~ TRANSBILA YER DISTRIBUTIONS OF RED CELL MEMBRANE PHOSPHOLIPIDS IN UNILAMELLAR VESICLES *

Transcription

1 Bichimica et Biphysica Acta, 769 (1984) Elsevier 419 BBA ~ TRANSBILA YER DISTRIBUTIONS OF RED CELL MEMBRANE PHOSPHOLIPIDS IN UNILAMELLAR VESICLES * AJA Y KUMAR and C.M. GUPTA.. Divisin f Biphysics, Central Drug Research Institute, Lucknw-22600I (India) (Received July lith, 1983) Key wrds: Lipid asymmetry; Phsphlipase A2; (Erythrcyte membrane) The phsphlipid rganizatin in unilamellar vesicles cmprised f varius purified phsphlipid cmpnents f mnkey erythrcyte membrane was ascertained using phsphlipase A2 and trinitrbenzenesulfnic acid as external membrane prbes. The vesicles were frmed by snicatin r detergent dialysis and fractinated by centrifugatin r gel permeatin chrmatgraphy. Experiments were dne t cnfirm that the phsphlipase A2 treatments did nt cause lysis r induce fusin f the vesicles. This enzyme hydrlysed nly the glycerphsphlipids in the uter surface f the vesicles. The amunts f the external phsphlipids determined by this enzymatic methd were verified using the chemical prbe, trinitrbenzenesulfnic acid. The chline-cntaining phsphlipids and phsphatidylethanlamine lcalized randmly in the tw surfaces f snicated vesicles (uter diameter, abut 30 nm), whereas phsphatidylserine preferentially distributed in the inner mnlayer. This phsphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (uter diameter, abut 45 nm). Furthermre, inclusin f chlesterl in bth the types f vesicles resulted in mre randm glycerphsphlipid distributins acrss the plane f vesicles bilayer, presumably due t the chlesterl-induced increases in the size f vesicles. These results demnstrate that the transbilayer distributin f erythrcyte membrane phsphlipids in unilamellar vesicles are cntrlled mainly by the surface curvature rather than by interlipid interactins, and therefre suggest that phsphlipid-phsphlipid and phsphlipid-chlesterl interactins shuld nt play any significant rle in determining the membrane phsphlipid asymmetry in red cells. It is prpsed that this asymmetry primarily riginates frm differential bindings f phsphlipids with membrane prteins in the tw leaflets f the membrane bilayer. Intrductin Varius membrane cmpnents are asymmetrically lcalized in tw halves f membrane bilayer [1,2]. Generally, the prteins are distributed with an abslute asymmetry while the varius classes f.4 Cmmunicatin N frm Central Drug Research Institute, Lucknw, India... T whm all crrespndence shuld be addressed. Abbreviatins: PC, phsphatidylchline; PE, phsphatidylethanlamine; SM, sphingmyelin; phsphatidylserine; PI, phsphatidylinsitl; TNBS, trinitrbenzenesulfnic acid. phsphlipids are present in bth mnlayers, albeit in unequal amunts. In erythrcyte membrane, phsphatidylchline (PC) and sphingmyelin (SM) are lcalized mainly in the uter mnlayer whereas phsphatidylethanlamine (PE) and phsphatidylserine (PS) are present almst exclusively in the inner mnlayer [3]. Several investigatrs have attempted t understand the intermlecular interactins that might induce asymmetric distributin f phsphlipids acrss the plane f red cell membrane bilayer by studying binary mixtures f phsphlipids in unilamellar vesicles [4-9]. It has been shwn that PE /84/$03.00 c[j1984 Elsevier Science Publishers B.V.

