Bic~chimica et Biophysics Acts, 1044 (1990) 305-314 305 Elsevier

BBALIP 53412

Asymmetr i c extraction of membrane l ipids by C H A P S +

P r o b a l B a n e r j e e , J o h n T . B u s e a n d G l y n D a w s o n

Departments o f Pediotries. Biochemist O, and ,~lolecula, BtologA'. Joseph P. Kennedy', J r . ~.f~'nral Retardation (. t ,,' : ,~. Unwer~tty o f Chtcago, Chwago IL ( U. S. A.)

(Received 15 November 1989)

Key words: Membrane lipid; Lipid extraction; CHAPS; Serotonin 5-HTIA; Cosolubilizauon

We hgve characterized and quantitated the lipids which are cosolubilized with serotonin 5-HTIA sites from sheep brain using 3- |(3-cholamidopropyl)di~i~thylammoniol-l-prolnmc~ulfonate (CHAPS) . Dialysis of the C H A P S e x ~ a c t pj~- duced a [3HIS-hydroxy(2-di-n-propylamino)tetralin (13HIS-OH-DPAT) binding vesicular preparation of the protein. Quantitative analysis of the lipids present in the C H A P S extract by H P T I ~ and transmittance-densitometr~- revealed extraction of phosphatidylethanolamine (PE), phosphatic|ylcholine (PC), phospltatidylinositot (Pl ) , phosphatidyl se- ine (PS) and phosphatidic acid (PA) in striking preference over cholesterol, galactosylceramides, sulfatides and sphingo- myelin. All lipids present in the clear CHAPS-extract were coeluted with the 13HIS-OH-DPAT binding sites during gel-filtration chromatography. W h e n the vesicles present in the post-dialysis | 3 H ] 8 - O H - D P A T binding preparation were separated by centrifugation, 95 -100% of the 13HIS-OH-DPAT binding protein w&s retained in the vesicle-containing pellet. The supernatant contained small atnounts of cholesterol. PE and PC, but virtually no PS , PI, or PA, whereas the vesicular pellet contained all the lipids mentioned, indicating that PS , Pl and PA are more tightly bound to the vesicles than PE, PC and cholesterol S D S - P A G E analysis of the pellet revealed two major protein bands, at 58 kDa and 33.5 kDa, respectively. Our report outl ines a simple and improved densitometrle assay used for the first detailed analysis of iipids cosolubilized with an active, membrane protein, and also, a simple assay for C H A P S .

In l roduc t ion

Menlbr~,ne p ro te ins a~e o f t en c o n s i d e r e d in two sep- a ra te ca tegor ies b a s e d on thei r m o d e of ass,~ciation wi th the p h o s p h o l i p i d b i l ayer [1,2]. Ext r ins ic or pe r iphera l p ro t e in s are those which are read i ly solubi l ized by high

Abbreviations: CHAPS, 3-[(3-cholarmdopropyl~limethylammoniol-l- propanesulfonate; SBH. sheep brain homogenate: SBP, sheer' brain- pellet; SBDS, sheep Brain detergent-soluble; SBDP. sheep brain deter- gent pellet; SBDSE, sheep brain detergent-soluble-25% ethylene- glycol-dialyzed; EGSIE, Extracti-OelXM-D-treated SBDS-dialyzed (di- alysis product of the sample loaded on the gel-filtration columnl; EGSP, Extracti-GelTM-D-treated SBDS-dialyze~ aqd pelleted: Bt"~SE, Bio-Gel exclu~ion-d:.alyzed (dialysis product of the exclusion peak f-ore gel-fiittation); BGSP. Bio-Gel exclusion-soluble-pelleted; 8- OH-DPAT, 8-hydroxy-2..(di-n-propylarmnc)tetralin; HPTI.C, I,igh- performance thin-layer chromatography; Sin, sphingomyelin; Sa, sulfatides, FFA, free fatty acid; C, cholesterol; buffer A, 50 mM Tris-HCI (pH 7.4)/0.32 M sucrose; buffer B, 50 mM Tris-HC, ~pH 7.4) containing 1 mM EGTA/5 mM MnCI2/5 mM ascorbie acid/t0 pM pargyline; buffer C. 25% ethylene glycol in buffer B; SOS-PAGE, sodium dodecyl sulfate-polTacrylamide gel electrophoresis.

Correspondence: G. Dawson, Department of Pediatrics, The Univer- sity of Chicago, 5841. S. Maryland Avenue, Box 82, Chicago, IL 60637, U.S.A

ionic s t rength , che la t ihg agents o r a l t e ra t ions in p H and do no t a p p e a r to be b o u n d d i rec t ly to the h y d r o c a r b o n oo r t i on of the l ipid b i layer . In t r ins ic o r integral p ro - teins, on the o t h e r hand, are mrJce dgh t ly b o u n d to the h y d r o c a r b o n in te r io r a n d so lub i l iza t iun o f the~e p ro- teins is b r o u g h t a b o u t on12~ wi th agents which d i s rup t the l ipid bi layer . In mesz cases, we do no t have i n fo rma- t ion as to the spec i f ic i ty o f i n t e r ac t i on with d i f f e ren t po r t i ons o f l ipid molecu les or the in f luence o f b o u n d lipid on the s t ruc tu re o f the pro te in . Several invest iga- t ions have s u p p o r t e d the idea that the t ight ly b o u n d e n d o g e n o u s l ipids dese rve special a t t en t i on {3-5]. S tud- ies on de t e rgen t soiubilize.t ion o f in t r ins ic m e m b r a n e p ro te ins have revealed tha t with an increase in de te r - g e n t / m e m b r a n e rat io, p ro t e in - l i p id -de t e rgen t com- plexes are re leased f rom the m e m b r a n e and at very h igh d e t e r g e n t / m e m b r a n e rat ios, l i p id -de te rgen t and p ro- t e in -de te rgen t comp lexes are f o u n d [6-8] . Since this is a thermc, d y n a m i c process , the c o n c e n t r a t i o n o f d e t e r g e n t requ i red to br ing a b o u t c o m p l e t e de l i p ida t i on o f m e m - b r a n e pro te ins a n d to to ta l ly sotubi l ize all l ipid mole- cu!es in de t e rgen t mieelles d e p e n d s on the re la t ive af f in- ities o f l ipid and de t e rgen t fo r p ro te in . Hc, wever, in o r d e r to ma in t a in the biological ac t iv i ty of a de tc rg - ,t- solubi l ized p ro te in it is des i rab le to use an o p t i m u m

0005-2760 /90 /$03 .50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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concentration of an appropriate detergent, in order to solubilize a protein lipid-detergent complex without re- moval of the endogenous lipid [8]. In the presence of a large molar excess of detergent (with respect ~o lipid). any lipid that remains bound to a membrane protein is clearly of higher affinity for that protein than the lipid which is readily displaced. After some initial concern [9,10], this subject was not investigated any further and no detailed analysis of the lipids which remain tightly bound to a protein during detergent-solubilization was made.

