y d e s c r i b e d [ 1 3 ] . R e a c t i o n p r o d u c t s w e r e scraped o f f t h e t h i n - l a y e r c h r o m a t o g r a p h y p l a t e and e i t h e r a s s a y e d for lipid phos phorus or s u s p e n d e d in Aquasol (New England Nuclear) and c o u n t e d in a liquid scintillation s p e c t r o m e t e r (Beckman LS-233) w i t h an e f f i c i e n c y o f 86%. Both m e t h o d s gave e s s e n t i a l l y t h e s a m e results.
Phosphatidylcholine
T m (°C)
Incubation temperature
Percent
(°C)
hydrolysis*
0
0 15
95 ~-5
Dimyristoyl phosphatidylcholine
23
15 23 31
"~-5 60 8
Dipalmitoyl phosphatidylcholine
41
31 41 58
9 52 7
23 31 41
~5 45 10
Dilauroyl phosphatidylchoHne
D i m y r i s t o y l / d i p a i m i t o y lp h o s p h a t i d y l c h o l i n e (1 : 1)
phospholipid
*The percent p h o s p h o l i p i d hydrolysis is t h e average o f t h r e e d e t e r m i n a t i o n s . Triplicate assays w e r e reproducible within 5%.
233 intermediate temperature (31°C) which is predicted to be the approximate mid-point of the phase transition for an equimolar mixture [15,16]. There was correspondingly little hydrolysis of the mixture at either 23°C (Tin (dimyristoyl phosphatidylcholine)) or 41°C (T_m_(dipalmitoyl phosphatidylcholine)). We conclude that the unusual temperature requirements are a property of the substrate, and not the enzyme. The presence of cholesterol in phosphatidylcholine liposomes markedly reduced the rate of hydrolysis (Table II). At a cholesterohphospholipid mol ratio of 0.33, hydrolysis of dipalmitoyl phosphatidylcholine was negligible as all temperatures measured. Lower Concentrations of cholesterol reduced the rates of hydrolysis, although maximal hydrolysis still occurred at the transition temperature. The observation that cholesterol containing membranes are poor substrates for the toxin's phospholipase activity may allow us to reconcile earlier findings with the toxin. Mitochondria with a cholesterol:polar lipid mol ratio of 0.03 [17], are inactivated by a 5 min exposure at 25°C to 2 nM /3-bungarotoxin [18], sarcoplasmic reticulum membranes (tool ratio = 0.05) show reduced calcium accumulation after exposure to as little as 1 nM ~bungarotoxin for 20 min [19]; bacterial membranes which presumably lack cholesterol are good enzyme substrates [12] ; red blood cells with a high cholesterol content (mol ratio = 1.0) are not lysed by 4 h exposure to 5000 nM toxin at 37°C [ 14]. For comparison, neuromuscular transmission is blocked when the frog neuromuscular junction is exposed to 10 nM ~bungarotoxin, for 2 h at 25°C (Strong and Kelly, unpublished observations). Schwann cell membranes (cholesterol:polar lipid mol ratio = 0.95) have an unaltered morphology even in the presence of 1000--5000 nM/3-bungarotoxin for two hours; the nerve terminal membranes disintegrate under these conditions [14,20]. Pancreatic phospholipase A2 and ~-bungarotoxin are similar in that both prefer deoxycholate-solubilized substrates, but will nevertheless hydrolyze phospholipids in liposomes provided that the phospholipids are near their transition temperatures, and that cholesterol is absent [6,7]. Since the natural substrates of pancreatic phospholipase A2 are probably bile salt-solubilized phospholipids, the physiological significance of this anomalous temperature dependence in the case of the pancreatic enzyme is unclear. The natural substrate of ~-bungarotoxin is however almost certainly an intact membrane [13, T A B L E II EFFECT OF CHOLESTEROL ON fl-BUNGAROTOXIN HYDROLYSIS PHATIDYLCHOLINE LIPOSOMES
OF DIPALMITOYLPHOS-
T h e e x p e r i m e n t a l p r o t o c o l w a s i d e n t i c a l t o t h a t for T a b l e I e x c e p t t h a t c h o l e s t e r o l and d i p a l m i t o y l p h o s p h a t i d F l c h o l i n e w e r e d i s s o l v e d in C H C 1 3 : C H 3 O H ( 2 : 1 ) , e v a p o r a t e d t o d r y n e s s and t h e p r o c e s s r e p e a t e d t w i c e b e f o r e h a n d s h a k e n l i p o s o m e s w e r e p r e p a r e d b y t h e usual p r o c e d u r e .
