Bic~chimica et Biophysics Acts,

1044 (1990) 305-314

305

Elsevier BBALIP 53412

A s y m m e t r i c extraction of m e m b r a n e lipids by C H A P S + Probal

Banerjee,

John

T. Buse and Glyn

Dawson

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

(Received 15 November 1989)

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

W e 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 ( C H A P S ) . Dialysis of the C H A P S e x ~ a c t pj~duced a [3HIS-hydroxy(2-di-n-propylamino)tetralin ( 1 3 H I S - O H - D P A T ) 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 o f phosphatidylethanolamine (PE), phosphatic|ylcholine (PC), phospltatidylinositot (Pl), phosphatidyl se-ine ( P S ) and phosphatidic acid (PA) in striking preference over cholesterol, galactosylceramides, sulfatides and sphingomyelin. All lipids present in the clear C H A P S - e x t r a c t were coeluted with the 1 3 H I S - O H - D P A T 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, 9 5 - 1 0 0 % of the 1 3 H I S - O H - D P A T binding protein w&s retained in the vesicle-containing pellet. The supernatant contained small atnounts of cholesterol. PE and PC, but virtually no P S , PI, or PA, whereas the vesicular pellet contained all the lipids mentioned, indicating that P S , Pl and PA are more tightly bound to the vesicles than PE, P C and cholesterol S D S - P A G E analysis of the pellet revealed two major protein bands, at 58 k D a and 33.5 kDa, respectively. Our report outlines 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 .

Inlroduction Menlbr~,ne p r o t e i n s a~e o f t e n c o n s i d e r e d in t w o sepa r a t e c a t e g o r i e s b a s e d o n t h e i r m o d e o f ass,~ciation w i t h t h e p h o s p h o l i p i d b i l a y e r [1,2]. E x t r i n s i c o r p e r i p h e r a l p r o t e i n s ar e t h o s e w hi ch a r e r e a d i l y s o l u b i l i z e d by high

Abbreviations: CHAPS, 3-[(3-cholarmdopropyl~limethylammoniol-lpropanesulfonate; SBH. sheep brain homogenate: SBP, sheer' brainpellet; SBDS, sheep Brain detergent-soluble; SBDP. sheep brain detergent pellet; SBDSE, sheep brain detergent-soluble-25% ethyleneglycol-dialyzed; EGSIE, Extracti-OelXM-D-treated SBDS-dialyzed (dialysis 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; 8OH-DPAT, 8-hydroxy-2..(di-n-propylarmnc)tetralin; HPTI.C, I,ighperformance 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 University of Chicago, 5841. S. Maryland Avenue, Box 82, Chicago, IL 60637, U.S.A 0005-2760/90/$03.50 ©

ionic s t r e n g t h , c h e l a t i h g a g e n t s o r a l t e r a t i o n s in p H a n d d o n o t a p p e a r to be b o u n d d i r e c t l y to t h e h y d r o c a r b o n o o r t i o n of t he l i pi d b i l a y e r . I n t r i n s i c o r integral p r o teins, on t h e o t h e r h a n d , are mrJce d g h t l y b o u n d t o th e h y d r o c a r b o n i n t e r i o r a n d s o l u b i l i z a t i u n o f the~e p r o teins is b r o u g h t a b o u t on12~ w i t h a g e n t s w h ic h d i s r u p t the lipid bilayer. In mesz cases, we d o n o t h a v e i n f o r m a tion as to t he s p e c i f i c i t y o f i n t e r a c t i o n with d i f f e r e n t p o r t i o n s o f lipid m o l e c u l e s o r t h e i n f l u e n c e o f b o u n d lipid o n the s t r u c t u r e o f t he p r o t e i n . S e v e ra l in v e stig a t i o n s have s u p p o r t e d t h e idea th a t th e tig h tly b o u n d e n d o g e n o u s lipids d e s e r v e special a t t e n t i o n {3-5]. S t u d ies o n d e t e r g e n t soiubilize.tion o f i n t r i n s i c m e m b r a n e p r o t e i n s h a v e r e v e a l e d t h a t w ith a n i n c r e a s e in d e t e r gent/membrane ratio, p r o t e i n - l i p i d - d e t e r g e n t c o m pl exes are r e l e a s e d f r o m t he m e m b r a n e a n d a t v e r y h i g h detergent/membrane ratios, l i p i d - d e t e r g e n t a n d p r o t e i n - d e t e r g e n t c o m p l e x e s are f o u n d [ 6 - 8 ] . S in c e this is a thermc,d y n a m i c p r o c e s s , t he c o n c e n t r a t i o n o f d e t e r g e n t required to bring about complete delipidation of memb r a n e p r o t e i n s a n d to t o t a l l y s o t u b i l i z e all lipid m o l e cu!es in d e t e r g e n t mieelles d e p e n d s o n t h e r e l a t i v e a f f i n ities o f lipid a n d d e t e r g e n t f o r p r o t e i n . Hc, wever, in o r d e r t o m a i n t a i n the b i o l o g i c a l a c t i v i t y o f a d e t c r g - ,ts o l u b i l i z e d p r o t e i n it is d e s i r a b l e to use an o p t i m u m

1990 Elsevier Science Publishers B.V. (Biomedical Division)

306 concentration of an appropriate detergent, in order to solubilize a protein lipid-detergent complex without removal 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 m e m b r a n e 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}, C H A P S has been used in the solubilization o f 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], G A B A A receptor [19], sex h o r m o n e - b i n d i n g globulin receptor [20], and adenosine receptor A t [21], to n a m e but a few. However, there is a universal problem with most of these receptors: the loss of binding activity in the purified protein preparation. In m o s t studies reported so far, the lipid requirement of a functional protein has been tested by adding specific lipids to solubilized and delip:,dated proteins [22,23]. Our investigation starts from the other end, by identifying the lipids which a protein remains associated with d u r i n g optimal solubilization and centrifugal separation o f 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 a n d PS, which are enriched during C H A P S extraction of the [ 3 H ] 8 - O H - D P A T binding protein, are probabiy responsible for maintaining the appropriate vesicular environment required by the reconstituted and functional receptor. Ma(erials and M e t h o d s

naleri~.,s C H A P S and Extracti-GdXM-D were from Pierce (Rockford, IL). H P T L C plates (10 x 10 cm) o f the H P K t)o, ru~ 00191.6) with 2 crn-wide p r e a d s o r b e n t layer were ,+,btained from W h a t m a n Chemical Separation (Clifton, NJ). The standard lipids (PE, PS, PI, G a l C e r and phrenosin from bovine brain; PA and PC from egg yolk) were obtained from Sigma (St. Louis, MO); densitometric analysis of the H P T L C plates were conducted u.,,ing a G S 300 densitometcr ( H o e f e r Scientific Instrument.~, San Fra" .isco, C A ) All t. reagents a n d 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 1 5 0 ° C for 1 h before use.