Apart from the serotonin receptors [I1,12}, CHAPS has been used in the solubilization of a broad spectrum of functional proteins, like the nitrendipine receptor [13], vasopressin receptor [14], net-+otensin receptor [15], caleitonin gene-related peptide binding protein [16], substance P binding protein [17], prostacyclin receptor [18], GABA A receptor [19], sex hormone-binding globulin receptor [20], and adenosine receptor A t [21], to name but a few. However, there is a universal prob- lem with most of these receptors: the loss of binding activity in the purified protein preparation. In most studies reported so far, the lipid requirement of a func- tional protein has been tested by adding specific lipids to solubilized and delip:,dated proteins [22,23]. Our in- vestigation starts from the other end, by identifying the lipids which a protein remains associated with dur ing optimal solubilization and centrifugal separation of the vesicles containing the reconstituted, functional protein.

We present here evidence indicating that specific phospholipids, particularly those associated with the cytoplasmic face of the membrane, such as PE, PI and PS, which are enriched during CHAPS extraction of the [3H]8-OH-DPAT binding protein, are probabiy re- sponsible for maintaining the appropriate vesicular en- vironment required by the reconsti tuted and functional receptor.

Ma(erials and Methods

naleri~.,s CHAPS and Extracti-GdXM-D were from Pierce

(Rockford, IL). HPTLC plates (10 x 10 cm) of the H P K t)o, ru~ 00191.6) with 2 crn-wide preadsorbent layer were ,+,btained from Whatman Chemical Separation (Clifton, NJ). The standard lipids (PE, PS, PI, GalCer and phrenosin from bovine brain; PA and PC from egg yolk) were obtained from Sigma (St. Louis, MO); densitometric analysis of the HPTLC plates were con- ducted u.,,ing a GS 300 densi tometcr (Hoefer Scientific Instrument.~, San Fra" .isco, C A ) All t. reagents and solvents were of analytical grade and the H t 'TL C plates were prewashed by developing with c h l o r o f o r m / m e t h a n o l / w a t e r (60 : 40 : 10, v / v ) and activated at 150°C for 1 h before use.

Storage of lipid mixture Each standard lipid or extracted lipid mixture was

weighed and dissolved in a specific volume of chloro- f o r m / m e t h a n o l (1 :1 , v / v ) ( C / M ) and stored under nitrogen in a Teflon seal-screw cap vial at - 2 0 ° C until u s e .

Membrane preparation and solubilization Sheep brain gray matter, obtained from freshly

slaughtered sheep, was carefully separated from white matter and polyt ron-homogenized (dial setting "4") in 10 vols. of buffer A (50 mM Tris-HCi (pH 7.4), 0.32 M sucrose) at 4 ° C and the homogena te (SBH) was centri- fuged for I0 rain at 1 0 0 0 x g (4°C) . The pellet was resuspended in buffer A, incubated at 37 ° C for 15 rain and then centr ifuged at 100000 x g for 20 rain (4°C) . The pellet obta ined was washed twice with 4 ° C de- ionized water (each time, followed by centr i fugat ion at 100000 × g) and resuspended in cold deionized water at a protein concentra t ion o f 10 rag /m! . This e n n c h e d membrane preparat ion (SBP), which could be stored indefinitely at - 7 0 ° C without loss of activity, was extracted with C H A P S (2%) in buffer B (50 m M Tris- HCI (pH 7.4), 1 mM EGTA, 5 m M ascorbic acid, 5 m M MnC! 2 and 1 ~tM pargy l ine )con ta in ing 1 ~M serotonin at 4 ° C for 20 rain at a protein concent ra t ion 10 m g / m l ( C H A P S / p r o t e i n , 2 : 1 (w/w) ) . The solubilized pre- paration (SBDS) obta ined was separated from the mem- branes by centr ifugation at 1 0 0 0 0 0 x g for 30 min (4 ° C) and then dialyzed against buffer C (25% ethylene glycol in buffer B) at 4 ° C for 20 h (change o f buffer after 2 h, 4 h and 16 h f rom the start). The stable, reconst i tu ted receptor p r epa ra t i on thus ob t a ined (SBDSE) (containing vesicles of endogenous phospho- lipids) was enriched by centr ifugal separation of the vesicles from 2-ml port ions of SBDSE at 100000 x g (5 min) and gentle resuspen~ion of the pellet in buffer C containing 0.3% CHAPS (2 ml) by vortexing and pipet- ting at 4 ° C. This enriched vesicle preparat ion (SBDSP) could be stored in plastic conta iners without freezing at - 20 ° C.

Binding assay o f the receptor activity The 8 -OH-DPAT binding activity was assayed by

incubation of a receptor preparat ion (protein _~ 1 mg) in buffer B a (20 mM Tris-HCI (pH 7.4), 0.4 mM EGTA, 2 mM MnC! 2, 2 mM ascorbic acid and 0.4 ~ M pargyline) at 2 3 ° C in the pre~,~ttce of 9 nM [3H]8-OH-DPA'r (100000 cpm) for 20 rain (total volume 1 ml). Non- specific binding was de te rmined in the presence of 10 pM serotonin. The bound ligand was separated f rom the free by filtration using 0.3% PEl-soaked G F / B filter strips and a Brandel M 12R cell harvester following by washes with cold (40C) 10 m M Tris-HCi (pH 7.4), 0.2 mM EGTA (3 X 6 ml) [24].

307

Sheep Brain Homogenate (SBHI

, / ~ 1000 x g,

Supernatant (SBS) ~ 37 "C & washes

Sheep Brain Enriched Pellet fSBP)

. ~ 1 ) 2% CHAPS, 1 laM sera.*onin Pellet (SBDP) 2) 100 000 x cJ

Supernatant (SBDS)

{Complete r e m o v a j of CFL~PS) . ~ Extraeti-

f Gel-D

D / ~ . . B i o 1 .Sm

Vestcles {EGSE) Dialysi~ Vesicles

Pc l~e7 100 000 x g ---I Pellet

(EGSP)

fBGSE)

(BGSP)

~ l l y D j'~ I'~ zecl agatnst Buffzr B ÷ 25% ethylene

col

SBDSE (vesicles)

Supernalant (SBDSS)

Pellet (SBDSP)

Fig. 1. Gchematic representation of the prcxedure to obtain the var~ou~ fractions which were analyzed for lipid content and [ ~H]8-OH-DPAT binding activity.