2 420 in small unilamellar vesicles cnsisting f PC and PE (> 10 ml%) distributes preferentially in the inner mnlayer [4,5] while SM in PC/SM vesicles prefers the uter mnlayer [6]. Furthermre, PS in small vesicles prefers the inner mnlayer [6], at least at lw PS cncentratins [7]. T evaluate the significance f the abve findings in terms f the factrs that induce membrane phsphlipid asymmetry in red cells, we have studied the trans bilayer distributins f mnkey erythrcyte membrane phsphlipids in unilamellar vesicles. The vesicles were prepared in the absence as well as in the presence f 40 weight % chlesterl frm the phsphlipid mixture cnsisting f PC, SM, PE and PS in a rati similar t that bserved in erythrcytes, and were fractinated by centrifugatin r gel permeatin chrmatgraphy. Phsphlipase A2 and trinitrbenzenesulfnic acid (TNBS) were used as external membrane prbes t study the phsphlipid rganizatin in vesicles bilayer. The experiments were carefully cntrlled t assure that the reagents did nt lyse r permeate the vesicles. The results f these studies shw that the transbilayer distributins f erythrcyte membrane phsphlipids in unilamellar vesicles are determined mainly by the packing cnstraints impsed by the surface curvature rather than by sme specific interlipid interactins. Materials and Methds 6-Carbxyflurescein and TNBS were frm Eastman Kdak Cmpany and Sigma Chemical Cmpany, respectively. Phsphlipase A2 was purified frm Naja naja snake venm (Haeffkine Institute, Bmbay) by the methd f Blecher [10], t a prtein cncentratin f apprx. 1 mg/ml. [3H]Chlesterl and [3H]inulin were purchased frm the Radichemical Center, Amersham, U.K., and New England Nuclear, respectively. Egg [methyl-14c]pc (30 ILCi/lLml) was prepared as described by Gupta and Bali [11]. Sephadex LH-20 ( ILm beads) and Sephadex G-50 (20-80 ILm beads) were btained frm Pharmacia Fine Chemicals. Bi-Gel A-50m was frm Bi-Rad Labratries. Pre-cated silica gel 60F-254 TLC plates (20 X 20cm, 0.2 mm thickness) were bught frm E. Merck. Silica gel ( mesh, activity 2-3) was purchased frm Sisc Research Labratries, Bmbay, India. The assay f radiactive istpes was carried ut in a Packard tri-carb 3330 liquid scintillatin spectrmeter with 2,5-diphenylxazle (4.0 g), 1.4- bis[2-(4-methyl-5-phenylxazlyl)]benzene (0.2 g), 2-methxyethanl (500 ml) and tluene (500 ml) as the scintillatr. Islatin and purificatin f phsphlipids frm mnkey erythrcyte membrane Lipids frm erythrcytes f freshly drawn rhesus mnkey bld were islated by the prcedure f Rse and Oklander [12]. Individual phsphlipid cmpnents frm the mixture were separated by chrmatgraphy ver silica gel (Table I) and Sephadex LH-20. The Sephadex LH-20 chrmatgraphy was perfrmed as described earlier [13]. Althugh chrmatgraphy ver silica gel clumn separated mst f the phsphlipid cmpnents, further chrmatgraphy f each phsphlipid cmpnent n Sephadex LH-20 was necessary t separate phsphatidylinsitl (PI) frm traces f PE, PS frm the presumed glyclipid cmpnent, and traces f clred impurities frm PE. Pure PC, PE, PS and PI were btained by this prcedure. Hwever, SM culd nt be made free frm PC by this methd. Therefre, the impure SM was first treated in diethyl ether with methanlic slutin f tetra-n-butylammnium hydrxide and then, after remval f the slvents, the mixture was chrmatgraphed n Sephadex LH-20. This led t the islatin f pure SM in reasnable amunts. Purity f phsphlipids was examined by TLC n silica gel G-60 plates using chlrfrm/ methanl/water (65: 25 : 4, viv) as the develping slvent system. The spts were identified after staining the plate with idine vapr fllwed by mlybdenum-blue spray [14]. All the samples exhibited single spts. Each phsphlipid cmpnent was characterized by its c-chrmatgraphy with the analytical samples, prepared as reprted earlier [15]. These were stred as slutins in chlrfrm/methanl (1: 1, v/v) at -20 C under N2. Separatin f phsphlipids by TLC Separatin f varius phsphlipids was dne by tw-dimensinal TLC n E. Merck silica gel TLC plates as described by Pllet et al. [16]. Spts