Storage of lipid mixture Each standard lipid or extracted lipid mixture was weighed a n d dissolved in a specific volume o f chlorof o r m / m e t h a n o l ( 1 : 1 , v / v ) ( C / M ) a n d stored u n d e r nitrogen in a Teflon seal-screw cap vial at - 2 0 ° C until use.

Membrane preparation and solubilization Sheep brain gray matter, o b t a i n e d from freshly slaughtered sheep, was carefully separated from white matter and p o l y t r o n - h o m o g e n i z e d (dial setting "4") in 10 vols. of buffer A (50 m M Tris-HCi ( p H 7.4), 0.32 M sucrose) at 4 ° C a n d the h o m o g e n a t e (SBH) was centrifuged for I 0 rain at 1 0 0 0 x g ( 4 ° C ) . T h e pellet was resuspended in buffer A, i n c u b a t e d at 37 ° C for 15 rain and then centrifuged at 100000 x g for 20 rain ( 4 ° C ) . The pellet o b t a i n e d was washed twice with 4 ° C deionized water (each time, followed by c e n t r i f u g a t i o n at 100000 × g ) and r e s u s p e n d e d in cold deionized w a t e r at a protein c o n c e n t r a t i o n o f 10 r a g / m ! . This e n n c h e d m e m b r a n e preparation (SBP), which c o u l d be stored indefinitely at - 7 0 ° C w i t h o u t loss o f activity, was extracted with C H A P S (2%) in buffer B (50 m M TrisHCI ( p H 7.4), 1 m M E G T A , 5 m M ascorbic acid, 5 m M MnC! 2 and 1 ~tM p a r g y l i n e ) c o n t a i n i n g 1 ~ M s e r o t o n i n at 4 ° C for 20 rain at a protein c o n c e n t r a t i o n 10 m g / m l ( C H A P S / p r o t e i n , 2 : 1 ( w / w ) ) . T h e solubilized preparation (SBDS) o b t a i n e d was separated from the m e m branes by centrifugation at 1 0 0 0 0 0 x g for 30 min (4 ° C) and then dialyzed against buffer C (25% e t h y l e n e glycol in buffer B) at 4 ° C for 20 h (change o f b u f f e r after 2 h, 4 h and 16 h f r o m the start). The stable, r e c o n s t i t u t e d r e c e p t o r p r e p a r a t i o n thus o b t a i n e d (SBDSE) (containing vesicles o f e n d o g e n o u s p h o s p h o lipids) was enriched by centrifugal separation of the vesicles from 2-ml portions of S B D S E at 100000 x g (5 min) and gentle resuspen~ion of the pellet in buffer C containing 0.3% C H A P S (2 ml) by vortexing a n d pipetting at 4 ° C. This enriched vesicle p r e p a r a t i o n (SBDSP) could be stored in plastic c o n t a i n e r s without freezing at - 20 ° C. Binding assay o f the receptor activity The 8 - O H - D P A T binding activity was assayed by incubation o f a receptor p r e p a r a t i o n (protein _~ 1 mg) in buffer Ba (20 m M Tris-HCI (pH 7.4), 0.4 m M E G T A , 2 m M MnC! 2, 2 m M ascorbic acid and 0.4 ~ M pargyline) at 2 3 ° C in the pre~,~ttce of 9 n M [ 3 H ] 8 - O H - D P A ' r (100000 c p m ) for 20 rain (total v o l u m e 1 ml). N o n specific binding was d e t e r m i n e d in the presence o f 10 p M serotonin. T h e b o u n d ligand was separated f r o m the free by filtration using 0.3% P E l - s o a k e d G F / B filter strips a n d a Brandel M 12R cell harvester following by washes with cold ( 4 0 C ) 10 m M Tris-HCi (pH 7.4), 0.2 m M E G T A (3 X 6 ml) [24].

307 Sheep Brain Homogenate (SBHI , / ~ Supernatant (SBS)

1000 x g, ~ 37 "C & washes

Sheep Brain Enriched Pellet fSBP) .~1) Pellet (SBDP)

2% CHAPS, 1 laM sera.*onin 2) 100 000 x cJ

Supernatant (SBDS) agatnst ~ l l y D j'~I'~zecl Buffzr B ÷ 25% ethylene col

{Complete r e m o v a j . ~ Extraetif Gel-D

of CFL~PS)

D

/

~

.

.B

Vestcles {EGSE)

i

o

1.Sm

SBDSE (vesicles)

Dialysi~ Vesicles fBGSE)

Supernalant (SBDSS)

Pellet (SBDSP) ---I Pellet (EGSP) (BGSP) 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. Pc l~e7