M o l e ratio cholesterol: dipalmit oyl phosphatidylcholine
P e r c e n t dip.almitoyl p h o s p h a t i d y l c h o l i n e h y d r o l y s i s
31°C
4 1 ° C (T m )
58°C
(No cholesterol) 0.18 0.33
9 5 <5
52 10 <5
7 <5 <5
234 14]; it is reasonable to enquire therefore what might be the significance of the toxin's enzymatic preference for membranes undergoing a phase transition. Hydrolysis of the 2-fatty acyl side chain of the phospholipid requires that the enzyme penetrate at least partially into the hydrophobic region of the membrane. Phillips et al. have shown preferential insertion of the hydrophobic protein, casein, into monolayers at their transition temperature [21]. In addition, van der Bosch and McConnell [22] have suggested that the hydrophobic region of the membrane may be partially exposed at boundaries between solid and fluid phases. Exposure of hydrophobic regions at phase boundaries might facilitate phospholipase action by allowing access to fatty acid chains. While few of the phospholipids in natural membranes may be at their transition temperatures at physiological temperatures, integral membrane proteins can induce phase boundaries in fluid membranes by having an annulus of tightly bound phospholipid, the hydrocarbon chains of which are not free to move. In natural membranes such phase boundaries might be the site of fusion [22] or of phospholipase A2 action. We have not yet succeeded in discovering why the plasma membranes of motor nerve terminals, in contrast to the surrounding Schwann cel] merr brane and postsynaptic muscle membranes, are highly sensitive to ~-bungar¢ toxin. Possibilities include an unusually low level of cholesterol in nerve terminals, an unusually high concentration of phase boundaries or, as suggested previously [23], a protein recognition site to which the toxin binds. After binding, the toxin could hydrolyze phospholipids in the vicinity of this recognition site, perhaps at the phase boundary between the annular lipids of the recognition site and the surrounding lipid bilayer. The irreversibility of the toxin's action, saturation effects at high toxin concentrations [10,20] and the saturable binding of iodinated/3-bungarotoxin to synaptosomes [23] encourage us to favor the latter possibility. We thank Ignacio Caceres for excellent technical assistance and our colleagues for helpful discussions. This work was supported by a National Institutes of Health grant, NS-09878-2 (to RBK). PNS is a fellow of the Muscular Dystrophy Association of America. References 1 2 3 4
I b r a h i m , S.A. a n d T h o m p s o n , R.H.S. ( 1 9 6 5 ) Biochim. Biophys. A c t a 9 9 , 3 3 1 - - 3 4 1 Gul, S. a n d S m i t h , A.D. ( 1 9 7 2 ) Biochim. Biophys. A c t a 2 8 8 , 2 3 7 - - 2 4 0 L o w , M.G., L i m h r i c k , A.R. a n d Finean, J.B. ( 1 9 7 3 ) FEBS L e t t . 34, 1 - - 4 Colley, C.M., Zwaat, R . F . A . , R o e l o f s e n , B. a n d V a n Deenen, L.L.M. ( 1 9 7 3 ) Biochim. BiophYs. Acta 307, 74--82 5 Zwaal, R . F . A . , R o e l o f s e n , B. a n d Colley, C.M. ( 1 9 7 3 ) Biochim. Biophys. A c t a 3 0 0 , 1 5 9 - - 1 8 2 6 0 P d e n K a m p , J . A . F . , de Gier, J. a n d V a n Deenen, L.L.M. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 4 5 , 253--256 7 0 p den K a m p , J.A.F., Kauerz, M.Th. and van Deenen, L.L.M. (1975) Biochim. Biophys. Acta 406, 169--177 8 L i n d e n , C.D., Wright, K.L., McConnell, H,M. a n d F o x , C.F. ( 1 9 7 3 ) Proe, Natl. A c a d . Sci. U.S. 70, 2 2 7 1 - - 2 2 7 5 9 C h a n g , C.C., Chen, T.F. a n d Lee, C.Y. ( 1 9 7 3 ) J. P h a r m a c o l . Exp. Ther. 1 8 4 , 3 3 9 - - 3 4 5 10 Kelly, R.B. a n d B r o w n , III, F.R. ( 1 9 7 4 ) J. Neuxobiol. 5, 1 3 5 - - 1 5 0 11 Kelly, R.B., Oberg, S.G., S t r o n g , P.N. a n d Wagner, G.M. ( 1 9 7 5 ) Cold Spring H a r b o r Syrup. Q u a n t . Biol. 40, 1 1 7 - - 1 2 5 12 Wernicke, J , F . , V a n k e r , A.D. a n d H o w a r d , B.D° ( 1 9 7 5 ) J. N e u r o c h e m . 25, 4 8 3 - - 4 9 6 13 S t r o n g , P.N., G o e r k e , J., Oberg, S.G. a n d Kelly, R.B, ( 1 9 7 6 ) Proc. Natl. Acad, Sci. U.S. 73, 1 7 8 - - 1 8 2