3 421 TABLE I SEPARATION OF MONKEY ERYTHROCYTE MEMBRANE PHOSPHOLIPIDS ON SILICA GEL COLUMN The clumn (0.6x4O em, 5.0 g silica gel) was washed with 100 ml f chlrfrm (free f ethanl) prir t applying the sample. Abut 250 mg f the crude mixture was applied in a ttal vlume f abut 2.0 ml f chlrfrm. The flw rate was mijmin. 5 ml fractins were cllected. The fractins were analysed by TLC. Slvent Slvent cmpsitin Vlume f N. rati (v Iv) eluate Chlrfrm Methanl (ml) Lipid eluted Neutral lipids Clred impurities PE PI + traces f PE Traces f sme unknwn lipid PS Sme glyclipid + traces f PS PC SM + traces f PC N ther phsphlipid fr different phsphlipids were identified after staining the plate with idine vapr fllwed by ninhydrin spray. These were remved and eluted with a mixture f methanl and chlrfrm (1 : 1, viv) fr several times. Ttal phsphrus present in each spt was determined as described by Ames and Dubin [17]. The recveries f varius phsphlipids frm silicagel were ver 95%. Preparatin f uni/amellar vesicles by snicatin A slutin f PC, PE, SM and PS in the absence as well as in the presence f 40 weight % chlesterl in chlrfrm/methanl (1: 1, viv) mixture was evaprated in a glass tube under a slw jet f N2, resulting in the frmatin f a thin lipid film n the wall f the tube. Final traces f slvents were remved by leaving the tube in vacu fr 3-4 h. The lipid mixture was dispersed in Tris-buffered r phsphate-buffered saline (10 mm in 0.9% NaCl, ph 7.4) s as t achieve a cncentratin f abut /lml lipid P/ml buffer. It was vrtexed fr 5-10 min at rm temperature. The lipid dispersin s btained was carefully transferred t a cuvette and snicated at ODC (r ~ 20DC) under N2 using a prbe-type snicatr (MSE 150 W, 20 KHz) t give an ptically clear suspensin (30-45 min). The snicated preparatins were centrifuged at X g (Ti-50 fixed angle rtr) fr 60 min at lodct affect the remval f titanium particles as well as prly dispersed lipids. Only the vesicles fund in the tp tw-thirds f the supernatant were used. Preparatin f uni/amellar vesiclesby detergent dialysis The dried phsphlipid mixture was dispersed in Tris-buffered r phsphate-buffered saline (10 mm in 0.9% NaCl, ph 7.4) cntaining 2% chlic acid and 0.02% NaN3 (wiv) s as t achieve the chlate t phsphlipid mlar rati f abut tw. It was vrtexed and then dialysed against 10 mm Tris r phsphate (ph 7.4) cntaining 0.9% NaCI and 0.02% NaN3 (4 X 500 ml) at 20DC.After dialysis, the vesicles were fractinated by centrifugatin as described abve. The amunt f chlate that culd nt be remved by dialysis was determined by including trace amunts f [3H]chlic acid in the lipid/detergent mixture. The mlar rati f phsphlipid t chlate in vesicles was apprx Determinatin f vesicle size distributin Hmgeneity f the vesicle preparatins was analysed by clumn chrmatgraphy n Bi-Gel A-50m at 20DC. A dwnward flwing clumn (1 X 50 em) f Bi-Gel A-50m maintained at cnstant hydrstatic pressure was equilibrated with 10 mm Tris-HCI buffer (ph 7.4). A measured aliqut

4 422 (0.5 ml) f the vesicle preparatin was applied t the clumn and eluted with the same buffer at 8 ml/h. Fractins were analysed by measuring ttal phsphrus. Determinatin f lipid cmpsitins f vesicle Lipids frm vesicles were extracted by Bligh- Dyer extractin prcedure [18].After remval f slvents frm the extracts, the dried lipid mixture was disslved (apprx. 4 ILglipid P/ILI) in chlrfrm/methanl (1 : 1, viv) mixture. This slutin was applied t E. Merck silica gel TLC plates (25-50 ILl/plate) and different phsphlipid species were separated as described abve. The percentage incrpratin f chlesterl in the bilayerswas ascertainedby includingabut 5 ILCif eh]chlesterl in the lipid mixture and then determining the rati f lipid P t 3H in vesicles. At least 90% f the added amunt f chlesterl was fund t be incrprated in phsphlipid bilayers. Preparatin f vesicles cntaining trapped f3hjinulin (r carbxyflurescein) Unilamellar vesicles cntaining trapped [3H]inulin r carbxyflurescein were prepared by dispersing the lipid mixture in buffer cntaining eh]inulin (apprx. 5 ILCi/ml) r carbxyflurescein (0.2 M). Free and trapped [3H]inulin (r carbxyflurescein) were separated by gel filtratin n Bi-Gel A-50m (1 X 50 cm clumn). The fractinated vesicles were cncentrated t abut 1 ml in an Amicn Centrifl CF-25 cne. Hydrlysis f vesicle phsphlipids by phsphlipase A] Vesicles (2-4ILmllipid P) were incubated with 20 ILgf phsphlipase A2 in Tris-buffered saline (10 mm in 0.9% NaCl cntaining 10 mm CaCI2. 