100 000 x g

Bio-Gel A 1.5 m gel-filtration chromatography T h e solubilized p r e p a r a t i o n S B D S (10 ml) was treated at 4 ° C w i t h 5 mi o f Extracti-GelTM-D f o r c o m p l e t e r e m o v a l o f C H A P S a n d a p a r t (8 ml) o f the d e t e r g e n t froc f r a c t i o n thus obtaine, d w a s s u b j e c t e d to gel filtration over 250 ml o f B i o - G e l A 1.5 m using b u f f e r B c o n t a i n i n g 0.1 M K O A c a n d 0.1% C H A P S [11]. T h e e x c l u s i o n (Vo) a n d total ( V i) v o l u m e s were d e t e r m i n e d u s i n g Blue D e x t r a n 2 0 0 0 0 0 0 a n d [ 3 H I g - O H - D P A T ( 1 0 0 0 0 0 c p m ) , respectively. A p a r t o f the s a m p l e l o a d e d (2 ml) w a s d i a l y z e d a g a i n s t b u f f e r C at 4 ° C to o b t a i n a [ 3 H ] 8 - O H - D P A T b i n d i n g fraction ( E G S E ) w h i c h gave an i d e a a b o u t the activity l o a d e d o n the c o l u m n . T h e c o l u m n f r a c t i o n s were d i a l y z e d against b u f f e r C a n d a s s a y e d for [ 3 H ] 8 - O H - D P A T b i n d i n g activity. T h e fraction (exclusion) c o n t a i n i n g the p e a k o f activity ( B G S E ) w a s s u b j e c t e d to lipid analysis a l o n g with E G S E . O n an average, the r e c o v e r y of [ 3 H ] 8 - O H - D F A T b i n d i n g activity a f t e r c o l u m n c h r o m a t o g r a p h y w a s 40%. C~.nf~rmation o f vesicle formation by electron microscopy T h e vesicles in S B D S E were c e n t r i f u g e d at 200 000 x g for 20 rain to o b t a i n a pellet w h i c h w a s fixed with 2% g l u t a r a l d e h y d e in 0.12 M T r i s - H C l ( p H 7 ~) a n d then t r e a t e d with 1% OsOa. F o l l o w i n g this, the pellet w a s e m b e d d e d in e p o x y resin, c a r e f u l l y c u t a n d m a d e into t h i n - s e c t i o n s w h i c h w e r e t r e a t e d with u r a n y l a c e t a t e a n d R e y n o l d s lead citrate. E l e c t r o n m i c r o s c o p y w a s carried at 6 0 k e V using a Philips 201 e l e c t r o n m i c r o s c o p e .

Spectrophotometric determination of C H A P S A s t a n d a r d curve w a s o b t a i n e d b y m e a s u r i n g abs o r b a n c e s of v a r i o u s c o n c e n t r a t i o n s ( 0 - 1 . 2 m g / m l ) o f C H A P S in d e i o n i z e d w a t e r at 2 " 0 nm. In o r d e r to m o n i t o r the r e m o v a l o f C H A P S d u r i n g dialysis o f the d e t e r g e n t - s o l u b i l i z e d fraction S B D S . a parallel experim e n t w a s run, in which several 3-ml p o r t i o n s of a 2% solution o f C H A P S in w a t e r w a s dialyzed against w a t e r at 4 ° C . Dialysis bags w2rc r e m o v e d after the i n d i c a t e d t i m e p e r i o d s of dialysis (Fig. 2b) a n d following 20-fold dilution o f 100-#1 aliquots, ultraviolet a b s o r b a n c e o f the fractions m e a s u r e d at 210 nm. Next, from the s t a n d a r d curve, it was p o s s i b l e to d e t e r m i n e the c o n c e n t r a t i o n profile o f C H A P S o b s e r v e d d u r i n g dialysis. Similar e x p e r i m e n t s were not p o s s i b l e in the p r e s e n c e o f b u f f e r C o r b u f f e r B, w h i c h a b s o r b strongly at 210 nm. Exiraction of lipids and H P T L C anah'sis T h e entire p r o c e s s o f m e m b r a n e p r e p a r a t i o n , so!ubilization and lipid analysis is o u t l i n e d in Fig. 1. T h e solubilized p r e p a r a t i o n ( S B D S ) w a s t r e a t e d with Extracti-GeiTM-D (3 ml wet v o l u m e o f gel for 10 ml o f S B D S ) at 4 ° C to r e m o v e m o s t of the C I t A P S p r e s e n t (so that C H A P S did n o t m a s k the o t h e r b a n d s d u r i n g H P T L C analysis) a n o then m i x e d with c h l o r o f o r m / m e t h a n o l (1 : 1, v / v ) ( C / M ) (15 ml for every 2.5 ml of E x t r a c t i - G e l - t r e a t e d SBDS). T h e m i x t u r e was stirred o v e r n i g h t in the dark u n d e r nitrogen in a sealed screwc a p t u b e at rot, m t e m p e r a t u r e ( 2 3 - 2 5 ° C ' and t)-:'):

