Vol. 182, No. 2, 1992 January 31, 1992
DIFFERENTIAL UNILAMELLAR
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 682-688
SUSCEPTIBILITY OF PHOSPHATIDYLCHOLINE SMALL VESICLES TO PHOSPHOLIPASES AZ, C AND D IN THE PRESENCE OF MEMBRANE ACTIVE PEPTIDES N.MADHUSUDHANA
RAO
Centre for Cellular and Molecular Biology Uppal road,Hyderabad, India 500 007 Received
November
21,
1991
Activities of phospholipases C and D along with A2 were followed on egg phosphatidylcholine small unilamellar vesicles in the presence of membrane active peptides melittin, gramicidin S and alamethicin. Decrease in the activity of phospholiapse C and D and enhancement of phospholipses A2 activity suggest that these enzymes are sensitive to alterations in the lipid packing in the membranes in the presence of these peptides. Phospholipase C and D,which have not been used to study peptide - membrane interactons, have potential use in 0 1992 studying membrane perturbations, since their activities are very sensitive to lipid packing. kademic
Press,
Inc.
Packing of lipids in the membrane is susceptible to physical and chemical perturbations (1,2). Altered lipid packing in a bilayer leads to nonbilayer phases in the membrane such as inverted hexagonal ( HII),cubic phases etc.( 3-5). In recent years, the information gathered with pure lipid systems suggested that lipid composition dependent polymorphic phase behavior of membranes has a crucial role to play in the cell metabolism (3). Studies on inverted hexagonal phases (HII) led to a cogent explanation for some cell biological phenomenon such as fusion, exoand endo-cytosis (1,7) , and inter / transbilayer movement of the lipids (8). 31P NMR (1) and small angle X-ray diffraction (9,lO) methods have been extensively used to monitor the lipid packing and in recent times fluorescent probes also have been used to monitor liquid crystalline ( Lo) to inverted hexagonal phase( HII) transitions (11,12 ). Sen er al (13) reported that phospholipase A2 activities enhance when there is a packing stressfor lipids in the membrane on the onset of bilayer to nonbilayer transitions. Enhancement of PLA2 activities occurs in situations where nonbilayer phases are induced by diacylglycerol,
membrane active peptides etc. (2,14). Since altered lipid packing changes the accessiblity of diferent lipid bonds to the bulk aqueous
Abbreviations DMSO, dimethyl sulfoxide; LUVET, large unilamellar vesicles made by extrusion technique; MLV, multilamellar vesicles; PC, phosphatidylcholine; PB, phosphatidylethanolamine; PLA2, phospholipase A2; PLC, phospholipase C; PLD, Phospholipase D; Ri, lipid to peptide ratio; SUV, small unilamellar vesicles. 0006-291X/92 Copyright All rights
$1.50
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
682
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
phase, the activities of phospholipase C and D are also expected to be dependent on the lipid packing. Susceptibility of egg phosphatidylcholine small unilamellar vesicles to phospholiapses A2,C and D in the presence of membrane active peptides was studied to know the dependence of activity on the lipid packing. The observations reported here suggest that following the activities of PLC and PLD is a very sensitive method of monitoring the peptide induced perturbations and, more importantly, in combination with PLA2 gives insight into the nature of lipid packing in the membrane.
Materials
and Methods
Materials: Dioleoyl phosphatidylcholine , dipahnitoyl phosphatidylcholine, phosphatidylcholine (PC) from egg yolk, Melittin, Gramicidin S, Alamethicin, Phospholipase C (Bacillus cereus, EC 3.1.4.3), bovine serum albumin and Tris were obtained from SigmaJJSA. Phospholipase A:! ( hog pancreas, EC 3.1.1.4) and phospholipase D (cabbage, EC 3.1.4.4) from Boehringer,W.Germany. All the three enzymes showed a single band on SDS-PAGE. All the other reagents were of analytical ade. L-3-phosphatidyl (N-methyl-t4C) choline,Z dipahnitoyl, L-3-phosphatidyl (N-methyl - 1PC ) choline,Zdioleyol and 1-palmitoyl-Z( 1-14C)palmitoyl phosphatidylcholine ,both 58 mCi/mmole, were obtained from Amersham, England. Egg PC was purified from the crude egg PC supplied by Sigma, by the method given by Bergelson (15) and found to be pure by TLC. Commercial melittin ( 91 % ) was purified by the method (16) and its purity was confiied by FPLC and the PLA2 contamination was not detected even at 2 mg/ml concentration and at five times the normal incubation time i.e., for 50 min. Concentrations of the peptide stocks were calculated from the amino acid composition ( LKB 4151 Alpha plus amino acid analyser) after complete hydrolysis of an aliquot of the sample.