Bio-Gel A 1.5 m gel-filtration chromatography T h e solubi l ized p r epa ra t i on S B D S (10 ml) was t reated

at 4 ° C wi th 5 mi o f Extrac t i -GelTM-D for comple t e r emova l o f C H A P S and a par t (8 ml) o f the de te rgen t froc f rac t ion thus obtaine, d was sub jec ted to gel filtra- t ion over 250 ml o f Bio-Gel A 1.5 m using buf fe r B con ta in ing 0.1 M K O A c and 0.1% C H A P S [11]. The exclus ion (Vo) and total ( V i) vo lumes were d e t e r m i n e d us ing Blue D e x t r a n 2 0 0 0 0 0 0 and [ 3 H I g - O H - D P A T ( 1 0 0 0 0 0 cpm) , respect ively . A pa r t o f the sample loaded (2 ml) was d ia lyzed agains t bu f f e r C at 4 ° C to ob t a in a [ 3 H ] 8 - O H - D P A T b ind ing f rac t ion ( E G S E ) which gave an idea a b o u t the ac t iv i ty loaded on the co lumn . The c o l u m n f rac t ions were d ia lyzed against b u f f e r C and a s sayed for [ 3 H ] 8 - O H - D P A T b ind ing activi ty. The frac- t ion (exclusion) c o n t a i n i n g the peak o f act iv i ty ( B G S E ) was sub jec t ed to l ipid analys is a long wi th EGSE. O n an average, the recovery of [ 3 H ] 8 - O H - D F A T b ind ing activ- i ty af te r c o l u m n c h r o m a t o g r a p h y was 40%.

C~.nf~rmation o f vesicle formation by electron microscopy The vesicles in S B D S E were cen t r i fuged at 200 000 x

g for 20 rain to o b t a i n a pel let wh ich was f ixed with 2% g lu t a r a ldehyde in 0.12 M Tr i s -HCl ( p H 7 ~) and then t rea ted wi th 1% OsOa. Fo l lowing this, the pellet was e m b e d d e d in e p o x y resin, carefu l ly cu t and m a d e into th in-sec t ions which were t rea ted with uranyl ace ta te and R e y n o l d s lead ci trate. Elec t ron m i c r o s c o p y was carr ied at 60 keV using a Phil ips 201 e lec t ron mic roscope .

Spectrophotometric determination of C H A P S A s t anda rd curve was o b t a i n e d b y measur ing ab-

so rbances of var ious concen t r a t i ons ( 0 - 1 . 2 m g / m l ) of C H A P S in de ionized water at 2"0 nm. In o rder to mon i t o r the removal o f C H A P S dur ing dialysis o f the de te rgen t - so lub i l i zed f ract ion SBDS. a parallel experi- men t was run, in which several 3-ml po r t ions of a 2% solut ion o f C H A P S in wa te r was d ia lyzed against water at 4 ° C . Dialysis bags w2rc r e move d after the indica ted t ime pe r iods of dialysis (Fig. 2b) and fol lowing 20-fold d i lu t ion o f 100-#1 al iquots , u l t raviole t a b s o r b a n c e o f the f rac t ions measured at 210 nm. Next , f rom the s t anda rd curve, it was poss ib le to d e t e r m i n e the concen t r a t i on prof i le o f C H A P S obse rved dur ing dialysis. Similar expe r imen t s were not poss ib le in the p resence of buf fe r C or bu f fe r B, which a b s o r b s t rongly at 210 nm.

Exiraction of lipids and H P T L C anah'sis The ent ire process o f m e m b r a n e p repara t ion , so!u-

bi l izat ion and lipid analysis is ou t l ined in Fig. 1. The solubi l ized p repa ra t ion (SBDS) was t rea ted with Ex- t ract i -GeiTM-D (3 ml wet vo lume o f gel for 10 ml of S B D S ) at 4 ° C to r emove mos t of the C I t A P S present (so that C H A P S did no t mask the o the r b a n d s dur ing H P T L C analysis) ano then mixed with c h l o r o f o r m / me thano l (1 : 1, v / v ) ( C / M ) (15 ml for every 2.5 ml of Ex t rac t i -Ge l - t r ea ted SBDS). The mix ture was st i rred overnight in the dark under ni t rogen in a sealed screw- cap t ube at rot, m t empera tu re ( 2 3 - 2 5 ° C ' and t)-:'):

308

cell tr ifuged at 2500 rpm using a table- top centrif ,Jge to separa te the prec ip i ta ted proteins. The superr .a tant ,~'as evapo ra t ed at 3 0 ° C , tbe residue dissolved in 5 ml of C / M and the protein precipi ta te ob ta ined was sep- a ra ted by centr i fugat ion. Evapora t ion of this super - na tan t p r o d u c e d a mixture of lipids which dissolved comple te ly in C / M . The soluiic, n was quan t i t a t ive ly t ransferred to a pre-weighed, T,:flon seal screw-cap vial and evapora ted by blowing d ry n i t r oge~ T h e res idue was weighed dissolved in a specific vo lume ol C / M in o rde r to make a s t andard solu t ion and then the so lu t ion was s tored at - 2 0 ° C . The m e m b r a n e - b o u n d prepara - t ions (SBH and SBP) were ex t rac ted in the same w a y (wi thou t the Ext rac t i -Gel t rea tment ) using 15 ml of C / M for 2.5 ml of SBH or SBP.

The vesicular p repara t ion ( S B D S E ) (1 ml) was cent r i fuged at 1 0 0 0 0 0 x g for 1 h at 4 ° C and a f te r separa t ing the supe rna t an t (SBDSS) , the pellet ( S B D S P ) con ta in ing the vesicles was ext rac ted with 5 ml of C / M . Centr i fugal separa t ion of the prec ip i ta ted p ro te ins at 2500 rpm, evapora t ion o f the supe rna t an t and reprecipi- ta t ion o f pro te ins af ter C / M solubi l izat ion of the re- s idue p r o d u c e d a pur i f ied mixture o f iipids re ta ined in the vesicles. The supe rna tan t ( S B D S S ) was ex t rac ted three t imes (3 × 2 ml) with c h l o r o f o r m and the poo led extract was washed once with wa te r and then e v a p o r a t e d to ob ta in a res idue which was ana lyzed for the p resence of lipids and C H A P S . Al iquots (1-ml each) of the Bin-Gel A 1.5 m exclu- ion fract ion ( B G S E ) a n d dialysis p r o d u c t (E(3SE) of the s ample loaded on the co lumn were cen t r i fuged as for S B D S E and the pel lets o b t a i n e d ( B G S P and EGSP , respect ively) were ex t rac ted in the s ame way with C / M iv ob ta in the lipids re ta ined in the vesicles.