2H20, ph 8.5) in a ttal vlume f 2.0 ml fr 2-3 h at C. The reactins were terminated by adding 50 mm EDT A (1.0 rni). The lipids were extracted and the intact and hydrlyzed phsphlipids were separated by tw-dimensinal TLC. Kinetics f hydrlysis f vesicle PC by phsphlipase A] Fr studying the kinetics f hydrlysis f 'vesicle PC, unilamellar vesicles were prepared after including trace amunts f egg [14C]PC (abut 1 ILCi) in the lipid mixture. The vesicle preparatin was divided in tw equal prtins. After adding Tritn X-100 (0.5%) r bvine serum albumin (9.2.1O-5M) t ne f the prtins, bth the prtins were incubated with phsphlipase A 2 essentially under the cnditins as described abve. Aliquts remved at different time intervals were treated with equal vlumes f 50 mm EDT A. Lipids were extracted. The extracts, after cncentrating under N2, were applied t silica gel G-60 TLC plates that were develped in chlrfrm/methanl/water (65: 25 : 4, viv). PC and lyspc were visualized by autradigraphy. The spts crrespnding t PC and lyspc were remved and assayed fr radiactivity. Labeling f vesicles with TN BS Unilamellar vesicles were prepared frm the lipid mixture in phsphate-buffered saline (10 mm in 0.9% NaCI, ph 7.4) by snicatin r detergent dialysis as described abve. The ph f the vesicle preparatin was raised t 8.2. T a part f this preparatin (2-4 ILmllipid P) was added a slutin f TNBS in aqueus NaHC03 (ph 8.2) s as t give a final cncentratin f 20 mm f the reagent in a ttal vlume f 2.0 ml. The mixture was incubated at 20:!::2 C fr min. After this perid, the reactins were terminated by adding 1 M HCI till ph f the reactin mixture was abut 4.0. The lipids were extracted. The labeled and unlabeled phsphlipids after separatin by tw-dimensinal TLC were estimated as described by Gupta and Misra [19]. Kinetics f TNBS labeling f vesicles Kinetics f TNBS labeling f aminphsphlipids in vesicles was studied by mdifying the publishedprcedure [8].T an aliqut (40 ILml) f vesicle preparatin, cntaining nt mre than 0.25 ILmlf aminphsphlipids,were added 10 mm phsphate buffer (ph 8.5; 560 ILl) and 0.8 M NaHC03 (200 ILl).After vrtexing the mixture, a 20 ILl aliqut f 1.5% TNBS slutin in 0.8 M NaHC03 was added and the mixture was incubated at 20:!::2 C in dark fr different time intervals. The reactin was terminated by adding 1.2% Tritn X-100 (400 ILl) in 1.5 M HCl. Absrbance at 410 nm was read within 1 h after terminating the reactin. The ttal aminphsph-

5 423 lipid cntent in vesicles was determined as fllws: an aliqut (40 ILl) f the same vesicle preparatin as abve was diluted t a final vlume f 0.6 ml with buffer. T this was added 200 ILl f 1.6% Tritn X-100 in 0.8 M NaHC03 (ph 8.5). The sample was mixed. A 20 ILl aliqut f the 1.5% TNBS slutin was added and the mixture was incubated in dark at 20:t 2 C fr the same time intervals as abve. After incubatin, 0.4% Tritn X-100 in 1.5 M HCI (400 ILl) was added. The absrbance at 410 nm was read within 1 h f acidificatin. The absrbance at 410 nm was linear with cncentratin t at least 0.8 absrbance units. Results and Discussin The varius phsphlipid cmpnents f mnkey erythrcyte membrane were purified as described in Materials and Methds. The pure majr cmpnents (PC, PE, SM and PS) were mixed s as t apprximate their rati in the erythrcyte membrane. Unilamellar vesicles frm the phsphlipid mixture were prepared in the "Of E 0.5 c 0 N CI:) III. II) II: 0 VI III «0 f.l 0.5 A B At Fig. 1. Elutin patterns f vesicles frm Bi-Gel A-50m clumn. (A) Snicated vesicles. (B) Detergent dialysed vesicles. Multilamellar vesicles were used as vid vlume (V) marker. absence as well as in the presence f 40 weight % chlesterl by snicatin r detergent dialysis, and fractinated by centrifugatin r gel filtratin. Hmgeneity f the vesicle preparatins was rutinely analysed by gel permeatin chrmatgraphy n Bi-Gel A-50m. These vesicles invariably eluted with the included vlume f the Bi-Gel clumn (Fig. 1). As may be seen in Fig. 1, the detergent dialysed vesicles eluted faster than the snicated vesicles frm the clumn. Apart frm this difference between the tw types f vesicles, the peak width at half height was smaller fr the detergent dialysed vesicles (Fig. 1). These bservatins clearly suggest that the vesicles frmed by detergent dialysis are lesser hetergeneus and larger in size than that frmed by snicatin. This was further cnfirmed by measuring the uter diameters f bth the types f vesicles, using negative staining electrn micrscpy. The average uter diameters f the snicated and the detergent dialysed vesicles were abut 30 nm (size range nm) and 45 nm (size range nm), respectively. Als, these average sizes f vesicles were cnsistent with the amunts f trapp~d slutes. Inclusin f 40 weight% chlesterl in the phsphlipid bilayers did nt change cnsiderably the size range f bth the types f vesicles, as evidenced by their elutin patterns frm the Bi- Gel clumn, but appeared t induce an appreciable increase in the average size f vesicles. The relative rati f the varius phsphlipid cmpnents in the vesicles bilayer was als determined and fund t be similar t that bserved in the erythrcyte membrane (Table II). The