Metha: Small unilamellar vesicles were prepared by drying the lipid film, under nitrogen or in a Savant speedvac, containing the radiolable (co.08 mole%, approximately giving 25,CO0 cpm/ assay) and swelling in 50 mM buffer (Tris.Cl,pH 7.4 for PLC, pH 8 for PLA2 and acetate buffer pH 5.4 for PLD). Hydrated lipid was sonicated to clarity with a microtip probe fitted to a Branson sonfier (B 30). Care was taken to avoid heating of the sample. Multi lamellar vesicles were prepared by the same procedure except the sonication step was avoided. Large unilamellar vesicles by extrusion technique were made using Lipex extruder with 0.1 pm polycarbonate filters (17). Lipid concentrations were calculated from the inorganic phosphate content in the vesicles.Phosphate was estimated after complete hydrolysis of the sample (18). Peptide probes were added to the liposomes while vortexing and preincubated before the addition of the enzyme. All the peptides stocks were made in DMSO except melittin, which was made in water.
Enzyme assays : Phospholipase C was assayed by addition of enzyme (0.15 units/assay) to SUV (2 mM) in 50 mM Tris .Cl (pH 7.2) containing 10 mM CaC12 (19). PLA2 assay was performed similar to PLC except that Tris.Cl buffer of pH 8 was used with 0.7 units of enzyme per assay . The product was extracted as given earlier (20). PLD assay was performed in 50 mM acetate buffer (pH 5.4) with 0.18 units /assay (21). Reaction was stopped by the addition of bovine serum albumin (0.75 %,W/V final )and perchloric acid (8%,V/V,final) and the product was extracted as given in PLC assay. All assays were performed in a shaking water bath at 37OC. Radioactivity was counted in a Packard Tricarb 1500 liquid scintillation analyser with Bray’s cocktail. Activities were expressed as percent hydrolysis i.e.,cpm released / total cpm incorporated. Total counts in each vesicle preparation was obtained to correct for variation in the extent of incorporation,if present. 683
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BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Results
. . . of DhQSPhPLUZaSes . in Studvof actwhes on eu PC vesicles
the
uresence of
Fig 1 presents the data on the percent activities compared to the control of PLA2,PLC and PLD on egg PC WV in the presence of melittin,gramicidin
S and alamethicin. In these radiometric
determination of the activities all the three peptides either enhance ( PLA2) or decrease (PLC and PLD) the activity of the phospholipases in a concentration dependent manner. PLA2 activity was enhanced 3-4 fold with all the peptides in a lipid to peptide
ratio (Ri) of 1000 -50, in case of
melittin and gramicidin S and 2000-200 in case of alamethicin. Alamethicin did not enhance the activity of PLA2 further at Ri c 200 and, in fact, the activity returned to the control value. PLC activity decreases to 15-50 % of the initial activity dependent on the peptide used, whereas the activity of PLD decreases to < 15 % of the control activity with all the three peptides. Thus PLD activity was more sensitive at high Ri values compared to PLC. Comparison of the effects of the three peptide with the activities of three phospholipases
suggests that melittin and alamethicin
were more effective than gramicidin S. Initially, linearity in the activities with respect to time and the protein was established with all the three phsopholipases. DMSO even at 2% (v/v) did not have any effect on the activity of phospholipases.Though the activities of phospholipases on pure liposomes in the absence of peptides showed some variation, the activity profiles in the presence of the peptides were identical among the replicates.This was confirmed four times with melittin.
4
c
3
3 2
y 0.4 F 5 2 O.O 0
2
lliz, 20
40
PEPTIDE,
F$J.4&&ies
60
60
FM
0
PEPTIDE,
20
40
PEPTIDE,
,uM
60
,uM
60
0
4
PEPTIDE,
6
12
,uM
of phospholipaseson egg PC SUV in the presence of melittin,gramicidin S and
Day-to-day variation was seenin control activity values,sothe activities with the peptideswere expressedtaking sameday control as one. Range of control activitiesin 10 min were as follows : PLA2,3-5% ( i.e.,6-10 nmoles ) ; PLC, 2834% ( i.e.,56-78 nmoles ); PLD, 3-5% ( 6-IOnmoles) of substrate hydrolysed.PLC ( A ) , PLD ( B ) and PIA2 activitieswere plotted againstmelittin ( 0 ),gramicidin D( 0 ) and alamethicin (A). PI& activity in the presenceof alamethicin was presentedin ( D ), sinceit was effective at even lower concentrations.Lipid peptide ratios (Ri) in theseexperiments were varied : melittin,1050-50; gramicidin D, 1000-50; alamethicin , 800-20 with PLC and PLD and 2000-200 with PLA2. 684
Vol.