235
14 Strong, P.N., Heuser. J.E. and Kelly, R.B. (1977) In: Cellular iY~urobiology, (Hall, Z., Kelly, R.B. and Fox, C.F., eds.), Alan R. Liss, New York, in press 15 Phillips, M.C., Ladbrooke, B.D. and Chapman, D. (1970) Biochim. Biophys. Acta 196, 35---44 16 Shimschick, E.J. and MeConnell, H.M. (1973) Biochemistry 12, 2351--2360 17 Parsons, D.F. and Yano, Y. (1967) Bioehim. Biophys. Acta 135, 362--364 18 Wagner, G.M., Mart, P,E. and Kelly, R.B. (1974) Biochem. Biophys. Res. Commun. 58,475--481 19 Lau, Y.H., Chiu, T.H., Caswell, A.H. and Potter, L.T. (1974) Bioehem. Biophys. Res. Commun. 61, 510--516 20 Abe, T., Li~abrick, A.R. and Mlledi, It. (1976) Proc. R. Soc. L o n d o n B. 194, 545---553 21 Phillips, M.C., Graham, D.E. and Hauser, H. (1975) Nature 254, 154--155 22 Van der Bosch, J, and MeConnell, H.M. (1975) Proe. Natl. Aead. Sci. U.S. 72, 4409--4413 23 Oberg, S.G. and Kelly, R.B, (1976) Bioehim. Biophys. Aeta 433, 662~673
Biochimica et Biophysica Acta, 469 (1977) 231--235 © Elsevier/North-Holland Biomedical Press
BBA Report BBA 71307
MEMBRANES UNDERGOING PHASE TRANSITIONS ARE PREFERENTIALLY HYDROLYZED BY BETA-BUNGAROTOXIN
PETER N. STRONG and REGIS B. KELLY
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, Calif. 94143 (U.S.A.) (Received April 13th, 1977 )
Summary ~-Bungarotoxin preferentially hydrolyzes choline phospholipids (dilauroyl, dimyristoyl, dipalmitoyl) at their respective gel to liquid crystalline phase transition temperatures. Cholesterol markedly reduces the rate of phospholipid hydrolysis; at 0.33 mol percent cholesterol:phospholipid, the toxin's phospholipase activity is completely inhibited. Certain hydrolytic enzymes (e.g. proteases, nucleases) have long been powerful tools for the structural biochemist. In comparison, the potential of phospholipases as probes of membrane structure is only just being realized. Studies with phospholipase A2 have provided some of the most convincing evidence for the existence of an asymmetric distribution of phospholipid across the erythrocyte membrane [1--5]. In addition op den Kamp et al. [6, 7] have shown that pancreatic phospholipase A2 preferentially hydrolyzes phosphatidylcholine liposomes at the transition temperature (Tin) of the phospholipid. They have explained the p h e n o m e n o n using the concept of lateral compressibility [8] suggesting that the pancreatic enzyme requires widely spaced lipid molecules at the interface, a situation that is maximally realized at the transition temperature. ~-Bungarotoxin, the presynaptic neurotoxin isolated from the venom of the krait Bungarus multicinctus [9,10] has recently been shown to possess phospholipase activity [11--13]. Although phospholipases do not normally modify transmitter release [13,14], electrophysiological and anatomical evidence indicates that ~-bungarotoxin affects transmitter release by selective hydrolysis of nerve terminal phospholipids [13,14]. We have studied the substrate requirements of ~-bungarotoxin to gain some insight into the possible structure of presynaptic plasma membranes. We have found that ~-bungarotoxin