308 celltrifuged at 2500 r p m using a table-top centrif,Jge to s e p a r a t e the p r e c i p i t a t e d proteins. T h e superr.atant ,~'as e v a p o r a t e d at 3 0 ° C , tbe residue dissolved in 5 ml of C / M a n d the protein precipitate o b t a i n e d was sepa r a t e d b y centrifugation. E v a p o r a t i o n of this s u p e r n a t a n t p r o d u c e d a m i x t u r e of lipids which dissolved c o m p l e t e l y in C / M . T h e soluiic, n was q u a n t i t a t i v e l y transferred to a pre-weighed, T,:flon seal s c r e w - c a p vial a n d e v a p o r a t e d b y blowing d r y n i t r o g e ~ T h e residue w a s weighed dissolved in a specific v o l u m e ol C / M in o r d e r to m a k e a s t a n d a r d solution a n d then the s o l u t i o n w a s stored at - 2 0 ° C . The m e m b r a n e - b o u n d p r e p a r a tions ( S B H a n d SBP) were e x t r a c t e d in the s a m e w a y ( w i t h o u t the E x t r a c t i - G e l t r e a t m e n t ) using 15 ml o f C / M for 2.5 ml o f S B H or SBP. T h e vesicular p r e p a r a t i o n ( S B D S E ) (1 ml) w a s c e n t r i f u g e d at 1 0 0 0 0 0 x g for 1 h at 4 ° C and a f t e r s e p a r a t i n g the s u p e r n a t a n t ( S B D S S ) , the pellet ( S B D S P ) c o n t a i n i n g the vesicles was e x t r a c t e d with 5 ml of C / M . C e n t r i f u g a l s e p a r a t i o n o f the p r e c i p i t a t e d p r o t e i n s at 2500 rpm, e v a p o r a t i o n o f the s u p e r n a t a n t a n d reprecipitation o f p r o t e i n s after C / M solubilization o f the residue p r o d u c e d a purified m i x t u r e o f iipids retained in the vesicles. T h e s u p e r n a t a n t ( S B D S S ) w a s e x t r a c t e d three times (3 × 2 ml) with c h l o r o f o r m and the p o o l e d extract w a s w a s h e d o n c e with w a t e r a n d then e v a p o r a t e d to o b t a i n a residue which was a n a l y z e d for t h e p r e s e n c e of lipids a n d C H A P S . A l i q u o t s (1-ml e a c h ) of the Bin-Gel A 1.5 m e x c l u - i o n fraction ( B G S E ) a n d dialysis p r o d u c t (E(3SE) o f the s a m p l e l o a d e d on the c o l u m n were c e n t r i f u g e d as for S B D S E a n d the pellets o b t a i n e d ( B G S P a n d E G S P , respectively) were e x t r a c t e d in the s a m e w a y with C / M iv o b t a i n the lipids retained in the vesicles. H P T L C analysis of lipids w a s c o n d u c t e d b y s p o t t i n g s t a n d a r d a m o u n t s o f lipids on the s a m e plate along the S B H - , SBP-, S B D S P - a n d SBDSS-iipids. T h e plates were d e v e l o p e d u p to 5 c m from the origin with ethyl acetate/1-propanol/chloroforro/methanol/0.25% KC1 (25 : 25 : 25 : 10 : 9, v / v ) , dried at r o o m t e m p e r a t u r e for a b o u t 15 min a n d then d e v e l o p e d full length w i t h hexane/diethyl ether/acetic acid ( 7 5 : 2 1 : 4 , v/v) (solvent c o m b i n a t i o n I). This a f f o r d e d better s e p a r a t i o n o f the b a n d s slower m o v i n g t h a n PC, b u t the s e p a r a t i o n b e t w ~- " ~ ~" -._'.ds f,~ PI a n d PS w a s not e n o u g h for reliable, den., ,,~metric e s t i m a t i o n . In o r d e r to i m p r o v e r e s o l u t i o n o ; PI a n d PS, the c o m p o s i t i o n o f the first solvent was c h a n g e d to ethyl -cetate/1-propanol/ ch!oroform/methanol/0.25% KCI (25 : 25 : 20 : 15 : 9, v / v ) (solvent c o m b i n a t i o n It). A f t e r dry:p~ t h e plates, the b a n d s were viewed b y s p r a y i n g cuprit, p h o s p h o r i c acid charring reag~-nt (10% C u S O 4 in 8% P l 3 r / , ) a n d heating at 1 8 0 ° C for 10 rain [251.

Densitometry and quantitation Each lane w a s scamaed in at least three s e p a r a t e lines

using a G S 300 dens, t o m e t e r in the t r a n s m i t t a n c e m o d e with a visual light b e a m ~ 5 7 0 - 6 0 0 rim) the d i m e n s i o n o f which was 0.25 x 2.54 mrra. T h e s c a n s p e e d was set at 4 c m / m i n a n d the p e a k a r e a s ( + S.E.) o b t a i n e d using (3aussian i n t e g r a t i o n were p l o t t e d using C r i c k e t G r a p h 1.2 (Cricket S o f t w a r e , M a l v e r n , PA 19355). Even t h o u g h r e p r e s e n t a t i v e H P T L C p l a t e s are s h o w n here, e a c h q u a n t i t a t i o n w a s c a r r i e d o u t three times with f r a c t i o n s o b t a i n e d f r o m three d i f f e r e n t b a t c h e s o f fresh a d u l t sheep brain g r a y m a t t e r . T h e m a s s o f e a c h lipid p r e s e n t in a lipid m i x t u r e (e.g., S B P - e x t r a c t , Fig. 6a, a n d S B D S P - e x t r a c t , Fig. 6b) w a s d e t e r m i n e d f r o m s t a n d a r d curves (e.g., Fig. 6a a n d b) o b t a i n e d b y s p o t t i n g increasing m a s s e s o f s t a n d a r d lipids a n d t h e n t h e results w e r e c r o s s - c h e c k e d 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 p r o f i l e s o b t a i n e d b y s c a n n i n g the l a n e s o n the H P T L C p l a t e s s h o w n in Fig. 5 ~. a n d b, Fig. 7a a n d b a n d Fig. 8.

Measurement of protein P r o t e i n w a s d e t e r m i n e d u s i n g t h e B r a d f o r d a s s a y [26] or a m o d i f i e d L o w r y a s s a y [27].

O.1% SDS-PA GE of proteins S a m p l e s w e r e held in b o i l i n g w a t e r in t h e p r e s e n c e o f dithiothreitol (20 m M ) a n d 2% ~odium d o d e c y l s u l f a t e ( S D S ) for 2 m i n a n d then a n a l y z e d b y o n e - d i m e n s i o n a l gel e l e c t r o p h o r e s i s o n h o m o g e n e o u s , 10% p o l y a c r y l a m i d e gels c o n t a i n i n g 0.1% S D S . F r a c t i o n s l o a d e d w e r e n o r m a l i z e d to p r o t e i n (12 /.tg) e x c e p t in l a n e 1, w h i c h received 4 # g of p r o t e i n , s i n c e a larger v o l u m e o f this fraction ( S B P ) w a s f o u n d to c a u s e e x c e s s i v e b a c k g r o u n d staining. E l e c t r o p h o r e s i s w a s c a r r i e d o u t at 120 V (constant) with w a t e r c o o l i n g a n d t h e gels w e r e s t a i n e d using a B i o - R a d st!vet stain kit. A f t e r staining, t h e gel w a s t r e a t e d for 15 rain w i t h A m p l i f y to e n h a n c e the 14C-labeled s t a n d a r d b a n d s a n d t h e n d r i e d at 8 0 ° C for 1 h, f o l l o w i n g w h i c h the s t a n d a r d lane w a s cut o u t a n d i n c u b a t e d with an X - r a y film at - 7 0 ~ C for 3 d a y s . T h e a u t o r a d i o g r a m strip o b t a i n e d was aligned with t h e rem a i n i n g p o r t i o n o f the gel to d e t e r m i n e m o l e c u l a r w e i g h t of the p r o t e i n b a n d s o b t a i n e d . Results