182,
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C
O-6 5.0
5 0.4 F u ii oz o-o
0
0.4k
3.0
20
40
MELITTIN,
60
SC,
I.0 -0
,uM
20
40
MELITTIN,
60
60
..i 0
,uM
2
DPPC,
mole
3
%
F&& Activities of PLA2 and PLC on MLV, LUVET and WV in the presence of melittin. Activities of PLC ( A ) and PLAz ( B ) on SUV ( 0 ), MLV ( 0 ) and LUVET (a) were plotted against melittin concentration. C. Activities of PLA:! ( 0 ) and PLC ( 0 ) on egg PC WV with varying amounts of cold DPPC. SUV with 0.08 mole% DPPC was considered as control.
Order of addition of
enzyme,peptide and the vesicles did not have any effect on the
activity
profiles indicating that the effects observed were independent of order of addition.
Effect of melittin unilamellar
on the activities
of PLA2 and PLC on
multilamellar
and large
vesicles:
To establish that the effects of peptide observed on the activities of PLC and PLA2 are not exclusive to the unilamellar vesicles, PLC and PLA2 activities were also monitored on MLV and LUVET in the presence of melittin. Fig.2A shows the effect of
melittin on activity profiles of
PLC on MLV and LUVET along with SUV from fig. 1. The activity profiles of PLC on the three vesicle preparations were similar in the presence of melittin though the extent of hydrolysis in the absence of melittin ( control) was 18% less in LUVET
compared to MLV and SUV ( Fig 2A)
( refer fig. 1 legend). In all the vesicle preparations the activity profile of PLA2 was similar though the extent of
hydrolysis of the lipid was maximal in case of LUVET and LUVET
most sensitive preparation since at very low melittin
seem to be
concentration significant stimulation was
seen (Fig.2B). Hydrolysis of lipid by PLA2 in the absence of melittin was 1.4%, 3%, and 8.7% of the total lipid in LUVET, MLV and SUV respectively. dioleoyl phosphatidylcholine
Further, these effects were identical in
SUV as well. These results suggest that the activity profiles of
phospholipases in the presence of melittin are independent of the lamellarity, size and curvature of the vesicle. In our radiometric assays radioactive DPPC at c 0.08 mole % in egg PC was used. To eliminate the possibility that DPPC at such low concentrations is effecting the phospholipases activity (22), we prepared SUV with
increasing amounts of cold DPPC upto 3 mole% of PC in
addition to 0.08 mole % of radioactive DPPC and used these vesicles to study the activities of PLC and PLA2 in the absence of any peptide. Fig.2C demonstrates there was a decrease in the 685
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activities of both PLA2 and PLC by about 15% in varying amounts of cold DPPC compared to the control i.e., 0.08 mole% DPPC. This data suggests that the effects of the peptides on the vesicles are exclusively due to their presence in the membrane and not a measurement artifact.
Discussion Activities of PLC and PLD in the presence of melittin, gramicidin S and alamethicin, decrease in a concentration dependent manner in contrast to the enhancement of activity of PLA2 on egg PC SUV vesicles. Similarity of the activity profiles of PLA2,PLC and PLD on egg PC SUV in the presence of these peptides suggests that the perturbations
caused by these peptides in the
membrane were also similar in nature. 90% of the observed responses seen in the activity profiles occur in Ri of 1000 to 200.
Alamethicin effects the PLA2 activity at Ri =2000, which means
approximately 2-3 molecules of alamethicin were enough per vesicle to enhance the PLA2 activity (11). Alkaline phosphatase and lysozyme signals along with their mutants,which are known to be perturbatory in assays such as fusion and carboxyfluorescene release (23), showed similar activity profiles with these phospholipases. However , h receptor signal neither effected phospholipases activity nor showed perturbatory nature in other assays (data not reported). Thus monitoring the activities of phospholipases to study membrane perturbations is very sensitive compared to other method such as alteration of solute permeabilities,which occur at much lower Ri values (24). We also observed that the effects of peptides on the enzyme activities were not dependent on vesicle lamellarity. Except PLA2, other phospholipases were not used to characterize the perturbations caused in the membrane by the membrane active presence of peptides
peptides. The changes in activities in the
may not be attributed to charge differences in the peptides,
since it was
shown that activities of PLA2 and PLC do not show any dependence on the surface charge of the liposomes (25). In SUV the primary site of interaction of these peptides will be the outer leaflet of the membrane. Interaction of these peptides with the membranes has been studied by several techniques (24,26) and at lower Ri values these peptides were shown to induce HII phases in the membrane (2).