H P T L C analysis of l ipids was c o n d u c t e d b y spo t t ing s t anda rd a m o u n t s of l ipids on the same pla te a long the SBH- , SBP-, SBDSP- and SBDSS-i ip ids . T he pla tes were deve loped up to 5 cm from the origin with ethyl a c e t a t e / 1 - p r o p a n o l / c h l o r o f o r r o / m e t h a n o l / 0 . 2 5 % KC1 (25 : 25 : 25 : 10 : 9, v / v ) , dr ied at r oom t empera tu re for a b o u t 15 min and then deve loped full length wi th h e x a n e / d i e t h y l e t h e r / a c e t i c acid ( 7 5 : 2 1 : 4 , v / v ) (so lvent c o m b i n a t i o n I). This a f fo rded bet ter separa t ion of the bands s lower mov ing than PC, bu t the separa t ion b e t w ~- " ~ ~" -._'.ds f,~ PI and PS was not enough for reliable, den., ,,~metric es t imat ion . In order to improve resolu t ion o ; PI and PS, the c o m p o s i t i o n o f the first solvent was changed to e thyl -cetate/1-propanol/ c h ! o r o f o r m / m e t h a n o l / 0 . 2 5 % KCI (25 : 25 : 20 : 15 : 9, v / v ) (solvent c o m b i n a t i o n It). Af ter dry:p~ the plates, the b a n d s were v iewed by spray ing cuprit, phosphor i c acid charr ing reag~-nt (10% C u S O 4 in 8% P l 3 r / , ) and heat ing at 1 8 0 ° C for 10 rain [251.

Densitometry and quantitation Each lane was scamaed in at least three separa te lines

using a GS 300 dens, t ome t e r in the t r ansmi t t ance m o d e with a visual light b e a m ~570-600 rim) the d i m e n s i o n o f which was 0.25 x 2.54 mrra. The scan speed was set at 4 c m / m i n and the peak a reas ( + S.E.) o b t a i n e d us ing (3aussian in tegra t ion were p lo t t ed us ing Cr icke t G r a p h 1.2 (Cr icket Sof tware , Malvern , PA 19355). Even t h o u g h representa t ive H P T L C pla tes are s h o w n here, each quan t i t a t ion was car r ied ou t three t imes with f rac t ions ob t a ined f rom three d i f fe ren t ba tches o f fresh adu l t sheep brain gray mat ter . T he mass o f each lipid p resen t in a lipid mixture (e.g., SBP-ext rac t , Fig. 6a, and SBDSP-ex t rac t , Fig. 6b) was d e t e r m i n e d f rom s t a n d a r d curves (e.g., Fig. 6a and b) o b t a i n e d by spo t t ing increas- ing masses o f s t anda rd l ipids and then the resul ts were c ross -checked b y c o m p a r i s o n of d e n s i t o m e t r i c prof i les ob t a ined by scann ing the lanes on the H P T L C pla tes shown in Fig. 5 ~. and b, Fig. 7a and b and Fig. 8.

Measurement of protein Prote in was de t e rmined us ing the B r a d f o r d assay [26]

or a mod i f i ed Lowry assay [27].

O. 1% SDS-PA GE of proteins Samples were held in boi l ing w a t e r in the p resence o f

d i th io thre i to l (20 m M ) and 2% ~odium dode c y l su l fa te (SDS) for 2 min and then ana lyzed by o n e - d i m e n s i o n a l gel e l ec t rophores i s on h o m o g e n e o u s , 10% po lyac ry l - amide gels con ta in ing 0.1% SDS. F r a c t i o n s l oaded were normal ized to p ro te in (12 /.tg) except in lane 1, wh ich received 4 # g of pro te in , s ince a larger v o l u m e o f this f ract ion (SBP) was f o u n d to cause excess ive b a c k g r o u n d staining. E lec t rophores i s was ca r r ied ou t a t 120 V (con- stant) with wa te r coo l ing and the gels were s t a ined using a B i o -R ad st!vet stain kit. A f t e r s taining, the gel was t rea ted for 15 rain wi th A m p l i f y to e n h a n c e the 14C-labeled s t anda rd b a n d s and then d r ied at 8 0 ° C for 1 h, fo l lowing which the s t anda rd lane w a s cut o u t a nd incuba ted with an X- ray fi lm at - 7 0 ~ C for 3 days . The a u t o r a d i o g r a m strip o b t a i n e d was a l igned with the re- main ing por t ion of the gel to d e t e r m i n e molecu la r weight of the p ro te in bands ob t a ined .

Results

Evidence for vesicle formation and retention of [31-118- OH-DPA T binding activity in the vesicles

Elect ron m i c r o s c o p y of the pel let o b t a i n e d b y cen- t r i fugat ion of S B D S E reveal,-d the p re sence o f vesicles, d imens ions o f which r anged f rom 200 to 2000 / i (elec- t ron mic rog raph no t shown) . Af t e r cen t r i fuga l sep- a ra t ion of the vesicles f rom S B D S E a nd S D S - P A G E analysis o f the enr iched p repa ra t i on , S B D S P (as de- sc r ibed in Mate r ia l s and M e t h o d s ) , it was f o u n d chat S B D S P d i sp layed two m a j o r p o l y p e p t i d e b a n d s at 58 k D a and 33.5 k O a respect ive ly (Fig. 2, lanes 3 a n d 4). [ 3H]8 -OH-DPA°F b ind ing assays of S B D S E (Fig. 2, l ane

2 3 4 5 6

MrxlO "3

ql2.5

t;9

4 6

3 0

I Spectflc ectlvlt g of [ZH] 8-OH-DPAT binding SBP SBDSE SBDSP

l fmol/mg 70 153 300

Fig. 2. SDS-PAGE analysis of solubfzed and reconstituted serotonin 5-HT1A rcceptc.,r preparations. The membrane-bound preparation (lane 1) was compared with the solubilized and reconstituted receptor preparation SBDSE (lane 2), the enriched preparation SBDSP (lane 3 and 4) and the inactive supematant obtained (SBDSS, lane 5) after centrifugal separation of the vesicles from SBDSE. Except for lane t (which received 4 pg of protein), 12 pg of protein was loaded ~n each lane and the polypeptide bands were compared ,,-ith ~4C-!abeled protein standards (lane 6). Inset: speciGc activity (f'r,o!/mg of [3HJS-

OH-DPAT binding) of SBDSE and SBDSE

309

2) a n d S B D S P s h o w e d t h a t 05 100% of the b i n d i n g ac t iv i t y w as r e t a i n e d in the ~esicles (Fig. 2. inse t ) w h e r e a s the s u p e r n a t a n t ( S B D S S , Fig. 2, l a n e 5) lef t a f t e r s e p a r a t i o n o f the ves ic les e x h i b i t e d n o b i n d i n g ac t iv i ty .