Evidence for vesicle formation and retention of [31-118OH-DPA T binding activity in the vesicles Electron m i c r o s c o p y of t h e pellet o b t a i n e d b y cent r i f u g a t i o n of S B D S E reveal,-d the p r e s e n c e o f vesicles, d i m e n s i o n s o f w h i c h r a n g e d f r o m 200 to 2000 / i (electron m i c r o g r a p h n o t s h o w n ) . A f t e r c e n t r i f u g a l sepa r a t i o n of the vesicles f r o m S B D S E a n d S D S - P A G E analysis o f the e n r i c h e d p r e p a r a t i o n , S B D S P (as des c r i b e d in M a t e r i a l s a n d M e t h o d s ) , it w a s f o u n d chat S B D S P d i s p l a y e d t w o 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 a n d 33.5 k O a r e s p e c t i v e l y (Fig. 2, lanes 3 a n d 4). [ 3 H ] 8 - O H - D P A ° F b i n d i n g a s s a y s o f S B D S E (Fig. 2, l a n e

309

2

3

4

5

6 MrxlO "3

ql2.5

2) a n d S B D S P s h o w e d t h a t 05 100% o f t h e b i n d i n g a c t i v i t y w a s r e t a i n e d in the ~esicles (Fig. 2. i n s e t ) w h e r e a s t h e s u p e r n a t a n t ( S B D S S , Fig. 2, l a n e 5) left a f t e r s e p a r a t i o n o f the v e s i c l e s e x h i b i t e d n o b i n d i n g activity.

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 n e a r u p t o 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) with a first-order plot allowed estimation of this deterg e n 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 l y s i s a g a i n s t d e i o n i z e d w a t e r ( F i g . 3b). T h e h y p e r b o l i c n a t u r e o f t h e profile, which showed that the CHAPS content of 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 t h e 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 i o n o f vesicles) a f t e r 3 - 4 h o f d i a l y s i s .

t;9

46

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

30

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) w a s 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 [3HJSOH-DPAT binding) of SBDSE and SBDSE

Comparison with standard lipids by HPTLC showed t h a t c h o l e s t e r o l (C), P E , PS. p h o s p h a t i d y l i n o s i t o l ( P I ) . PC and GalCer were the major components of 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 a n e 5) w h i c h a l s o contained phosphatidic acid (PA) and sulfatides (Su) ( F i g . 4a). I n 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 b e s e e n to b e 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 , S u a n d c h o l e s t e r o l ( C ) ( F i g . 4a, l a n e 4). A l k a l i n e m e t h a n o l y s i s o f S B P , 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 and HPTLC analysis of the products confirmed the v i r t u a 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 i g . 4b. l a n e 1). A f t e r d i a l y s i s o f STUDS, w h e n C H A P S c o n t e n t w a s r e d u c e d t o 0.04% ( w / v ) , a s m a 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 t h e vesicles o b t a i n e d ¢ S B D S P , Fig. 5a, l a n e 2). G a l a c t o s y l c e r a m ~ d e s ( H F A - G a l C e r or phrer=o.dn, 8.6% a n d N F A - G a l C e r . 3.8q~ o f l i p i d s ) in S B P (Fig. 5a. l a n e 2), w e r e r~resent in m a r k e d l y r e d u c e d

y = - 6 5 2 1 2 e - 2 . | 7331x R'2 =09.97

E

2.0

J

f

EL

1

~

1.0

,0

0

.

0.0

(a)

.

.

.

,

.

.

0.5

.

.

T

1.0

.

.

.

.

! .S

O,C . . . . . . . . 0 1 0

T, 2o

--

(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. CHAPS

310 TABLE I

Proportions o f lipids pr~'sent in the membrane-bound and reconstitutedsoluble preparations T h e p e r c e n t c h a n g e in each lipid w i t h respect to S B P is n o t e d in p a r e n t h e s e s . T h e p r o p o r t i o n o f each l i p i d was o b t a i n e d w i t h r e s p e c t t o t h e sum o f masses o f all t h e m a j o r a n d m i n o r lipid c o m p o n e n t s as o b t a i n e d f r o m d e n s i t o m e t r i c assay. T h e values p r e s e n t e d h e r e a r e the a v e r a g e of t~-ree diffe, e n t e x p e r i m e n t s c a r r i e d o u t w i t h t h r e e 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 a n d e v e n t h o u g h t h e t o , a l m a s s o f lipids v a r i e d w i t h i n 3%, t h e p r o p o r t i o n of e a c h lipid varied "~pithin o n l y 1%. ( w / w ) o f e s t i m a t e d Upids

Cholesterol NFAGalCer HFAGalCer (phrenosin) Sphingomyelin Phosphatidylet hanolamine Phosphatidylinositol Phosphatidylserine Phosphatidylcholine CHAPS

~-.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 c o e x t r a c t e d lipids w i t h s t a n d a r d s . ( a ) Lipids f r o m S B P (25 lag) ( l a n e 5) a n d S B D S (25 ~tg) ( l a n e 4) were c o m p a r e d with PE, PC ( l a n e 1, 10 ~tg each)), C, PS ( l a n e 2, 10 ~tg each), Pl 0 a n e 3, 10 lag) P A ( l a n e 6, 2 0 lag), C H A P S ( C H , l a n e 7, 50 ~xg)) a n d s u l f a t i d e s (su) ( l a n e g, 5 lag). ( b ) A l k a l i n e m e t h a n o l y s i s p r o d u c t s of S B D S ( l a n e 1, 100 lag), S B H ( l a n e 2, 100 lag) a n d S B P ( l a n e 3, 100 lag) were c o m p a r e d w i t h glycolipid s t a n d a r d s ( l a n e 4, 100 lag), L a c C e r (5 lag) a n d G b O s e 3 ( G b 3 , 10 lag) ( l a n e 5), galactosyl c~_-~ramides ( G a l C e r , 10 ~tg) a n d a s i a l o G M I ( G g 4 , 2 lag) ( l a n e 6), p h r e n o s i n ( h y d r o x y fatty acid g a l a c t o s y i c e r a m i d e , 5 lag) a n d C H A P S (50 jag) (lame 7). B o t h plates were d e v e l o p e d u s i n g s o l v e n t c o m b i n a t i o n !1.