Since on La to HII phase transitions the interfacial curvature
tends to be
concave it is expected that the head groups would be less accessible compared to the acyl chains, hence correspondingly the activities of PLC and PLD are expected to decrease and PLA2 activity would be enhanced. Several reports have observed that on induction of nonbilayer phases either by nonbilayer preferring lipids, such as diacylglycerol(14),
phosphatidylethanolamine
(1) etc.
or by peptides such as gramicidin S/D (8,27) or signal peptides (28), ‘interfacial activation’ of
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1992
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PLA2 activity, ability to fuse and inter/trans bilayer movement of lipids have been observed. (2). In such situations it is expected that PLC and PLD would be ‘inter-facially inhibited’,since the accessibility of hydrolysable bonds by PLC and PLD would be less. Preliminary results on activities
of PLA2
and PLC on vesicles made from varying
phosphatidylethanolamine
amounts
mole % of
in PC suggest that these enzymes respond to the change in the lipid
packing during La to HII transitions. Phospholipases apparently are able to sense these alterations in the packing and thereby report these transitions. Monitoring activities of phsopholipases is simplecombined with lower lipid and peptide requirement makes them very useful to study the perturbatory nature of peptides. Enzymes by virtue being extremely sensitive and specific, several improvements can be affected in the detection of activity. Thus use of phospholipases not only offers a very sensitive method of monitoring peptide-membrane interactions
but also gives
insight into the nature of the perturbation.
References 1.
Cullis,P.R., de Kruijff,B., Hope,M., Verkleij, A.J., Nayar,R., Farren,S.B., Tilcock,C., Madden,T.D., and Bally,M.B. (1983) in Membrane fluidity in Biology ( Aloia,R.C.,ed.) vol. 1,pp 39-8 1, Academic Press.
2.
Tournois, H.. and de Kruijff,B.
3.
Seddon,J.M. (1990) Biochim.Biophys.Acta
4.
Gruner,S.M. (1989) J.Phys.Chem. 93,7562-7570.
5.
Siegel,D.P.(1986) Bi0phys.J. 49,1155-l 170.
6
Siegel,D.P.(1986) Bi0phys.J. 49,1171-1183.
7.
Burger,K.N.J., and Verkleij,A.J. (1990) Experientia 46, 631644 .
8.
Classen,J., Haest, C.W.M., Tournois, H., and 6604-6612.
9.
Luzatti,V. (1968) in Biological Membranes (Chapman,D.,ed) Press, New York.
(1991) Chem.Phys.Lipids 57,327-340. 1031, l-69.
Deuticke,B.(1987)
Biochemsitry
pp. 71-123, Academic
10.
Caffrey,M., Nagin,R.L., Hummel,B., and Zhang,J. (1990) Bi0phys.J. 58, 21- 29.
11.
Huang,C., and Mason,J.T. (1978) Proc.Natl.Acad.Sci. USA 75,
13.
Sen,A., Isac,T.V., and Hui, S-W (1991) Biochemistry 30,4516-4521.
14.
Ellens,H., Siegel,D.P., Alford,D.,Yeagle,P.L., Bentz,J. (1989) Biochemistry 28,3692-3703 .
15.
Bergelson,L.D. Amsterdam.
16.
King,T.P., Sobotka, A.K.,Kochoumian,L., Biophys.Biochem. 172,661-671.
(1980) Lipid Biochemical
687
Boni,L., preparations,
26,
308-310.
Lis,L.J.,
Quinn,P.J.,
Elsevier/North
and Lichtenstein,
L.M.
(1976)
and
Holland, Arch.
Vol.
182,
No.
BIOCHEMICAL
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AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
17.
Hope,M.J., Bally,M.B., 55-65.
Webb,G., and Cullis,P.R. (1985) Biochem.Biophys.Acta
18.
McC1areC.W.F. (197 1) Anal.Biochem. 39,527-530.
19.
Klug,E.L., and Kent,C. (1981) in Meth.Enzymol. 72, 351, Academic Press, New York.
20.
Argiolas,A., and Pisan0,J.J. (1985) J.Biol.Chem. 260,
21.
Kates,M., and Sastry,P.S. (1969) in Meth. Enzymol.14 203, Academic Press,New York.
22.
Gabriel,N.E., Agman,N.V., and Roberts,M.F. (1987) Biochemistry 26,7409-7418.
23.
Nagaraj,R., Joseph,M. and Reddy,G.L (1987) Biochem.Biophys.Acta
24.
Bemheimer,A.W., and Rudy,B. (1986) Biochim.Biophys.Acta
25.
Gruner,S.M., Cullis,P.R., Hope,M.J., and Tilcock,T.P.S Biophys.Chem. 14,21 l-238.
26.
Dempsey,C.E. (1990) Biochim.Biophys. Acta 1031, 143-161.
27.
Killian,J.A., 812,21-26
28.
Killian,J.A., de Jong,A.M.Ph., Bijvelt,J., Verkleij, A.J. and de Kruijff,B. EMBO J. 9,815-819.
Verkleij,A.J., Bijvelt,J., and de
688
812,
(J.M.Lowenstein, ed.) pp. 3471437-1444. (J.M.Lowenstein,ed.)
pp. 197-
903,465-472.
864,123-14124.
(1985) Ann. Rev. Biophys.