Removal o f C H A P S by dialysis T h e a b s o r b a n c e o f C H A P S a t 210 n m was l i nea r u p

to 1.2 m g / m l , b e y o n d w h i c h s c a t t e r i ~ r r e a s - - "~: •

T h e r e a s o n a b l e c o r r e l a t i o n (0 .987, C r i c k e t G r a p h 1.2) wi th a f i r s t - o r d e r p lo t a l l o w e d e s t i m a t i o n o f th i s d e t e r - gen t r e m a i n i n g in a l i q u o t s a f t e r d i a lys i s aga in s t de- i o n i z e d w a t e r (F ig . 3b). T h e h y p e r b o l i c n a t u r e o f the p ro f i l e , w h i c h s h o w e d t h a t t he C H A P S c o n t e n t o f a s o l u t i o n d r o p p e d t o c m c (0 .5%) in 4 h, c o r r o b o r a t e d the o b s e r v a t i o n tha.: S B D S g r e w c l o u d y ( i n d i c a t i n g f o r m a - t ion o f ves ic les) a f t e r 3 - 4 h o f d ia lys i s .

Depletion in GalCer and other glycolipids during C H A P S extraction o f the enriched, 1000 x g membrane-pellet (SBP)

C o m p a r i s o n w i t h s t a n d a r d l ip ids b y H P T L C s h o w e d tha t c h o l e s t e r o l (C) , PE , PS. p h o s p h a t i d y l i n o s i t o l (P I ) . PC a n d G a l C e r were the m a j o r c o m p o n e n t s o f the m e m b r a n e p r e p a r a t i o n ( S B P , Fig. 4a, l ane 5) wh ich a l so c o n t a i n e d p h o s p h a t i d i c ac id ( P A ) a n d su l f a t i de s (Su) (F ig . 4a) . In c o n t r a s t , t h e C H A P S - e x t r a c t ( S B D S ) c a n be s een to be d r a m a t i c a l l y d e p l e t e d in G a l C e r , Su a n d c h o l e s t e r o l (C ) (F ig . 4a, l ane 4). A l k a l i n e m e t h a n o l y s i s o f SBP, S B H a n d S B D S in 0.2 M N a O H in m e t h a n o l a n d H P T L C a n a l y s i s o f the p r o d u c t s c o n f i r m e d the v i r t ua l a b s e n c e o f g l y c o l i p i d s f r o m S B D S (F ig . 4b. l ane 1). A f t e r d i a ly s i s o f STUDS, w h e n C H A P S c o n t e n t was r e d u c e d to 0 .04% ( w / v ) , a s ma l l a m o u n t o f G a l C e r c o u l d b e d e t e c t e d in the ves ic les o b t a i n e d ¢ S B D S P , Fig. 5a, l ane 2). G a l a c t o s y l ce r am~des ( H F A - G a l C e r o r phrer=o.dn, 8.6% a n d N F A - G a l C e r . 3.8q~ o f l ip ids) in S B P (Fig. 5a. l ane 2), w e r e r~resent in m a r k e d l y r e d u c e d

E

,0

2.0

y = - 65212e -2 . | 7331x R'2 =09.97

J f

. . . . , . . . . T . . . .

0 .5 1.0 ! .S

E L

1 ~ 1.0

0 O,C . . . . . . . . T , -- 0.0 0 1 0 2o

(a) CHAPS (mo/m,) (b ) T,me (h) Fig. 3. Removal of CIIAPS by dialysis from a concentration (2%) higher than cmc. (a) Standard curve obtained by following concentration

dependence of ~bsorbanc¢ at 210 rim. (b) Dialysis profile of CHAPS obtained by monitoring absorbance at 210 am.

310

~-.Phre -,..-Su "-"Lc2 ~Gib3

1 2 3 4 5 6 7

Fig. 4. C o m p a r i s o n o f coex t r ac t ed l ipids wi th s t anda rds . (a ) Lipids f rom S B P (25 lag) ( lane 5) and S B D S (25 ~tg) ( l ane 4) were c o m p a r e d wi th PE, PC ( lane 1, 10 ~tg each)) , C, PS ( l ane 2, 10 ~tg each), Pl 0 a n e 3, 10 lag) PA ( l ane 6, 20 lag), C H A P S ( C H , l ane 7, 50 ~xg)) and su l fa t ides (su) ( l ane g, 5 lag). (b) Alka l ine m e t hano ly s i s p r o d u c t s of S B D S ( lane 1, 100 lag), S B H ( lane 2, 100 lag) a n d S B P ( lane 3, 100 lag) were c o m p a r e d wi th glycol ip id s t a n d a r d s ( lane 4, 100 lag), LacCer (5 lag) a n d G b O s e 3 (Gb3 , 10 lag) ( l ane 5), galactosyl c~_-~ramides (Ga lCer , 10 ~tg) a n d as ia lo G M I (Gg4, 2 lag) ( lane 6), p h r e n o s i n ( h y d r o x y fat ty acid ga lac tosy iceramide , 5 lag) a n d C H A P S (50 jag) (lame 7). Bo th

plates were deve loped us ing so lven t c o m b i n a t i o n !1.

T A B L E I

Proportions o f lipids pr~'sent in the membrane-bound and reconstituted- soluble preparations

T h e pe rcen t change in each l ipid w i th respect to SBP is n o t e d in pa ren theses . T h e p r o p o r t i o n o f each l ip id was o b t a i n e d wi th respec t to the sum o f masses o f all t h e major a n d m i n o r l ipid c o m p o n e n t s as o b t a i n e d f r o m dens i t ome t r i c assay. T h e values p r e s e n t e d here a r e the ave rage of t~-ree diffe , en t e x p e r i m e n t s ca r r i ed o u t wi th t h r ee s e p a r a t e b a t c h e s of s h e e p b r a i n and e v e n t h o u g h the to ,a l mass o f l ipids va r i ed wi th in 3%, t he p r o p o r t i o n of e ach l ipid var ied "~pithin o n l y 1%.