transbilayer distributins f glycerphsphlipids in these vesicles were asc.;:rtained using phsphlipase A 2 and TNBS as the external membrane prbes [4,20]. Experiments were dne t cnfirm that the structural integrity f the vesicles is retained during r after their hydrlysis with phsphlipase A2. Vesicles cntaining [3H]inulin r carbxyflurescein as the trapped slute were used in these experiments. The vesicle stability was ascertained by (a) measuring the efflux f the entrapped slutes, and (b) gel filtratin f the hydrlysed vesicles. Leakage f the trapped slutes remained unaffected by phsphlipase A 2 treatments f the vesicles (Figs. 2 and 3). Als, the elutin prfiles f the hydrlysed vesicles frm

6 424 TABLE II MEMBRANE PHOSPHOLIPID COMPOSITIONS Each value is a mean f determinatins:ts.d.. The values shwn fr PC include the amunts f the crrespnding Iys derivative als. The amunts f this Iyslipid in the lipid extracts f red cells and vesicles were 0-2% and 1-3%, respectively. N ther Iyslipid was present in these extracts. Membrane % f ttal phsphlipids PC PE PS SM PI Mnkey red cell 38.8:t :t :t :t :t 0.4 Vesicles 39.5:t :t :t :t Bi-Gel A-50m were similar t that bserved fr intact vesicles (Fig. 3, A and B). These findings cmpletely exclude the pssibility f vesicle lysis during the enzyme hydrlysis. Mrever, the bserved similar elutin patterns f the vesicles befre and after the enzyme (r Ca2+) treatment UJ VI «~ 20 UJ a: ~ 40 I..L TIME (MINUTE) Fig. 2. Efflux f the entrapped 6-carbxyflurescein frm vesicles during phsphlipase A2 treatments. Fully quenched cncentratins f 6-carbxyflurescein (38) were entrapped in vesicles. Free and the trapped dye were separated by gel permeatin chrmatgraphy (see Materials and Methds). After this chrmatgraphy, 20-25% f the ttal dye eluted with vesicles was characterized as free 6-carbxyflurescein. N attempts were, hwever, made t remve this amunt f free dye frm the vesicles. These vesicles were incubated separately with buffer (0), 10 mm Ca2+ (L», phsphlipase A2 (5 I1g prtein/l1ml lipid P) and 10 mm Ca2+ (X), and phsphlipase A2 and 5 mm EDT A ('7) at C. ph f the incubatin mixture was abut 8.5. Measured aliquts frm these incubatin mixtures were withdrawn at specified perids f time and the amunts f ttal and free dye determined by measuring flurescence in the presence as well as in the absence f Tritn X-l00 (1%final cncentratin) n an Aminc SPF-5 flurimeter using excitatin and emissin wavelengths f 490 and 520 nm, respectively. Percent 6-carbxyflurescein release was calculated frm 100XDyer/Dye" where Dyer and Dye, dente free and ttal dye, respectively. Degradatin f phsphlipids in the vesicles that were treated with phsphlipase A2 in the presence f Ca2+ was established by TLC analysis. (Fig. 3, A-C) als rule ut the pssibility f the vesicle fusin in these experiments. This was further established by ur bservatin that the vesicle size, as measured by negative staining electrn micrscpy, was nt much affected after treating the vesicles with phsphlipase A 2' Earlier studies have revealed that phsphlipase A 2 hydrlyses nly the phsphlipids in the uter leaflet f the vesicle bilayer [20,21]. In rder t verify these findings in the present case, the kinetics f the enzyme hydrlysis f the vesicle PC was examined as described in Materials and Methds. The results shwn in Fig. 4 indicate that during the initial phase f the enzyme reactin the hydrlysis in the intact vesicles prceeded much faster than that in the vesicles lysed with Tritn X-100. In the first 30 min apprx. 70% f the ttal vesicle PC was hydrlysed. After this perid, there were n further increases in the amunts f the hydrlysed PC upt 6 h. Mrever, these amunts were nt affected even by including M bvine serum albumin in the incubatin mixtures, excluding the pssibility f inhibitin f the enzyme activity by the reactin prducts [21]. Hwever, additin f methanl! ether (2: 98, viv) t these reactins led t the cmplete hydrlysis f all the glycerphsphlipids. These experiments strngly indicate that phsphlipase A 2 hydrlyses nly the phsphlipids in the exterir side f the unilamellar vesicles, withut affecting the vesicle structural integrity. Therefre, this enzyme was used t study the trans bilayer phsphlipid distributins in varius vesicle preparatins. The results given in Table III indicate that the enzyme invariably degraded appreciably higher amunts f PC than that f PE and PS in vesicles. These differences in the amunts were nt