232 hydrolyzes phosphatidylcholine bilayers optimally at their transition temperature between liquid and gel phases. We have also demonstrated that the presence of cholesterol inhibits phospholipid hydrolysis, a finding which can explain some of the selectivity of toxin action. Table I demonstrates that fl-bungarotoxin preferentially hydrolyzed dilauroyl, dimyristoyl and dipalmitoyl phosphatidylcholines at their respective liquid crystalline transition temperatures. The lack of hydrolysis above the transition temperature of dipalmitoyl phosphatidylcholin e is not due to thermal inactivation of the enzyme. In the presence of the detergent sodium deoxycholate, (4 raM), which serves to disperse the lipid substrate [ 11], dipalmitoyl phosphatidylcholine was completely hydrolyzed by ~-bungarotoxin in 15 min at 58°C. In the absence of deoxycholate, the specific activity of the phospholipase was very low (1.9 gequiv./min/mg protein), compared with regular assay conditions in the presence of detergent (250 gequiv./min/mg protein) (Strong and Kelly, in preparation), using dimyristoyl phosphatidylcholine as a substrate. For reasons not yet understood, dimyristoyl phosphatidylcholine liposomes were the best substrate for the toxin when absolute rates of phospholipid hydrolysis were compared. Table I also demonstrates that fl-bungarotoxin preferentially hydrolyzed a binary mixture of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, not at their individual transition temperatures but at an TABLE I HYDROL YSIS OF PHOSPHATIDYLCHOLINE LIPOSOMES BY fl-BUNGAROTOXIN AT D I F F E R E N T T E MPERAT URES Dilauroyl, d i m y r i s t o y l , and d i p a i m i t o y l p h o s p h a t i d y l c h o l i n e w e r e o b t a i n e d f r o m Sigma Co. [1-14C]D i m y r i s t o y l p h o s p h a t i d y l c h o l i n e w a s o b t a i n e d f r o m Applied Science. [ 1-14C] D i p a l m i t o y l phosp l i a t i d y l c h o l i n e w a s o b t a i n e d f r o m N e w England Nuclear. Hand-shaken p h o s p h a t i d y l c h o l i n e liposomes (2/~mol p h o s p h o l i p i d / m l ) w e r e dispersed in 100 mM NaC1, 5 mM CaCI2, 0.05 mM EDTA and 100 mM Tris .HCI, pH 7.6. A 200/J1 aliquot of Hposomes (400 n m o l ) w a s i n c u b a t e d w i t h f l - b u n g a r o t o x i n (8 nmol) at t h e t e m p e r a t u r e s i n d i c a t e d for e i t h e r 15 min ( d i m y r i s t o y l phos pha t i dyl c hol i ne ), 30 rain ( d i p a i m i t o y l p h o s p h a t i d y l c h o l i n e , d i m y r i s t o y l p h o s p h a t i d y l c h o l i n e / d i p a i m i t o y lp h o s p b a t i d y l c h o l i n e (1:1)) or 2 h (dilauroyl p h o s p h a t i d y l c h o l i n e ) . I n c u b a t i o n s w e r e t e r m i n a t e d with 1 ml 0.5 M EDTA. If EDTA was added t o t h e i n c u b a t i o n m i x t u r e b e f o r e t o x i n , n o h y d r o l y s i s occurred. Phospholipids w e r e e x t r a c t e d and c h r o m a t o g r a p h e d as p r e v i o u s l y d e s c r i b e d [ 1 3 ] . R e a c t i o n p r o d u c t s w e r e scraped o f f t h e t h i n - l a y e r c h r o m a t o g r a p h y p l a t e and e i t h e r a s s a y e d for lipid phos phorus or s u s p e n d e d in Aquasol (New England Nuclear) and c o u n t e d in a liquid scintillation s p e c t r o m e t e r (Beckman LS-233) w i t h an e f f i c i e n c y o f 86%. Both m e t h o d s gave e s s e n t i a l l y t h e s a m e results.
Phosphatidylcholine
T m (°C)
Incubation temperature
Percent
(°C)
hydrolysis*
0
0 15
95 ~-5
Dimyristoyl phosphatidylcholine
23
15 23 31
"~-5 60 8
Dipalmitoyl phosphatidylcholine
41
31 41 58
9 52 7
23 31 41
~5 45 10
Dilauroyl phosphatidylchoHne
D i m y r i s t o y l / d i p a i m i t o y lp h o s p h a t i d y l c h o l i n e (1 : 1)
phospholipid
*The percent p h o s p h o l i p i d hydrolysis is t h e average o f t h r e e d e t e r m i n a t i o n s . Triplicate assays w e r e reproducible within 5%.