( w / w ) o f e s t i m a t e d Upids

S B P S B D S P E G S P

Choles te ro l 12.8 8.0 ( - 38) %7 ( -- 40 ) N F A G a l C e r 3.8 1.3 ( - 66) 0 .86 ( -- 77) H F A G a l C e r ( p h r e n o s i n ) 8.6 3.0 ( -- 65) 1.9 ( -- 78 ) S p h i n g o m y e l i n 5.8 < 0.1 < 0.1 P h o s p h a t i d y l e t h a n o l a m i n e 33.2 43 .3 ( + 30) 40.7 ( + 23) P h o s p h a t i d y l i n o s i t o l 4.6 8.6 ( + 87) 10.3 ( + 124) P h o s p h a t i d y l s e r i n e 19.3 10.5 ( - 46) 21.0 ( + 9) P h o s p h a t i d y l c h o l i n e 12.0 17.5 ( + 46) 17.6 ( -*- 47) C H A P S 0 7.8 < 0.I

amounts (3% and 1.3% of lipids, respectively) in SBDSP (Fig. 5a, lane 3) (Table I).

Depletion in sphingomyelin during C H A F S extraction In order to resolve the bands for sphingomyelin (Sin)

from the other bands, an H P T L C plate was developed using a solvent of higher polari ty ( c h l o r o f o r m / m e t h a n o l / a c e t i c a c i d / w a t e r : 65 : 25 : 8 .8:4 , v /v ) . We observed that nei ther sphingomyelin bands (conta ining long- and short-chain fat ty acids, respectively), present in SBH (Fig. 5b, lane 1) and SBP (lane 2), were ex- tracted by CHAPS (SBDSP, Fig. 5b, lane 4 and EGSP, lane 6) (Table i). In contrast , the detergent- insoluble pellet showed markedly increased content of Sm (1.4- times that in SBP by densi tometry, Fig. 5b, lane 3). The dark band below Sm in lane 3 is for C H A P S and the lower most bands (below Sin, which were stained yellow

{b) Fig. 5. Comparison of lipid profiles of membrane-bound anO solubilized fractions. (a) Lipids obtained from SBH (lane 1), SBP (lane 2), SBDSP (lane 3). SBDS (lane 4) (100 lag each) and standards (lane 5 and 6). The plate was developed using solvent combination I. (b) Lipids (200 ttg each) from SBH (lane 1), SBP (lane 2), SBDP (lane 3, note the enrichment in Sm), SBDSP (lane 4) and EGSP (lane 6) were compared with standard

sphingomyelin (20 pg, lane 5). The plale was developed using ch loroform/methanol /acet ic ac id /water (65 : 25 : 8.8 ; 4, v / v ) .

311

P E -

P S -

CH,..

1 2 3 4 5 6

t - - i y = 0.77'! 1S * 0.5d14~ - 4-71,11~1-3x"2 i / . a in .2= oJ.~

°1 x" ~ I - " , , , , ~ - ~ 1 / - / / " I I ' ' ° ' s ' * ' ° ~ " ~ . = m | ,J .~.-/ / I I " ~ m a , e [ Iv =. o~==~, ozamk a,'~. o .

O - 0 10 2 0 3 0

(a) Lip id (p.g)

"-C

-,,PC

I y ~ 1,1230 * 1.2161I - Lg~911-21"2

[y. -0.471~I.0~I11~t~ R"2=OIIPD I " otmi~am~ o ~ pm~ mj,1 ~oc~

1 lY = O ~ * O . t R'2 = Ores l " O-W~S ( v ~ m . ~ o ~

0 tO 2 0 3G

(b) m-h~kJ (~)

Fig. 6. Estimation of lipids present in SBP and SBDSP. (a) Lipids obtained from SBP (lane 4) (100 lag) were assayec~ by comparing with 2.275 ;*g (lane 1), 4.55 ~ag (lane 2), 9.1 pg (lane 3L 18.2 lag (lane 5), and 36.4 lag (lane 6) of standard lipids. (b) Lipids from SBDSP (lane 4) were assayed by comparing with 2 #g (lane l), 4 lag (lane 2), 8 /~g (lane 3), 16 jag (lane 5) and 32 lag (lane 6) of standard lipids. In either case, the plate was developed using solvent combination I1 and the band intensities were d,:terrmned by densitometry. The standard curves obtained are shown below

the respecti~,e plates.

"~nstead of being charred) in lanes 1 and 2 are for unidentif ied components of SBH and SBP.

Densi tometr ic quanti tat ion o f the lipids and C H A P S con- tent o f membrane extracts

The linear range of the s tandard curves for the phospholipids (0 -80 Fg) (only a part of the range is shown here) and CHAPS ( 0 - 4 0 / , g ) (two t~pical HPTLC plates shown in Fig. 6a and b) were used. The nature and gradient of the individual s tandard curves varied to a certain extent but those for GalCer (phrenosin), Sm and cholesterol usually followed second-order equa- tions. Variation of results obtained from different batches of sheep brain was always less than 1% and Table i is an average of results obta ined from three such experiments in which densi tometric scan of each lane was carried out in three to five dist2nct straight lines. At te r staining, each HPTLC plate was kept sealed f rom air and light and subjected to densi tometry within 24 h. The stain was found to fade considerably when the plates were left uncovered for 1 week.

The detergent-insoluble F "qet is depleted in PE, PA, t"I, P S a e d P C and is enriche.~ in GalCer, S m and cholesterol

A comparison of the lipid profiles of the original homogenate (SBH), the enriched 1000 x g membrane pellet (SBP), the C H A P S extract (SBDS), CHAPS-in-

soluble pellet (SBDP) and the vesicle containing pellets. SBDSP and EGSP (Fig. 1) revealed that the phospho- glycerides were coextracted with the serotonin [3H]8- O H - D P A T binding activity (in SBDSP mid EGSP) in preference to the sphingolipids (galactosylceramides and sphingomyelin) and cholesterol (Fig. 7a, compare SBP, lane 2, SBDSP, lane 4 and EGSP, lane 6). In contrast, Ga!Cers (1.5-times that in SBP, Fig. 8, lane 3), cholesterol and Sm (1.4-times that in SBP, Fig. 5b, lane 3) were enriched in ,he CHAPS-insoluble pellet at the expense of PE, PA, PI, PS and PC.