Kruijff,B.( 1985) Biochim.Biophys.Acta (1990) The
DIFFERENTIAL UNILAMELLAR
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 682-688
SUSCEPTIBILITY OF PHOSPHATIDYLCHOLINE SMALL VESICLES TO PHOSPHOLIPASES AZ, C AND D IN THE PRESENCE OF MEMBRANE ACTIVE PEPTIDES N.MADHUSUDHANA
RAO
Centre for Cellular and Molecular Biology Uppal road,Hyderabad, India 500 007 Received
November
21,
1991
Activities of phospholipases C and D along with A2 were followed on egg phosphatidylcholine small unilamellar vesicles in the presence of membrane active peptides melittin, gramicidin S and alamethicin. Decrease in the activity of phospholiapse C and D and enhancement of phospholipses A2 activity suggest that these enzymes are sensitive to alterations in the lipid packing in the membranes in the presence of these peptides. Phospholipase C and D,which have not been used to study peptide - membrane interactons, have potential use in 0 1992 studying membrane perturbations, since their activities are very sensitive to lipid packing. kademic
Press,
Inc.
Packing of lipids in the membrane is susceptible to physical and chemical perturbations (1,2). Altered lipid packing in a bilayer leads to nonbilayer phases in the membrane such as inverted hexagonal ( HII),cubic phases etc.( 3-5). In recent years, the information gathered with pure lipid systems suggested that lipid composition dependent polymorphic phase behavior of membranes has a crucial role to play in the cell metabolism (3). Studies on inverted hexagonal phases (HII) led to a cogent explanation for some cell biological phenomenon such as fusion, exoand endo-cytosis (1,7) , and inter / transbilayer movement of the lipids (8). 31P NMR (1) and small angle X-ray diffraction (9,lO) methods have been extensively used to monitor the lipid packing and in recent times fluorescent probes also have been used to monitor liquid crystalline ( Lo) to inverted hexagonal phase( HII) transitions (11,12 ). Sen er al (13) reported that phospholipase A2 activities enhance when there is a packing stressfor lipids in the membrane on the onset of bilayer to nonbilayer transitions. Enhancement of PLA2 activities occurs in situations where nonbilayer phases are induced by diacylglycerol,
membrane active peptides etc. (2,14). Since altered lipid packing changes the accessiblity of diferent lipid bonds to the bulk aqueous
Abbreviations DMSO, dimethyl sulfoxide; LUVET, large unilamellar vesicles made by extrusion technique; MLV, multilamellar vesicles; PC, phosphatidylcholine; PB, phosphatidylethanolamine; PLA2, phospholipase A2; PLC, phospholipase C; PLD, Phospholipase D; Ri, lipid to peptide ratio; SUV, small unilamellar vesicles. 0006-291X/92 Copyright All rights
$1.50
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
682
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
phase, the activities of phospholipase C and D are also expected to be dependent on the lipid packing. Susceptibility of egg phosphatidylcholine small unilamellar vesicles to phospholiapses A2,C and D in the presence of membrane active peptides was studied to know the dependence of activity on the lipid packing. The observations reported here suggest that following the activities of PLC and PLD is a very sensitive method of monitoring the peptide induced perturbations and, more importantly, in combination with PLA2 gives insight into the nature of lipid packing in the membrane.
Materials
and Methods
Materials: Dioleoyl phosphatidylcholine , dipahnitoyl phosphatidylcholine, phosphatidylcholine (PC) from egg yolk, Melittin, Gramicidin S, Alamethicin, Phospholipase C (Bacillus cereus, EC 3.1.4.3), bovine serum albumin and Tris were obtained from SigmaJJSA. Phospholipase A:! ( hog pancreas, EC 3.1.1.4) and phospholipase D (cabbage, EC 3.1.4.4) from Boehringer,W.Germany. All the three enzymes showed a single band on SDS-PAGE. All the other reagents were of analytical ade. L-3-phosphatidyl (N-methyl-t4C) choline,Z dipahnitoyl, L-3-phosphatidyl (N-methyl - 1PC ) choline,Zdioleyol and 1-palmitoyl-Z( 1-14C)palmitoyl phosphatidylcholine ,both 58 mCi/mmole, were obtained from Amersham, England. Egg PC was purified from the crude egg PC supplied by Sigma, by the method given by Bergelson (15) and found to be pure by TLC. Commercial melittin ( 91 % ) was purified by the method (16) and its purity was confiied by FPLC and the PLA2 contamination was not detected even at 2 mg/ml concentration and at five times the normal incubation time i.e., for 50 min. Concentrations of the peptide stocks were calculated from the amino acid composition ( LKB 4151 Alpha plus amino acid analyser) after complete hydrolysis of an aliquot of the sample.