SBP

SBDSP

EGSP

12.8 3.8 8.6 5.8 33.2 4.6 19.3 12.0 0

8.0 1.3 3.0 < 0.1 43.3 8.6 10.5 17.5 7.8

%7 ( -- 4 0 ) 0.86 ( -- 7 7 ) 1.9 ( -- 7 8 ) < 0.1 40.7 ( + 23) 10.3 ( + 124) 21.0 ( + 9) 17.6 ( -*-4 7 ) < 0.I

( - 38) ( - 66) ( -- 6 5 ) (+ (+ ((+

30) 87) 46) 46)

a m o u n t s (3% and 1.3% o f lipids, respectively) in S B D S P (Fig. 5a, lane 3) (Table I).

Depletion in sphingomyelin during C H A F S extraction In order to resolve the b a n d s for sphingomyelin (Sin) from the o t h e r bands, a n H P T L C plate was developed using a solvent of higher polarity ( 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 ) . W e observed that neither sphingomyelin b a n d s ( c o n t a i n i n g long- and short-chain fatty acids, respectively), present in SBH (Fig. 5b, lane 1) and SBP (lane 2), were extracted by C H A P S (SBDSP, Fig. 5b, lane 4 a n d EGSP, lane 6) (Table i). In contrast, the detergent-insoluble pellet showed m a r k e d l y increased c o n t e n t o f Sm (1.4times that in SBP by densitometry, Fig. 5b, lane 3). T h e dark b a n d below Sm in lane 3 is for C H A P S and the lower most b a n d s (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), S B D S P (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 c h l o r o f o r m / m e t h a n o l / a c e t i c acid/water (65 : 25 : 8.8 ; 4, v / v ) .

311

"-C

PEPS-,,PC

CH,..

i

1

2

3

4

t

5

6

-

-

y = 0.77'! 1S * 0.5d14~ - 4-71,11~1-3x"2

i

°1 1 |

/-

x"

/

~ //"

,J

.~.-//

10

20

I y ~ 1,1230 *

. a in.2= oJ.~

I- ",,,,~-~ I I''°'s'*'°~" ~.=m II" ~ ma,e [ Iv =. o~==~, ozamk a,'~. o.

1.2161I - Lg~911-21"2

[y. -0.471~I.0~I11~t~ R"2=OIIPD I" otmi~am~ o ~ pm~ mj,1 ~oc~ lY = O ~ * O . t R'2 = Ores l" O-W~S( v ~ m . ~ o ~

1

O-

0

(a)

30

L i p i d (p.g)

0

(b)

tO

20

3G

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 p g (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 c h a r r e d ) in lanes 1 and 2 are for unidentified c o m p o n e n t s of SBH and SBP. Densitometric quantitation o f the lipids a n d C H A P S content o f m e m b r a n e extracts T h e linear range of the s t a n d a r d curves for the phospholipids ( 0 - 8 0 Fg) (only a part of the range is shown here) and C H A P S ( 0 - 4 0 / , g ) (two t~pical H P T L C plates s h o w n in Fig. 6a and b) were used. T h e nature and gradient of the individual s t a n d a r d curves varied to a certain extent b u t those for G a l C e r (phrenosin), Sm and cholesterol usually followed second-order equations. Variation o f results obtained from different batches of sheep brain was always less than 1% a n d Table i is a n average of results o b t a i n e d from three such experiments in which densitometric scan of each lane was carried out in three to five dist2nct straight lines. A t t e r staining, each H P T L C plate was kept sealed f r o m air a n d light and subjected to d e n s i t o m e t r y within 24 h. T h e stain was f o u n d 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 a n d is enriche.~ in GalCer, S m a n d cholesterol A comparison of the lipid profiles of the original h o m o g e n a t e (SBH), the enriched 1000 x g m e m b r a n e pellet (SBP), the C H A P S extract (SBDS), C H A P S - i n -

soluble pellet (SBDP) a n d the vesicle containing pellets. SBDSP a n d E G S P (Fig. 1) revealed that the phosphoglycerides were coextracted with the serotonin [3H]8O H - D P A T binding activity (in SBDSP mid E G S P ) in preference to the sphingolipids (galactosylceramides and sphingomyelin) a n d cholesterol (Fig. 7a, c o m p a r e SBP, lane 2, SBDSP, lane 4 a n d 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 a n d PC. It was also noticed (Fig. 8) that the relatively less intense b a n d for free fatty acid (checked separately with respect to oleic acid, although the s t a n d a r d is not shown here), which moves faster than cholesterol, was enriched in the soluhilized fractiops (SBDSP, lane 4 a n d EGSP, lane 6) and completely absent in the pellet (SBDP, lane 3). Investigation o f the a p p a r e n t reduction o f PS (Table I a n d Fig. 7b) in SBDSP showed that a b o u t 5 0 ~ o f the solubilized PS a n d 17% of PI were dialyzed out along with C H A P S during the dialysis of SBDS. This depletion of coextracted PS a n d PI had no effect on [3H]8O H - D P A T binding activity o f the vesicles a n d did not occt;r when C H A P S was completely removed from SBDS by Extracti-GeLD t r e a t m e n t 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 e a t e d S B D S ( E G S P ) (Fig. 7b, lane 2) were retained, in t h e s a m e p r o p o r t i o n , a f t e r B i o - G e l A 1.5-m gel-filtration c h r o m a t o g r a p h y a n d dialysis o f the e x c l u d e d fraction w h i c h c o n t a i n e d the p e a k of r e c e p t o r activity ( B G S P , Fig. 7b, l a n e 3) c o n f i r m i n g that C H A P S (a C H A P S - P S / P I c o m p l e x is p r o b a b l y f o r m e d at higher C H A P S c o n c e n t r a t i o n s ) w a s n e c e s s a r y for the partial r e m o v a l of PS a n d PI. H P T L C analysis f o l l o w i n g C / M e x t r a c t i o n of the s u p e r n a t a n t ( S B D S S ) a n d the [ 3 H ] 8 - O H - D P A T b i n d i n g pellet ( S B D S P ) s e p a r a t e d f r o m S B D S E b y c e n t r i f u g a tion s h o w e d that a small a m o u n t o f C H A P S (50 # t g / m g of solid lipid mixture, e q u i v a l e n t to 0.04% C H A P S in S B D S E , w / v ) was retained in t h e vesicles, w h e r e a s the s u p e r n a t a n t ( S B D S S ) w a s totally d e v o i d o f t h e detergent.