233 intermediate temperature (31°C) which is predicted to be the approximate mid-point of the phase transition for an equimolar mixture [15,16]. There was correspondingly little hydrolysis of the mixture at either 23°C (Tin (dimyristoyl phosphatidylcholine)) or 41°C (T_m_(dipalmitoyl phosphatidylcholine)). We conclude that the unusual temperature requirements are a property of the substrate, and not the enzyme. The presence of cholesterol in phosphatidylcholine liposomes markedly reduced the rate of hydrolysis (Table II). At a cholesterohphospholipid mol ratio of 0.33, hydrolysis of dipalmitoyl phosphatidylcholine was negligible as all temperatures measured. Lower Concentrations of cholesterol reduced the rates of hydrolysis, although maximal hydrolysis still occurred at the transition temperature. The observation that cholesterol containing membranes are poor substrates for the toxin's phospholipase activity may allow us to reconcile earlier findings with the toxin. Mitochondria with a cholesterol:polar lipid mol ratio of 0.03 [17], are inactivated by a 5 min exposure at 25°C to 2 nM /3-bungarotoxin [18], sarcoplasmic reticulum membranes (tool ratio = 0.05) show reduced calcium accumulation after exposure to as little as 1 nM ~bungarotoxin for 20 min [19]; bacterial membranes which presumably lack cholesterol are good enzyme substrates [12] ; red blood cells with a high cholesterol content (mol ratio = 1.0) are not lysed by 4 h exposure to 5000 nM toxin at 37°C [ 14]. For comparison, neuromuscular transmission is blocked when the frog neuromuscular junction is exposed to 10 nM ~bungarotoxin, for 2 h at 25°C (Strong and Kelly, unpublished observations). Schwann cell membranes (cholesterol:polar lipid mol ratio = 0.95) have an unaltered morphology even in the presence of 1000--5000 nM/3-bungarotoxin for two hours; the nerve terminal membranes disintegrate under these conditions [14,20]. Pancreatic phospholipase A2 and ~-bungarotoxin are similar in that both prefer deoxycholate-solubilized substrates, but will nevertheless hydrolyze phospholipids in liposomes provided that the phospholipids are near their transition temperatures, and that cholesterol is absent [6,7]. Since the natural substrates of pancreatic phospholipase A2 are probably bile salt-solubilized phospholipids, the physiological significance of this anomalous temperature dependence in the case of the pancreatic enzyme is unclear. The natural substrate of ~-bungarotoxin is however almost certainly an intact membrane [13, T A B L E II EFFECT OF CHOLESTEROL ON fl-BUNGAROTOXIN HYDROLYSIS PHATIDYLCHOLINE LIPOSOMES
OF DIPALMITOYLPHOS-
T h e e x p e r i m e n t a l p r o t o c o l w a s i d e n t i c a l t o t h a t for T a b l e I e x c e p t t h a t c h o l e s t e r o l and d i p a l m i t o y l p h o s p h a t i d F l c h o l i n e w e r e d i s s o l v e d in C H C 1 3 : C H 3 O H ( 2 : 1 ) , e v a p o r a t e d t o d r y n e s s and t h e p r o c e s s r e p e a t e d t w i c e b e f o r e h a n d s h a k e n l i p o s o m e s w e r e p r e p a r e d b y t h e usual p r o c e d u r e .