7 )( 4 I \ A V 2 0 I I/) I/) > 50..J a: > :I: ~ TIME (HOUR) Fig. 4. Kinetics f hydrlysis f vesicle PC by phsphlipase A2. Vesicles cntaining trace amunts f egg [14C]PC were treated, in the absence (0) as well as in the presence f 0.5% Tritn X-1OO(8), with phsphlipase A2 and the amunts f the hydrlysed PC were determined, as described in Materials and Methds. E Q. U A 15 FRACTION C due t partial cleavage f the external phsphlipids because the amunts f degraded PC and PE in vesicles virtually remained cnstant between 1 h and 4 h after the enzyme treatments (Fig. 5). In rder t further examine the suitability f phsphlipase A 2 t prbe the phsphlipid rganizatin in the vesicles, we have studied the transbilayer distributin f aminphsphlipids in the tw selected types f vesicles (snicated vesicles, with and withut 40 weight % chlesterl) using the chemical prbe, TNBS. The impermeability f the reagent acrss the bilayer was cnfirmed by studying the kinetics f amin grup labeling in intact and lysed vesicles (Fig. 6). At all Fig. 3. Elutin prfile f phsphlipase A2 hydrlysed vesicles cntaining trapped [3H]inulin. Vesicles cntaining trapped inulin were incubated separately with buffer (A), phsphlipase A2 (5 p.g prteinjp.mllipid P) and 10 mm Ca2+ (B), and 10 mm Ca2+ alne (C) at C fr 60 min. ph f the incubatin mictures was 8.5. Measured aliquts f these incubatin mixtures were chrmatgraphed n Bi-Gel A-50m clumns (1.2 x 15 cm). Each fractin (0.5 mi) was analysed by measuring ttal phsphrus and radi activity. Fractins eluted after the fractin number 28 did nt cntain phsphrus. Vesicles that did nt receive the abve treatments were als chrmatgraphed n this clumn. The elutin patterns f these vesicles were similar t that shwn in the tp panel (A). The small peak eluting like inulin is presumably f free inulin which did nt separate at the initial purificatin step. Psitin f inulin was determined by passing free eh]inulin thrugh the clumn. TABLE III VESICLE PHOSPHOLIPID DEGRADATION BY PHOS- PHOLIPASE A2 Valuesare mean f 4-6 determinatins:f:s.d.;pl, phsphlipid; Chl, chlesterl (40 weight %). Samples PC PE PS (%) (%) (%) Snicated vesicles PL alne 73.4 :f: :f: :f:2.8 PLjChl 70.2:f: :f: :f: 1.9 Detergent dialysed vesicles PL alne 62.6:f: :f: :f: 1.7 PLjChl 59.0:f: :f: :f: 1.2

8 II) in ~ 50 > J: ~ 2 <4 TIME (HOUR) Fig. 5. Kinetics f phsphlipase A2 hydrlyses f PC and PE in vesicles. Phsphlipase A2 treatments f vesicles fr different perids f time were carried ut under the cnditins as given in Materials and Methds. After blcking the enzyme activity with EDTA, the hydrlysed vesicle lipids were extracted and the amunts f the degraded phsphlipids were determined. Values shwn fr PC (0) and PE (8) are mean frm duplicate experiments. The variatin was 2-5%. Hwever, % degradatin f PS at 30 min culd nt be determined with this accuracy. The amunts f hydrlysed PS in vesicles did nt increase after the 2 h treatment. time perids significantly higher amunts f the phsphlipids were labeled by TNBS in the lysed vesicles as cmpared t thse in the intact vesicles. A cntinuus increase in the labeling amunts E 0.75 I c: «1&1 u Z«CD a: 0.25 III «20 <40 60 TIME (MINUTE) Fig. 6. Kinetics f labeling f the vesicles with TNBS at 20 C. Vesicles were treated with TNBS in the absence (0) as well as in the presence f Tritn X-100 (8) as described in Materials and Methds. The amunts f amin grup labelings at different perids f time were determined by measuring absrbance fr yellw clr at 410 nm, after acidificatin. In case when the same experiment was carried ut at r abve 25 C the labeling curve fr intact vesicles did nt plateau upt 60 min. with time was bserved upt the first 30 min. N further increases in the amunts ccurred after this perid. These findings indicate that under the present experimental cnditins TNBS des nt permeate the vesicle bilayer and thus the amunts f PE and PS that were labeled by this reagent in intact vesicles (withut chlesterl: PE = 68 :t 2% and PS = 48:t 3%; with chlesterl: PE = 66:t 2% and PS = 54:t 2%) shuld represent the ttal amunts f these phsphlipids in the external mnlayer. As may be seen in Table III, the amunts f PE and PS that were hydrlysed by phsphlipase A2 are quite cmparable t thse mdified by TNBS in intact snicated vesicles, again indicating that the enzyme degraded nly the external phsphlipids. Several investigatrs have shwn that the transbilayer exchange (flip-flp) f phsphlipids in mdel membrane systems is a very slw' prcess [22]. Hwever, since rapid exchange can ccur under sme cnditins [23], the pssibility that inclusin f chlesterl in the phsphlipid bilayers may enhance the transbilayer exchange was excluded by studying the kinetics f aminphsphlipid labeling in the vesicles cntaining chlesterl. The results btained in these studies were almst identical t that shwn in Fig. 6, suggesting that chlesterl incrpratin in vesicles did nt induce phsphlipid exchange acrss the bilayer. i These bservatinsdemnstrate that the phsphlipase A 2 hydrlyses all the external glycerphsphlipids in intact vesicles, and that there is n rapid transmembrane exchange f phsphlipids acrss the vesicles bilayer. Thus the accessibility f the varius phsphlipids t the enzyme in intact vesicles can directly be crrelated with their lcalizatin in the external mnlayer. Hence, apprx. 