It was also noticed (Fig. 8) that the relatively less intense band for free fatty acid (checked separately with respect to oleic acid, although the s tandard is not shown here), which moves faster than cholesterol, was enriched in the soluhilized fractiops (SBDSP, lane 4 and EGSP, lane 6) and completely absent in the pellet (SBDP, lane 3).

Investigation of the apparent reduct ion of PS (Table I and Fig. 7b) in SBDSP showed that about 50~ of the solubilized PS and 17% of PI were dialyzed out along with CHAPS during the dialysis of SBDS. This deple- tion of coextracted PS and PI had no effect on [3H]8- O H - D P A T binding activity of the vesicles and did not occt;r when CHAPS was completely removed from SBDS by Extract i -GeLD t rea tment before dialysis (EGSP, Fig. 7b, lane 2). All lipids present in the Ex-

312

(a) 1 2 3 4 5 6 (b)- Fig. 7. Comparison of lipid profiles after resolution of Pl and PS. (a) Lipids (I00 pg each) from SBH (lane I), SBP (lane 2), SBDSP (lane 3), SBDS (lane 4) were compared with standards (lanes 5 and 6). (b) Lipids (25 lag each) from various solubilized fractions, SBDSP (lane 1) and SBDSS (lane 4), the vesicles from the sample chromatographed EGSP (lane 2) and the vesicles from the exclusion peak, BGSP (lane 3), were compared. In either

case the plate was developed using solvent combination II.

tracti Gel D- t r ea t ed S B D S ( E G S P ) (Fig. 7b, lane 2) were re ta ined, in the same p ropor t ion , a f te r Bio-Gel A 1.5-m gel-f i l t rat ion c h r o m a t o g r a p h y and dialysis o f the exc luded fract ion which c o n t a i n e d the p e a k of r ecep to r ac t iv i ty (BGSP, Fig. 7b, lane 3) con f i rming that C H A P S (a C H A P S - P S / P I complex is p r o b a b l y f o r m e d at higher C H A P S concen t ra t ions ) was necessary for the part ial r emova l of PS and PI.

H P T L C analysis fo l lowing C / M ex t rac t ion of the supe rna t an t (SBDSS) and the [ 3 H ] 8 - O H - D P A T b ind ing pel let ( S B D S P ) separa ted f rom S B D S E b y cent r i fuga- tion showed that a small a m o u n t o f C H A P S (50 # t g / m g of sol id l ipid mixture , equiva len t to 0.04% C H A P S in S B D S E , w / v ) was re ta ined in the vesicles, whe reas the supe rna t an t (SBDSS) was total ly devo id of the deter- gent.

1 2 3 4 5 C FiB. 8. Comparison of lipid profiles of SBP, SBDSP, '-GS r" and SBDP. 100 pg of each of the following lipids were analyzed- SBH (lane 1), $BP (lane 2), SBDP (lane 3, note the enrichment in GalCers and cholesterol) and EGSP (lane 6). GalCer (10 Fg) was spotted in

lane 5.

PS, PI and PA are retained completely by" the vesicular pellet (SBDSP)

T he s u p e r n a t a n t (SB DSS , Fig. 7b, lane 4), which con ta ined on ly 0.3 mg o f l ipid c o m p a r e d to 7.4 mg presen t in S B D S P (Fig. 7b, lane 1), was f o u n d to con ta in PE, P C and cho les te ro l bu t essen t ia l ly n o PI, P A or PS. This sugges ts tha t even t h o u g h s o m e loss of PS and PI o c c u r r e d du r ing dia lys is o f S B D S (which ha d no effect o n the [ 3 H ) 8 - O H - D P A T b i n d i n g act ivi ty) (Fig. 2, inset), t he rest o f PS a n d PI p resen t in S B D S P , a long wi th PA, were m o r e t ight ly b o u n d to the p ro t e in s a n d l ipids in the vesicles (SBDSP) .

Lipid-protein stoichiometry in the vesicles A key fea tu re in r econs t i t u t i on of func t iona l p ro t e in s

is the p r o p o r t i o n o f l i p i d : p r o t e i n in the r e c o n s t i t u t e d vesicles con t a in ing the en r i ched and act ive pro te in . A f t e r s epa ra t ion of the vesicles f r o m S B D S E by cen t r i fuga- t ion and r e suspens ion as d e s c r i b e d in the text, we o b t a i n e d S B D S P ( 2 - 2 . 5 m g / m l o f pro te in) , wh ich re- t a ined 9 5 - 1 0 0 % of the solubi l ized [ 3 H ] 8 - O H - D P A T b ind ing ac t iv i ty (p resen t in S B D S E ) a nd 7.4 m g of to ta l l ipid pe r ml. This va lue a b o u t the to ta l mass o f S B D S P - l ipids was o b t a i n e d b y s u m m a t i o n o f masses o f all the ma jo r and minor t ipids as d e t e r m i n e d b y d e n s i t o m e t r i c assay. Therefore , l i p i d : p r o t e i n s t o i ch iome t ry o f the vesicles con t a in ing the solubi l ized [ 3 H ] 8 - O H - D P A T b ind ing p ro te in was 3 - 4 : 1.

D i s c u s s i o n

T he ques t i on of so lubi l iz ing a func t iona l p ro t e in a long wi th the c losely a s soc i a t ed l ip ids has b e e n occa- s ional ly a d d r e s s e d [8]. Ind i rec t s tud ies have b e e n car r ied ou t [221, on de l i p ida t ed p ro t e in s in o r d e r to d e t e r m i n e

the lipids essential for reconstitution of activity. Micel- lar enzymology [28], the new trend in molecular biology involving catalysis by enzymes ent rapped in hydrated, reversed micelles composed of surfactants (phospho- lipids and detergents) in organic solvents, lends further support to studies and analysis of lipids specifically associated to functional proteins. The proteins of con- cern in all these studies were enzymes, but the same could be t rue for the receptors, such as the 5-HT~^ receptor protein. Dur ing optimization of conditions for detergent solubilization of the [ 3H]8-OH-DPAT binding activity, we have found that some detergents, like octy! glucoside, Nc-aidet P--40 and Tri ton X-100 produce weakly active solubilized preparat ions which contain very small amounts of lipids (unpublished observation). As a result of this, unlike in tne cas= of CHAPS extraction, it was not possible to obtain a highly active, [ 3H]8_OH.DPAT binding, vesicular preparat ion of the binding protein f rom these detergent extracts. Even though the exact lipid requirement of the receptor is yet unknown, in order to retain a receptor- t ransducer com- plex in a functionally active state, it appears essential to reconsti tute the appropr ia te components in the correct conformat ion. To initiate this we need to unders tand the lipid envi ronment surrounding the complex. In order to move towards this goal we have analyzed the lipids present in the membrane -bound form of a G-protein- coupled receptor, the 5-HT~A receptor, and compared the results with the lipid profiles obtained from various solubilized preparat ions which retain [3H]8-OH-DPAT binding activity.