Metha: Small unilamellar vesicles were prepared by drying the lipid film, under nitrogen or in a Savant speedvac, containing the radiolable (co.08 mole%, approximately giving 25,CO0 cpm/ assay) and swelling in 50 mM buffer (Tris.Cl,pH 7.4 for PLC, pH 8 for PLA2 and acetate buffer pH 5.4 for PLD). Hydrated lipid was sonicated to clarity with a microtip probe fitted to a Branson sonfier (B 30). Care was taken to avoid heating of the sample. Multi lamellar vesicles were prepared by the same procedure except the sonication step was avoided. Large unilamellar vesicles by extrusion technique were made using Lipex extruder with 0.1 pm polycarbonate filters (17). Lipid concentrations were calculated from the inorganic phosphate content in the vesicles.Phosphate was estimated after complete hydrolysis of the sample (18). Peptide probes were added to the liposomes while vortexing and preincubated before the addition of the enzyme. All the peptides stocks were made in DMSO except melittin, which was made in water.
Enzyme assays : Phospholipase C was assayed by addition of enzyme (0.15 units/assay) to SUV (2 mM) in 50 mM Tris .Cl (pH 7.2) containing 10 mM CaC12 (19). PLA2 assay was performed similar to PLC except that Tris.Cl buffer of pH 8 was used with 0.7 units of enzyme per assay . The product was extracted as given earlier (20). PLD assay was performed in 50 mM acetate buffer (pH 5.4) with 0.18 units /assay (21). Reaction was stopped by the addition of bovine serum albumin (0.75 %,W/V final )and perchloric acid (8%,V/V,final) and the product was extracted as given in PLC assay. All assays were performed in a shaking water bath at 37OC. Radioactivity was counted in a Packard Tricarb 1500 liquid scintillation analyser with Bray’s cocktail. Activities were expressed as percent hydrolysis i.e.,cpm released / total cpm incorporated. Total counts in each vesicle preparation was obtained to correct for variation in the extent of incorporation,if present. 683
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BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Results
. . . of DhQSPhPLUZaSes . in Studvof actwhes on eu PC vesicles
the
uresence of
Fig 1 presents the data on the percent activities compared to the control of PLA2,PLC and PLD on egg PC WV in the presence of melittin,gramicidin
S and alamethicin. In these radiometric
determination of the activities all the three peptides either enhance ( PLA2) or decrease (PLC and PLD) the activity of the phospholipases in a concentration dependent manner. PLA2 activity was enhanced 3-4 fold with all the peptides in a lipid to peptide
ratio (Ri) of 1000 -50, in case of
melittin and gramicidin S and 2000-200 in case of alamethicin. Alamethicin did not enhance the activity of PLA2 further at Ri c 200 and, in fact, the activity returned to the control value. PLC activity decreases to 15-50 % of the initial activity dependent on the peptide used, whereas the activity of PLD decreases to < 15 % of the control activity with all the three peptides. Thus PLD activity was more sensitive at high Ri values compared to PLC. Comparison of the effects of the three peptide with the activities of three phospholipases
suggests that melittin and alamethicin
were more effective than gramicidin S. Initially, linearity in the activities with respect to time and the protein was established with all the three phsopholipases. DMSO even at 2% (v/v) did not have any effect on the activity of phospholipases.Though the activities of phospholipases on pure liposomes in the absence of peptides showed some variation, the activity profiles in the presence of the peptides were identical among the replicates.This was confirmed four times with melittin.
4
c
3
3 2
y 0.4 F 5 2 O.O 0
2
lliz, 20
40
PEPTIDE,
F$J.4&&ies
60
60
FM
0
PEPTIDE,
20
40
PEPTIDE,
,uM
60
,uM
60
0
4
PEPTIDE,
6
12
,uM
of phospholipaseson egg PC SUV in the presence of melittin,gramicidin S and
Day-to-day variation was seenin control activity values,sothe activities with the peptideswere expressedtaking sameday control as one. Range of control activitiesin 10 min were as follows : PLA2,3-5% ( i.e.,6-10 nmoles ) ; PLC, 2834% ( i.e.,56-78 nmoles ); PLD, 3-5% ( 6-IOnmoles) of substrate hydrolysed.PLC ( A ) , PLD ( B ) and PIA2 activitieswere plotted againstmelittin ( 0 ),gramicidin D( 0 ) and alamethicin (A). PI& activity in the presenceof alamethicin was presentedin ( D ), sinceit was effective at even lower concentrations.Lipid peptide ratios (Ri) in theseexperiments were varied : melittin,1050-50; gramicidin D, 1000-50; alamethicin , 800-20 with PLC and PLD and 2000-200 with PLA2. 684
Vol.
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No.