M o l e ratio cholesterol: dipalmit oyl phosphatidylcholine
P e r c e n t dip.almitoyl p h o s p h a t i d y l c h o l i n e h y d r o l y s i s
31°C
4 1 ° C (T m )
58°C
(No cholesterol) 0.18 0.33
9 5 <5
52 10 <5
7 <5 <5
234 14]; it is reasonable to enquire therefore what might be the significance of the toxin's enzymatic preference for membranes undergoing a phase transition. Hydrolysis of the 2-fatty acyl side chain of the phospholipid requires that the enzyme penetrate at least partially into the hydrophobic region of the membrane. Phillips et al. have shown preferential insertion of the hydrophobic protein, casein, into monolayers at their transition temperature [21]. In addition, van der Bosch and McConnell [22] have suggested that the hydrophobic region of the membrane may be partially exposed at boundaries between solid and fluid phases. Exposure of hydrophobic regions at phase boundaries might facilitate phospholipase action by allowing access to fatty acid chains. While few of the phospholipids in natural membranes may be at their transition temperatures at physiological temperatures, integral membrane proteins can induce phase boundaries in fluid membranes by having an annulus of tightly bound phospholipid, the hydrocarbon chains of which are not free to move. In natural membranes such phase boundaries might be the site of fusion [22] or of phospholipase A2 action. We have not yet succeeded in discovering why the plasma membranes of motor nerve terminals, in contrast to the surrounding Schwann cel] merr brane and postsynaptic muscle membranes, are highly sensitive to ~-bungar¢ toxin. Possibilities include an unusually low level of cholesterol in nerve terminals, an unusually high concentration of phase boundaries or, as suggested previously [23], a protein recognition site to which the toxin binds. After binding, the toxin could hydrolyze phospholipids in the vicinity of this recognition site, perhaps at the phase boundary between the annular lipids of the recognition site and the surrounding lipid bilayer. The irreversibility of the toxin's action, saturation effects at high toxin concentrations [10,20] and the saturable binding of iodinated/3-bungarotoxin to synaptosomes [23] encourage us to favor the latter possibility. We thank Ignacio Caceres for excellent technical assistance and our colleagues for helpful discussions. This work was supported by a National Institutes of Health grant, NS-09878-2 (to RBK). PNS is a fellow of the Muscular Dystrophy Association of America. References 1 2 3 4
I b r a h i m , S.A. a n d T h o m p s o n , R.H.S. ( 1 9 6 5 ) Biochim. Biophys. A c t a 9 9 , 3 3 1 - - 3 4 1 Gul, S. a n d S m i t h , A.D. ( 1 9 7 2 ) Biochim. Biophys. A c t a 2 8 8 , 2 3 7 - - 2 4 0 L o w , M.G., L i m h r i c k , A.R. a n d Finean, J.B. ( 1 9 7 3 ) FEBS L e t t . 34, 1 - - 4 Colley, C.M., Zwaat, R . F . A . , R o e l o f s e n , B. a n d V a n Deenen, L.L.M. ( 1 9 7 3 ) Biochim. BiophYs. Acta 307, 74--82 5 Zwaal, R . F . A . , R o e l o f s e n , B. a n d Colley, C.M. ( 1 9 7 3 ) Biochim. Biophys. A c t a 3 0 0 , 1 5 9 - - 1 8 2 6 0 P d e n K a m p , J . A . F . , de Gier, J. a n d V a n Deenen, L.L.M. ( 1 9 7 4 ) Biochim. Biophys. A c t a 3 4 5 , 253--256 7 0 p den K a m p , J.A.F., Kauerz, M.Th. and van Deenen, L.L.M. (1975) Biochim. Biophys. Acta 406, 169--177 8 L i n d e n , C.D., Wright, K.L., McConnell, H,M. a n d F o x , C.F. ( 1 9 7 3 ) Proe, Natl. A c a d . Sci. U.S. 70, 2 2 7 1 - - 2 2 7 5 9 C h a n g , C.C., Chen, T.F. a n d Lee, C.Y. ( 1 9 7 3 ) J. P h a r m a c o l . Exp. Ther. 1 8 4 , 3 3 9 - - 3 4 5 10 Kelly, R.B. a n d B r o w n , III, F.R. ( 1 9 7 4 ) J. Neuxobiol. 5, 1 3 5 - - 1 5 0 11 Kelly, R.B., Oberg, S.G., S t r o n g , P.N. a n d Wagner, G.M. ( 1 9 7 5 ) Cold Spring H a r b o r Syrup. Q u a n t . Biol. 40, 1 1 7 - - 1 2 5 12 Wernicke, J , F . , V a n k e r , A.D. a n d H o w a r d , B.D° ( 1 9 7 5 ) J. N e u r o c h e m . 25, 4 8 3 - - 4 9 6 13 S t r o n g , P.N., G o e r k e , J., Oberg, S.G. a n d Kelly, R.B, ( 1 9 7 6 ) Proc. Natl. Acad, Sci. U.S. 73, 1 7 8 - - 1 8 2
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