73% PC, 67% PE and 46% PS are distributed in the uter surface f the snicated vesicles that were free f chlesterl (Table III). These amunts f the external glycerphsphlipids accunt fr nly abut 53% f the ttal vesicle phsphlipids. As the fractin f phsphlipid mlecules in the uter mnlayer f small vesicles is abut tw-third, apprx. 70% f ttal vesicle SM shuld be external. This means that in these small vesicles PC, SM and PE are randmly distributed in the tw mnlayers, whereas PS is

9 427. lcalized preferentially in the inner mnlayer. Hwever, this asymmetric distributin f PS virtually disappeared in larger size vesicles (uter diameter, abut 45 nm) that were frmed by detergent dialysis and fractinated by centrifugatin. Nt nly PS, but als PC, SM and PE were symmetrically distributed in the detergent dialysed vesicles (Table III), as the amunt f external phsphlipids in the vesicles having uter diameter abut 45 nm is apprx. 60% f the ttal vesicle phsphlipids [24]. The bserved transbilayer distributins f SM and PE in the snicated vesicles d nt seem t agree with the results f earlier studies which shwed that PE in PC/PE (> 10 ml %) vesicles prefers the inner mnlayer [4,5] and SM in PC/SM vesicles the uter mnlayer [6]. This discrepancy is prbably due t the marked differences between the lipid cmpsitins f small vesicles studied here and previusly [4-6]. Hwever, as bserved earlier in vesicles cmprised f binary mixtures f phsphlipids [6,7], PS in snicated vesicles f erythrcyte membrane phsphlipids distributed preferentially in the inner mnlayer. Nevertheless, this asymmetric distributin f PS virtually disappeared in larger size vesicles, suggesting that the PS asymmetry in small vesicles was enfrced mainly by the packing cnstraints impsed by the high degree f surface curvature [25]. It may therefre be inferred that erythrcyte membrane phsphlipids in unilamellar vesicles prefer the randm transbilayer distributin rather than the asymmetric distributin, as bserved earlier in the native erythrcyte membrane [19]. Van Deenen and c-wrkers have shwn that chlesterl, a majr lipid cmpnent f red cell membrane, interacts preferentially with PC in PC/PE mixtures [26] and with SM in SM/PC mixtures [27]. T evaluate the influence, if any, f these differential interactins f chlesterl with phsphlipids n the transbilayer distributins f the erythrcyte membrane phsphlipids, we have analysed the phsphlipid rganizatin in vesicles cnsisting f the phsphlipids and 40 weight % chlesterl. The effect f chlesterl n the vesicle asymmetry was ascertained by cmparing the relative ratis f external glycerphsphlipids. It was presumed that the higher affinity f chlesterl fr TABLE IV RELATIVE RATIOS OF EXTERNAL GL YCEROPHOS- PHOLIPIDS IN VESICLES The ratis f the external PC ([PC).) t the external PE ([PEl.) r the external PS ([PSI.) were calculated frm the mean value f the phsphlipid fractin that was accessible t phsphlipase A2 in intact vesicles (see Table III). PL, phsphlipid; Chl, chlesterl (40 weight %). Samples Snicated vesicles PL alne 1.09 PL/Chl 1.03 [PC)./[PEI. Detergent dialysed vesicles PL alne 1.03 PL/Chl 1.01 [Pc)./[PSI SM and PC may, in principle, lead t a decrease in the amunts f external aminphsphlipids and cnsequently an increase in the ratis f the external PC t the external PE (r PS). But Table IV shws that these ratis decreased upn incrprating chlesterl in either f the tw types f vesicles, suggesting that the chlesterl incrpratin leads t mre randm arrangements f phsphlipids acrss the vesicles bilayer. This decrease in the ratis can nt be due t preferential interactins f chlesterl with SM and PC but culd arise frm the chlesterl-induced increase in the size f vesicles. An increase in the vesicle size was quite evident frm the elutin prfiles f chlesterlcntaining vesicles frm the Bi-Gel clumn, which is in agreement with earlier reprts [28,29]. Cnclusin Present study indicates that the transbilayer distributins f red cell membrane phsphlipids in unilamellar vesicles are largely cntrlled by the packing cnstraints impsed by degree f surface curvature [25] rather than by interlipid interactins. The fact that the size f the red cell (abut 8 ILm) is much larger than that f the vesicles, these phsphlipids in prtein-free red cell membrane must be randmly distributed in the tw mnlayers. Therefre, the asymmetric distributin f phsphlipids in this membrane shuld be induced by sme factrs ther than the interlipid