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C
O-6 5.0
5 0.4 F u ii oz o-o
0
0.4k
3.0
20
40
MELITTIN,
60
SC,
I.0 -0
,uM
20
40
MELITTIN,
60
60
..i 0
,uM
2
DPPC,
mole
3
%
F&& Activities of PLA2 and PLC on MLV, LUVET and WV in the presence of melittin. Activities of PLC ( A ) and PLAz ( B ) on SUV ( 0 ), MLV ( 0 ) and LUVET (a) were plotted against melittin concentration. C. Activities of PLA:! ( 0 ) and PLC ( 0 ) on egg PC WV with varying amounts of cold DPPC. SUV with 0.08 mole% DPPC was considered as control.
Order of addition of
enzyme,peptide and the vesicles did not have any effect on the
activity
profiles indicating that the effects observed were independent of order of addition.
Effect of melittin unilamellar
on the activities
of PLA2 and PLC on
multilamellar
and large
vesicles:
To establish that the effects of peptide observed on the activities of PLC and PLA2 are not exclusive to the unilamellar vesicles, PLC and PLA2 activities were also monitored on MLV and LUVET in the presence of melittin. Fig.2A shows the effect of
melittin on activity profiles of
PLC on MLV and LUVET along with SUV from fig. 1. The activity profiles of PLC on the three vesicle preparations were similar in the presence of melittin though the extent of hydrolysis in the absence of melittin ( control) was 18% less in LUVET
compared to MLV and SUV ( Fig 2A)
( refer fig. 1 legend). In all the vesicle preparations the activity profile of PLA2 was similar though the extent of
hydrolysis of the lipid was maximal in case of LUVET and LUVET
most sensitive preparation since at very low melittin
seem to be
concentration significant stimulation was
seen (Fig.2B). Hydrolysis of lipid by PLA2 in the absence of melittin was 1.4%, 3%, and 8.7% of the total lipid in LUVET, MLV and SUV respectively. dioleoyl phosphatidylcholine
Further, these effects were identical in
SUV as well. These results suggest that the activity profiles of
phospholipases in the presence of melittin are independent of the lamellarity, size and curvature of the vesicle. In our radiometric assays radioactive DPPC at c 0.08 mole % in egg PC was used. To eliminate the possibility that DPPC at such low concentrations is effecting the phospholipases activity (22), we prepared SUV with
increasing amounts of cold DPPC upto 3 mole% of PC in
addition to 0.08 mole % of radioactive DPPC and used these vesicles to study the activities of PLC and PLA2 in the absence of any peptide. Fig.2C demonstrates there was a decrease in the 685
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activities of both PLA2 and PLC by about 15% in varying amounts of cold DPPC compared to the control i.e., 0.08 mole% DPPC. This data suggests that the effects of the peptides on the vesicles are exclusively due to their presence in the membrane and not a measurement artifact.
Discussion Activities of PLC and PLD in the presence of melittin, gramicidin S and alamethicin, decrease in a concentration dependent manner in contrast to the enhancement of activity of PLA2 on egg PC SUV vesicles. Similarity of the activity profiles of PLA2,PLC and PLD on egg PC SUV in the presence of these peptides suggests that the perturbations
caused by these peptides in the
membrane were also similar in nature. 90% of the observed responses seen in the activity profiles occur in Ri of 1000 to 200.
Alamethicin effects the PLA2 activity at Ri =2000, which means
approximately 2-3 molecules of alamethicin were enough per vesicle to enhance the PLA2 activity (11). Alkaline phosphatase and lysozyme signals along with their mutants,which are known to be perturbatory in assays such as fusion and carboxyfluorescene release (23), showed similar activity profiles with these phospholipases. However , h receptor signal neither effected phospholipases activity nor showed perturbatory nature in other assays (data not reported). Thus monitoring the activities of phospholipases to study membrane perturbations is very sensitive compared to other method such as alteration of solute permeabilities,which occur at much lower Ri values (24). We also observed that the effects of peptides on the enzyme activities were not dependent on vesicle lamellarity. Except PLA2, other phospholipases were not used to characterize the perturbations caused in the membrane by the membrane active presence of peptides
peptides. The changes in activities in the
may not be attributed to charge differences in the peptides,
since it was
shown that activities of PLA2 and PLC do not show any dependence on the surface charge of the liposomes (25). In SUV the primary site of interaction of these peptides will be the outer leaflet of the membrane. Interaction of these peptides with the membranes has been studied by several techniques (24,26) and at lower Ri values these peptides were shown to induce HII phases in the membrane (2).