The zwitterionic compound, CHAPS, was the deter- gent which gave op t imum solubilization of the receptor-lipid complex. The easy spectrophotometr ic as- say described by us for quant i ta t ing the amount of CHAPS can be used for a solution of the detergent in water and the densi tometric assay described gives a general method of assaying both detergents and lipids based on t ransmit tance measurements. The use of an inexpensive oensitometer, GS 300 (Hoefer), to obtain reproducible results was made possible by the measure- ment of t ransmit tance instead of reflectance, which is the more common ly used parameter for scanning thin- layer chromatograms [29]. The major advantage of this technique is that quant i ta t ion is linear up to 80 /Jg for the phospholipids. Using this method we could also determine the amoun t of CHAPS remaining in the vesicles obtained after dialysis of the solubilized recep- tor preparat ion, The method was sensitive enough to indicate that while a 20 h dialysis would completely remove CHAPS f rom an aqueous solution, the [3il]8- O H - D P A T binding protein and the associated proteins and iipids which form vesicles after reconstitution by dialysis were able to retain a small amount (0.04% of SBDSE, w / v ) of this detergent which obviously re- mained tightly associated with the rest of the lipids and

313

proteins. The mode of this a_-~ociation has been dis- cussed earlier by Tanford 18].

It may be argued that ~hc liptd composition dis- cussed here may be specific for sheep brain. To answer this question we have analyzed rat brain membrane samples prepared in the same way and found that it contains the same lipids and in very simdar proport ions as found in SBP (data not inciuded for tF, e sake of brevity). So, even though some vari,=t~on ~,: , ~ . - =.:_~,~ tent among different species is expected, yet the need for co-solubilization of lipids in order to conserve the native conformat ion of solubilized, functional proteins is independent of the source of the functional proteins.

We have compared the lipids present in the mem- brane-bound and solubilized preparat ions of the recep- tor and shown that some of those lipids were solubilized in dramat ic preference over others. The phospho- glycerides, phosphatidylcholine, phosphat idylethanol- amine, phosphatidic acid, phosphatidylinositol and free fatty acids were enriched dur ing solubilization and re- consti tution, whereas there was a marked depletion in cholesterol and a dramat ic decrease in sphingomyelin and galactosylceramides. Phosphatidylserine and some phosphatidylinositol were partia | ly removed, probably as CHAPS-complex dur ing dialysis of the detergent extract. However, this deplet ion did not affect [~H]8- O}i -DPAT binding activity of the vesicles. Centrifugal separation of the vesicles present in the reconsti tuted preparat ion showed that all PA, PI and PS were re- tained in the pellet, while small amounts of PE, PC and cholesterol could be released into the supernatant . This suggested that the remaiping port ions of the lipids PA, PI and PS were strongly bound to the p~oteins and lipids retained in the vesicles.

We believe that the lipid composit ion of the active receptor preparat ion (SBDSE) is a function of the prop- erties of both CHAPS (in solubilizing certain lipid-pro- tein complexes as a whole, without disruption) and the membrane proteins (e.g., the 5-HT~A receptor) which are responsible for the fine control of this l ipid-protein association.

Many detergents have been us*_d ,-,n ~.n ,-~pirical basis to solubilize l igand-binding activity frc~m mem- branes but no clear rationale has been provided for their selection. While a more detailed~ comparat ive analysis of extraction of lipids by various detergents is being undertaken, our present set of observations should be particularly useful in the s tudy of various functional proteins. The second question, which is important in developing reeonstitution procedures, is about removal of the detergent in order to forth vesicles from the extracted lipids. We have found that al though op t imum solubilization of lipids was achieved at 2% CHAPS, which is higher than its critical micellar concentra t ion (approx. 0.5%), the detergent could be removed without the use of any hydrophobic resins (which often adst, rb

314

hydrophob ic proteins), simply by dialysis. The 5-HT~A and related receptors are predic ted to

have seven t r ~ m e m b r a n e regions [30], based on the hydrophob ic i ty o f predicted amino acid sequences a n d there is no obvious reaso~ per se why the receptor p repara t i cn should be enr iched in those lipids which are p r edominan t l y associated with thc cy top lasmic face of the p lasma membrane . Our da t a suppor t the idea t ha t sphingolipids, together with a s ignif icant por t ion of the cholesterol pool m a y exist in doma ins which are discrete f rom the receptor proteins and have quite a d i f ferent type of functic, n i: the membrane . Tt-.e glycolipids and sphingomyel in are k n o w n to fo rm the outer layer or the ant igenic de te rminan t s o f a cell [31-33], whereas the phosphol i~ ids like PE, PS, PI and PAL p redomina te in the inner b o d y of the m e m b r a n e lipid bi layer and act as a source of second messengers; PC and cholesterol occur in both. Such a symmet r i c d is t r ibut ion of lipids specifically in bra in membranes and the effect of such env i ronmen t on the funct ional act ivi ty of some mem- brane proteins have been d o c u m e n t e d before [34-39]. The fact tha t we have observed preferential soluhil iza- t ion of the inner m e m b r a n e lipid e ~ m p o n e n t s (except for PC) a long with the [3HI°_, O I t - D P A T b ind ing pro- tein may suggest that p r o t e i n - p r o t e i n in teract ion be- tween the t r a n s m e m b r a n e regions might be more im- p o r t an t on the outs ide o f the cell, and lipids, m a n y of which (e.g., PI, PIP2 and PC) are the target o f receptor coupled enzymes (e.g., phosphol ipase C) and the source of second messengers released into the cytosol , m a y form more in tegra ted associa t ions on the inside of the cell.

Acknowledgements

Suppor ted by U S P H S G r a n t HD-06426. We are grateful to Drs. R.L. Rosenf ie ld and M a y a Medn ieks of Endocr ino logy for permi t t ing us to use their dens i tom- eter and Dr. S. Szuchet for providing fresh samples of sheep brain. This invest igat ion was suppor ted by U P H S G r a n t HD-06426.

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