10 428 interactins. We suggest that the phsphlipid asymmetry in red cells primarily arises frm differential bindings f phsphlipids with membrane prteins in the tw halves f the membrane bilayer. This suggestin finds strng supprt frm the earlier studies which shwed that red cell membrane skeletal prteins, especially spectrinand 4.1 plypeptide, differentially interact with phsphlipids in mnlayers, vesicles and erythrcyte ghsts [30-32]. That the interactins between the membrane skeletal prteins and the inner layer phsphlipids prbably maintain the phsphlipid asymmetry in the native erythrcyte membrane is amply demnstrated in the studies f the mammalian red cells that pssess abnrmal membrane skeletn [33-37]. Acknwledgements We thank Prfessr H. Gbind Khrana, M.I.T., Cambridge, U.S.A., fr permitting ne f us (C.M.G.) t carry ut sme f the experiments reprted here in his labratry, Dr. Nitya Nand, Directr, C.D.R.I., Lucknw, fr cnstant encuragement thrughut the curse f this wrk, and Cuncil f Scientific and Industrial Research, New Delhi, fr award f Junir Research Fellwship t A.K. We are als grateful t Drs. G.c. Misra fr his participatin in the preliminary stage f lipid purificatin and A.C. Shipstne fr electrn micrscpy. References 1 Etemadi, A.-H. (1980) Bichim. Biphys. Acta 604, Etemadi, A.-H. (1980) Bichim. Biphys. Acta 604, Verkleij, A.J., Zwaal, R.F.A, Relfsen, B., Cmfurius, P., Kastelijn, D. and Van Deenen, L.L.M. (1973) Bichim. Biphys. Acta 323, Litman, B.J. (1973) Bichemistry 12, Litman, B.J. (1974) Bichemistry 13, Berden, J.A., Barker, R.W. and Radda, G.K. (1975) Bichim. Biphys. Acta 375, Barsukv, L.I., Victrv, AV., Vasilenk, I.A., Evstigneeva, R.P. and Bergelsn, L.D. (1980) Bichim. Biphys. Acta 598, Nrdlund, J.R., Schmidt, C.F., Dicken, S.M. and Thmpsn, T.E. (1981) Bichemistry 20, Kumar, A. and Gupta, C.M (1983) Bichim. Biphys. Acta 730, Blecher, M. (1966) Bichem. Biphys. Res. Cmmun. 23, Gupta, C.M. and Bali, A. (1981) Bichim. Biphys. Acta 663, Rse, H.G. and Oklander, M. (1965) J. Lipid Res. 6, Gupta, C.M., Radhakrishnan R. and Khrana, H.G. (1977) Prc. Natl. Acad. Sci. U.S.A 74, Gswami, S.K. and Frey, C.F. (1971) J. Lipid Res. 12, Rck, R.C., Standefer, J.c., Ck, R.T., Little, W. and Sprinz, H. (1971) Cmp Bichem. Physil. B 38, Pllet, S., Ermidu, S., Le Saux, F., Mnge, M. and Bhu~ mann, B. (1978) J. Lipid Res. 19, Ames, B.N. and Dubin, D.T. (1960) J. BiI. Chern. 235, Bligh, E.G. and Dyer, W.J. (1959) Can. J. Bichem. Physil. 37, Gupta, C.M. and Misra, G.c. (1981) Science 212, Sundler, R., Alberts, A.W. and Vagels, P.R. (1978) J. BiI. Chern. 253, Kupferberg, J.P., Ykyama, S. and Kezdy, F.J. (1981) J. BiI. Chern. 256, Op den Kamp, J.A.F. (1979) Annu. Rev. Bichem. 48, Van Deenen, L.L.M. (1981) FEBS Lett. 123, Rseman, M., Litman, B.J. and Thmpsn, T.E. (1975) Bichemistry 14, Chrzeszczyk, A, Wishnia, A. and Springer, C.S., Jr. (1977) Bichim. Biphys. Acta 470, Van Dijck, P.W.M., De Kruijff, B., Van Deenen, L.L.M., De Gier, J. and Demel, R.A. (1976) Bichim. Biphys. Acta 455, Demel, R.A., Jansen, J.W.C.M., Van Dijck, P.W.M. and Van Deenen, L.L.M. (1977) Bichim. Biphys. Acta 465, De Kruijff, B., Cullis, P.R. and Radda, G.K. (1976) Bichim. Biphys. Acta 436, Gent, M.P.N. and Prestegard, J.H. (1974) Bichemistry 13, Mmbers, c., Verkleij, A.J., De Gier, J. and Van Deenen, L.L.M. (1979) Bichim. Biphys. Acta 551, Mmbers, C., De Gier, J., Demel, R.A and Van Deenen, L.L.M. (1980) Bichim. Biphys. Acta 603, Sat, S.B. and Ohnishi, S.l. (1983) Eur. J. Bichem. DO, Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Bichim. Biphys. Acta 509, Lubin, B., Chiu, D., Bastacky, J., Relfsen, B. and Van Deenen, L.L.M. (1981) J. Clin. Invest. 67, Gupta, C.M., Alam, A., Mathur, P.N. and Dutta, G.P. (1982) Nature 299, Williamsn, P., Bateman, J., Kzarsky, K., Mattcks, K., Hermanwicz, N., Che, H.-R. and Schlegel. R.A. (1982) Cell 30, Kumar, A. and Gupta, C.M. (1983) Nature 303, Weinstein, J.N., Yshikami, S., Henkart, P., Blumenthal, R. and Hagins, W.A. (1977) Science 195,