Since on La to HII phase transitions the interfacial curvature
tends to be
concave it is expected that the head groups would be less accessible compared to the acyl chains, hence correspondingly the activities of PLC and PLD are expected to decrease and PLA2 activity would be enhanced. Several reports have observed that on induction of nonbilayer phases either by nonbilayer preferring lipids, such as diacylglycerol(14),
phosphatidylethanolamine
(1) etc.
or by peptides such as gramicidin S/D (8,27) or signal peptides (28), ‘interfacial activation’ of
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PLA2 activity, ability to fuse and inter/trans bilayer movement of lipids have been observed. (2). In such situations it is expected that PLC and PLD would be ‘inter-facially inhibited’,since the accessibility of hydrolysable bonds by PLC and PLD would be less. Preliminary results on activities
of PLA2
and PLC on vesicles made from varying
phosphatidylethanolamine
amounts
mole % of
in PC suggest that these enzymes respond to the change in the lipid
packing during La to HII transitions. Phospholipases apparently are able to sense these alterations in the packing and thereby report these transitions. Monitoring activities of phsopholipases is simplecombined with lower lipid and peptide requirement makes them very useful to study the perturbatory nature of peptides. Enzymes by virtue being extremely sensitive and specific, several improvements can be affected in the detection of activity. Thus use of phospholipases not only offers a very sensitive method of monitoring peptide-membrane interactions
but also gives
insight into the nature of the perturbation.
References 1.
Cullis,P.R., de Kruijff,B., Hope,M., Verkleij, A.J., Nayar,R., Farren,S.B., Tilcock,C., Madden,T.D., and Bally,M.B. (1983) in Membrane fluidity in Biology ( Aloia,R.C.,ed.) vol. 1,pp 39-8 1, Academic Press.
2.
Tournois, H.. and de Kruijff,B.
3.
Seddon,J.M. (1990) Biochim.Biophys.Acta
4.
Gruner,S.M. (1989) J.Phys.Chem. 93,7562-7570.
5.
Siegel,D.P.(1986) Bi0phys.J. 49,1155-l 170.
6
Siegel,D.P.(1986) Bi0phys.J. 49,1171-1183.
7.
Burger,K.N.J., and Verkleij,A.J. (1990) Experientia 46, 631644 .
8.
Classen,J., Haest, C.W.M., Tournois, H., and 6604-6612.
9.
Luzatti,V. (1968) in Biological Membranes (Chapman,D.,ed) Press, New York.
(1991) Chem.Phys.Lipids 57,327-340. 1031, l-69.
Deuticke,B.(1987)
Biochemsitry
pp. 71-123, Academic
10.
Caffrey,M., Nagin,R.L., Hummel,B., and Zhang,J. (1990) Bi0phys.J. 58, 21- 29.
11.
Huang,C., and Mason,J.T. (1978) Proc.Natl.Acad.Sci. USA 75,
13.
Sen,A., Isac,T.V., and Hui, S-W (1991) Biochemistry 30,4516-4521.
14.
Ellens,H., Siegel,D.P., Alford,D.,Yeagle,P.L., Bentz,J. (1989) Biochemistry 28,3692-3703 .
15.
Bergelson,L.D. Amsterdam.
16.
King,T.P., Sobotka, A.K.,Kochoumian,L., Biophys.Biochem. 172,661-671.
(1980) Lipid Biochemical
687
Boni,L., preparations,
26,
308-310.
Lis,L.J.,
Quinn,P.J.,
Elsevier/North
and Lichtenstein,
L.M.
(1976)
and
Holland, Arch.
Vol.
182,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
17.
Hope,M.J., Bally,M.B., 55-65.
Webb,G., and Cullis,P.R. (1985) Biochem.Biophys.Acta
18.
McC1areC.W.F. (197 1) Anal.Biochem. 39,527-530.
19.
Klug,E.L., and Kent,C. (1981) in Meth.Enzymol. 72, 351, Academic Press, New York.
20.
Argiolas,A., and Pisan0,J.J. (1985) J.Biol.Chem. 260,
21.
Kates,M., and Sastry,P.S. (1969) in Meth. Enzymol.14 203, Academic Press,New York.
22.
Gabriel,N.E., Agman,N.V., and Roberts,M.F. (1987) Biochemistry 26,7409-7418.
23.
Nagaraj,R., Joseph,M. and Reddy,G.L (1987) Biochem.Biophys.Acta
24.
Bemheimer,A.W., and Rudy,B. (1986) Biochim.Biophys.Acta
25.
Gruner,S.M., Cullis,P.R., Hope,M.J., and Tilcock,T.P.S Biophys.Chem. 14,21 l-238.
26.
Dempsey,C.E. (1990) Biochim.Biophys. Acta 1031, 143-161.
27.
Killian,J.A., 812,21-26
28.
Killian,J.A., de Jong,A.M.Ph., Bijvelt,J., Verkleij, A.J. and de Kruijff,B. EMBO J. 9,815-819.
Verkleij,A.J., Bijvelt,J., and de
688
812,
(J.M.Lowenstein, ed.) pp. 3471437-1444. (J.M.Lowenstein,ed.)
pp. 197-
903,465-472.
864,123-14124.
(1985) Ann. Rev. Biophys.
Kruijff,B.( 1985) Biochim.Biophys.Acta (1990) The