PS, P I and PA are retained completely by" the vesicular pellet ( S B D S P ) T h e s u p e r n a t a n t ( S B D S S , Fig. 7b, lane 4), w h i c h c o n t a i n e d o n l y 0.3 m g o f lipid c o m p a r e d t o 7.4 m g p r e s e n t in S B D S P (Fig. 7b, l a n e 1), w a s f o u n d to c o n t a i n P E , P C a n d c h o l e s t e r o l b u t e s s e n t i a l l y n o PI, P A or PS. This suggests that e v e n t h o u g h s o m e loss of P S a n d PI o c c u r r e d d u r i n g dialysis o f S B D S ( w h i c h h a d n o effect o n the [ 3 H ) 8 - O H - D P A T b i n d i n g activity) (Fig. 2, inset), t h e rest o f PS a n d PI p r e s e n t in S B D S P , a l o n g w i t h PA, w e r e m o r e tightly b o u n d to the p r o t e i n s a n d lipids in t h e vesicles ( S B D S P ) . Lipid-protein stoichiometry in the vesicles A k e y f e a t u r e in r e c o n s t i t u t i o n o f f u n c t i o n a l p r o t e i n 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 c o n t a i n i n g the e n r i c h e d a n d active p r o t e i n . A f t e r s e p a r a t i o n of the vesicles f r o m S B D S E b y c e n t r i f u g a tion a n d r e s u s p e n s i o n as d e s c r i b e d in the text, w e o b t a i n e d S B D S P ( 2 - 2 . 5 m g / m l o f p r o t e i n ) , w h i c h ret a i n e d 9 5 - 1 0 0 % o f t h e solubilized [ 3 H ] 8 - O H - D P A T b i n d i n g activity ( p r e s e n t in S B D S E ) a n d 7.4 m g o f t o t a l lipid p e r ml. T h i s v a l u e a b o u t t h e total m a s s o f S B D S P lipids w a s o b t a i n e d b y s u m m a t i o n o f m a s s e s o f all the m a j o r a n d m i n o r tipids 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. T h e r e f o r e , l i p i d : p r o t e i n s t o i c h i o m e t r y o f the vesicles c o n t a i n i n g the solubilized [ 3 H ] 8 - O H - D P A T b i n d i n g p r o t e i n w a s 3 - 4 : 1. Discussion

1

2

3

4

5

C

FiB. 8. Comparison of lipid profiles of SBP, SBDSP, '-GS r" and SBDP. 100 p g of each o f 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.

T h e q u e s t i o n o f solubilizing a f u n c t i o n a l p r o t e i n a l o n g with the closely a s s o c i a t e d lipids has b e e n o c c a sionally a d d r e s s e d [8]. I n d i r e c t s t u d i e s h a v e b e e n c a r r i e d o u t [221, o n d e l i p i d a t e d p r o t e i n s in o r d e r t o d e t e r m i n e

313 the lipids essential for reconstitution of activity. Micellar e n z y m o l o g y [28], the new trend in molecular biology involving catalysis by enzymes e n t r a p p e d in hydrated, reversed micelles c o m p o s e d of surfactants (phospholipids and detergents) in organic solvents, lends further support to studies and analysis of lipids specifically associated to functional proteins. The proteins of concern in all these studies were enzymes, but the same could be true for the receptors, such as the 5-HT~^ receptor protein. D u r i n g optimization o f conditions for detergent solubilization o f the [ 3 H ] 8 - O H - D P A T binding activity, we have f o u n d that s o m e detergents, like octy! glucoside, Nc-aidet P--40 and Triton X-100 p r o d u c e weakly active solubilized preparations which contain very small a m o u n t s o f lipids (unpublished observation). As a result of this, unlike in tne cas= of C H A P S extraction, it was not possible to obtain a highly active, [ 3H]8_OH.DPAT binding, vesicular p r e p a r a t i o n of the binding protein f r o m these detergent extracts. Even though the exact lipid requirement of the receptor is yet u n k n o w n , in order to retain a receptor-transducer c o m plex in a functionally active state, it appears essential to reconstitute the a p p r o p r i a t e c o m p o n e n t s in the correct c o n f o r m a t i o n . To initiate this we need to u n d e r s t a n d the lipid e n v i r o n m e n t s u r r o u n d i n g the complex. In o r d e r to m o v e towards this goal we have analyzed the lipids present in the m e m b r a n e - b o u n d form o f a G-proteincoupled receptor, the 5-HT~A receptor, and c o m p a r e d the results with the lipid profiles o b t a i n e d from various solubilized preparations which retain [ 3 H ] 8 - O H - D P A T binding activity. T h e zwitterionic c o m p o u n d , C H A P S , was the detergent which gave o p t i m u m solubilization of the receptor-lipid complex. T h e easy spectrophotometric assay described by us for quantitating the a m o u n t o f C H A P S can be used for a solution of the detergent in water and the densitometric assay described gives a general m e t h o d of assaying both detergents and lipids based o n t r a n s m i t t a n c e measurements. The use of an inexpensive oensitometer, GS 300 (Hoefer), to obtain reproducible results was m a d e possible by the measurem e n t of transmittance instead o f reflectance, which is the m o r e c o m m o n l y used p a r a m e t e r for scanning thinlayer c h r o m a t o g r a m s [29]. The major a d v a n t a g e of this technique is that quantitation is linear up to 80 /Jg for the phospholipids. Using this m e t h o d we could also d e t e r m i n e the a m o u n t o f C H A P S remaining in the vesicles obtained after dialysis of the solubilized receptor preparation, T h e m e t h o d was sensitive enough to indicate that while a 20 h dialysis would completely remove C H A P S f r o m an aqueous solution, the [3il]8O 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 a m o u n t (0.04% o f SBDSE, w / v ) of this detergent which obviously rem a i n e d tightly associated with the rest of the lipids a n d

proteins. T h e m o d e of this a_-~ociation has been discussed earlier by T a n f o r d 18]. It may be argued that ~hc liptd composition discussed here may be specific for sheep brain. T o answer this question we have analyzed rat brain m e m b r a n e samples p r e p a r e d in the same way a n d f o u n d that it contains the same lipids a n d in very simdar proportions as f o u n d in SBP (data not inciuded for tF,e sake of brevity). So, even though some vari,=t~on ~,: , ~ . - =.:_~,~ tent a m o n g different species is expected, yet the need for co-solubilization of lipids in o r d e r to conserve the native c o n f o r m a t i o n of solubilized, functional proteins is i n d e p e n d e n t of the source of the functional proteins. W e have c o m p a r e d the lipids present in the m e m b r a n e - b o u n d and solubilized preparations of the receptor a n d shown that some of those lipids were solubilized in d r a m a t i c preference over others. The phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and free fatty acids were enriched d u r i n g solubilization and reconstitution, whereas there was a m a r k e d depletion in cholesterol and a d r a m a t i c decrease in sphingomyelin and galactosylceramides. Phosphatidylserine a n d some phosphatidylinositol were partia|ly removed, p r o b a b l y as C H A P S - c o m p l e x d u r i n g dialysis of the detergent extract. However, this depletion did not affect [~H]8O } i - D P A T binding activity of the vesicles. Centrifugal separation o f the vesicles present in the reconstituted preparation showed that all PA, PI a n d PS were retained in the pellet, while small a m o u n t s of PE, PC a n d cholesterol could be released into the supernatant. This suggested that the remaiping portions of the lipids PA, PI a n d PS were strongly b o u n d to the p~oteins and lipids retained in the vesicles. W e believe that the lipid composition of the active receptor p r e p a r a t i o n (SBDSE) is a function of the properties of both C H A P S (in solubilizing certain lipid-protein complexes as a whole, without disruption) and the m e m b r a n e proteins (e.g., the 5-HT~A receptor) which are responsible for the fine control of this lipid-protein association. M a n y detergents have been us*_d ,-,n ~.n ,-~pirical basis to solubilize ligand-binding activity frc~m membranes but no clear rationale has been provided for their selection. While a more detailed~ c o m p a r a t i v e analysis of extraction of lipids by various detergents is being undertaken, our present set of observations should be particularly useful in the study of various functional proteins. T h e second question, which is i m p o r t a n t in developing reeonstitution procedures, is about removal o f the detergent in o r d e r to forth vesicles from the extracted lipids. We have found that although o p t i m u m solubilization of lipids was achieved at 2% C H A P S , which is higher than its critical micellar c o n c e n t r a t i o n (approx. 0.5%), the detergent could be removed without the use of any h y d r o p h o b i c resins (which often adst, rb

314 h y d r o p h o b i c proteins), simply b y dialysis. T h e 5-HT~A a n d related receptors are p r e d i c t e d to have seven t r ~ m e m b r a n e regions [30], based on the h y d r o p h o b i c i t y o f predicted a m i n o acid sequences a n d there is no obvious reaso~ p e r se w h y the r e c e p t o r p r e p a r a t i c n should be e n r i c h e d in those lipids which are p r e d o m i n a n t l y a s s o c i a t e d with thc c y t o p l a s m i c face of the p l a s m a m e m b r a n e . O u r d a t a s u p p o r t the idea t h a t sphingolipids, t o g e t h e r with a significant p o r t i o n of t h e cholesterol pool m a y exist in d o m a i n s which are discrete f r o m the r e c e p t o r p r o t e i n s a n d have quite a d i f f e r e n t type of functic, n i: the m e m b r a n e . Tt-.e glycolipids a n d s p h i n g o m y e l i n are k n o w n to f o r m the o u t e r l a y e r or the antigenic d e t e r m i n a n t s o f a cell [31-33], w h e r e a s the p h o s p h o l i ~ i d s like PE, PS, PI a n d PAL p r e d o m i n a t e in the i n n e r b o d y o f the m e m b r a n e lipid bilayer a n d act as a source of s e c o n d messengers; PC a n d c h o l e s t e r o l o c c u r in both. Such a s y m m e t r i c d i s t r i b u t i o n of lipids specifically in b r a i n m e m b r a n e s and the effect o f such e n v i r o n m e n t on t h e f u n c t i o n a l activity of s o m e m e m b r a n e p r o t e i n s h a v e been d o c u m e n t e d b e f o r e [34-39]. T h e fact t h a t we have o b s e r v e d preferential soluhilization of the i n n e r m e m b r a n e lipid e ~ m p o n e n t s (except for PC) a l o n g w i t h the [3HI°_, O I t - D P A T b i n d i n g p r o tein m a y suggest t h a t p r o t e i n - p r o t e i n i n t e r a c t i o n bet w e e n the t r a n s m e m b r a n e regions m i g h t be m o r e imp o r t a n t o n the o u t s i d e o f the cell, a n d lipids, m a n y o f w h i c h (e.g., PI, PIP2 a n d PC) are the target o f r e c e p t o r c o u p l e d e n z y m e s (e.g., p h o s p h o l i p a s e C) and t h e source of s e c o n d messengers released into the cytosol, m a y f o r m m o r e i n t e g r a t e d a s s o c i a t i o n s on the inside of t h e cell.

Acknowledgements S u p p o r t e d b y U S P H S G r a n t HD-06426. We are grateful to Drs. R.L. R o s e n f i e l d a n d M a y a M e d n i e k s of E n d o c r i n o l o g y for p e r m i t t i n g us to use their d e n s i t o m eter a n d Dr. S. Szuchet for p r o v i d i n g fresh s a m p l e s of sheep brain. This i n v e s t i g a t i o n w a s s u p p o r t e d b y U P H S G r a n t HD-06426.

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