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Isolation of an organ specific protein antigen from cell-surface membrane rat liver
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BIOCHIMICAET BIOPHYSICAACTA
BBA 35173 ISOLATION OF AN ORGAN SPECIFIC P R O T E I N A N T I G E N FROM CELLSURFACE MEMBRANE OF RAT L I V E R
DAVID M. NEVILLE, JR. Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Md. (U.S.A.)
(Received August I4th. 1967)
SUMMARY
I. A single membrane protein accounting for roughly lO% of the total membrane protein has been isolated from alkali extracts of rat liver cell membranes by preparative disc electrophoresis at pH 2.7 in urea. 2. A Stokes radius of 58/~ and a molecular weight of 7 ° ooo has been estimated for the protein by gel filtration in 8 M urea. 3. Divalent cations are involved in the binding of the protein to the membrane matrix. Dialysis of membranes against ! mM EDTA solubilizes the protein at neutral pH. 4. Antisera prepared against the purified protein in sheep yield a single precipitin line after immunodiffusion with EDTA extracts of liver and liver cell membranes. 5. The protein is apparently organ specific not being detectable in rat plasma or E D T A extracts of erythrocytes, kidney, spleen, intestine, pancreas, muscle or adipose tissue. In addition the protein appears to be membrane specific not being detected in extracts of rat liver nuclei, mitochondria, endoplasmic reticulum or cell sap. The protein has been localized to the cell surface membrane of intact hepatocytes b y immunofluorescence. 6. The liver membrane protein could not be detected in extracts of Morris hepatoma but was found to be present in a highly differentiated second generation hepatoma induced by diacetyl amino fluorene.
INTRODUCTION This report describes the isolation and initial characterization of an organ specific protein extracted from cell-surface membranes of rat liver. Previous work from this laboratory has demonstrated that a variety of cell membrane proteins can be solubilized by dilute alkali and effectively fractionated b y disc electrophoresis at p H 2.7 in urea 1. Using these techniques on a preparative scale we now report the isolation of milligram quantities of a single membrane protein. By developing antibodies to the purified protein its localization has been studied. These studies demonstrate that the isolated protein is present in the cell membranes of intact Biochim. Biophys. Acta, 154 (1968) 54o-552
ORGAN-SPECIFIC CELL MEMBRANE PROTEIN
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hepatocytes and that it is organ specific for liver. The significance of these findings is discussed in terms of the role of the cell membrane in the processes of cellular organization and differentiation. MATERIALS AND METHODS
Cell membrane isolation Liver cell plasma membranes were isolated using a modification of the author's original technique 2. The method is described below in detail because attention to these details is necessary to insure reproducibility. In particular the sucrose concentrations are quite critical. These are given here in percent (w/w) at 20 ° and are checked b y an Abbe refractometer. All steps are carried out between 0-4 °. Media refers to o.ooi M NaHCOv (I) Decapitate eight IOO g rats and quickly excise the livers, trim free from connective tissue, place in an iced beaker and mince with scissors. (2) Place IO g of minced liver in large Dounce homogenizer (available from Blaessig Glass Spec. Co., Rochester, N.Y.). Add 25 ml media. Homogenize at 4 ° with 8 vigorous strokes of the loose pestle. Repeat × i. Add the pooled homogenates to 500 ml media (4 °) and stir for 3 rain, then filter first through 2 layers of cheesecloth, then 4 layers. (3) Distribute the filtered homogenate equally between four 25 ° ml glass centrifuge bottles (do not pre-chill the bottles) and spin 15oo × g m a x for IO min. Carefully pour off supernatant and while tube is upside down insert absorbant paper into neck to remove excess supernatant. Now pour off pellets into a large Dounce homogenizer. (4) Repeat steps 2-3 x i. (5) Homogenize pellets with 3 gentle strokes of the loose pestle. (6) Adjust 69 % stock sucrose solution to 69 ± o.I % using refractometer. Place 34 ml into a ioo ml cylinder and cool in ice bucket. Pour in homogenate from step 5. Add H~O to make 60 ml. Mix vigorously with rod until no Schlerin patterns are evident. Check in refractometer. Adjust with H20 or 69 % sucrose until the sucrose-homogenate reads 44.0 i o . I °/o. (7) Pour 20 ml into each of three S-25 tubes. (8) Carefully overlay with IO ml of 42.3 + o . I % sucrose (checked b y refractometer). (9) Balance all three tubes within i 0.05 g by adding 42.3 % sucrose. (io) Load tubes into pre-chilled S-25 rotor (4 °) and spin 25 ooo rev./min (9° ooo X g max) for 2 h, brake on. Handle tubes carefully to preserve the density interface. Make certain caps are well greased to keep tubes at I atm. (See Step 13 now.) (ii) Remove float with spatula. Add 8 ml media. Spin to pack sediment 25 ooo X g m a x for IO rain. (12) Add 4 ml media and resuspend pellet by squirting I x through No. 22 needle. (13) Into the bottom of 2 siliconized S-25 tubes place a 'cushion' of 4.1 ml of 50 + 1% sucrose. Over each form a linear sucrose gradient from 37-3%. (14) Overlay gradients with 2 ml of resuspended homogenate and spin 2000 rev./min (550 x g max) for I h with brake off. (15) Using a syringe and long blunt No. 20 needle remove material at cushion Biochim. Biophys. Acta, 154 (1968) 540-552
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interface. Check purity in phase scope. Count ratio of vesicles per membrane which should not exceed I. In order to increase the quantity of membranes produced the volume of the flotation rather than the tissue concentration must be increased. I f a three place 60 ml swinging bucket is available (Spinco S-25.2) steps 1- 4 m a y be repeated and the volumes of homogenate and sucrose in step 6 doubled. 4 ° ml are poured into each tube and overlayed with 20 ml of 42. 3 -6o.1% sucrose. Spin at 25 ooo rev./min (lO7 ooo × g max) for 15o min. Three gradients should now be used in step 13. When examined b y phase microscopy these membranes appeared similar to the initial preparation except for their larger size. Electron micrographs of this preparation compared to the initial method showed better preservation of the villous processes of the bile canaliculi and an occasional patch of rough endoplasmic reticulum. These differences reflect the substitution of a single zonal centrifugation for repeated washings b y ordinary centrifugation. Cell membranes were also isolated using a media of 22% sucrose plus I mM MgCIv After homogenizing as before the membranes were collected in a 13 ooo × g max, IO rain, pellet. This pellet was washed by recentrifuging 2 x , taken up in 43~/o sucrose + I mM MgC12and floated at 9 ° ooo x g max for 2 h. The float was resuspended and applied to a 37-15 ~o sucrose gradient, containing I mM MgC12, overlaying a 50% sucrose cushion. Centrifugation and collection of membranes was carried out as described in the first procedure. Membranes appeared identical to those harvested in I mM NaHCO 8 when examined by phase microscopy. However this procedure is not entirely reproducible. On some occasions large quantities of vesicles float at 43~/o sucrose, and these have high sedimentation rates and cannot be separated from the membranes by zonal centrifugation. It is our opinion that the density of the large vesicle population changes with the nutritional and physical states of the animal and we have not been successful in controlling these variables.
Preparative electrophoresis Preparative polyacrylamide gel disc electrophoresis was performed as described by JovlN, CHRAMBACHAND •AUGHTON 3 using the Buchler 'Poly-Prep' instrument. The p H 2.7 gel system 1 was used with the following modifications. Upper and lower buffers were made 5 M in urea. The membrane holder was filled with lower buffer. The elution buffer was identical to the lower gel buffer and 9 M in urea. All buffers were degassed prior to use. The lower and upper gels were photopolymerized with 0.05 ~o ammonium persulfate and riboflavin, 0.o0025% lower gel, and 0.0005 ~o upper gel. The high catalyst concentration is necessary because the polyethylene gel forming insert inhibits polymerization leaving a soft uneven gel surface. In part this can be overcome by purging the apparatus with N~ prior to casting the gel. The lower gel solution is overlayed with water saturated with pure oxygen to prevent polymerization of the curved meniscus of the lower gel solution. Dissymmetry of banding was noticed with this gel system until the upper electrode was made symmetrical b y wrapping platinum wire around the central cooling tube just below the upper buffer level. Using a I½ cm lower gel, a I cm upper gel and a flow rate of 3-4 1/min through each cooling jacket at 25°; 35 mA could be drawn without causing band dissymetry. A 2 cm 3 pellet of cell membranes obtained from 16o g of liver was extracted with Biochim. Biophys. Acta, 154 (1968) 540-552
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2o ml of o.o5 M K2CO3 as previously described 1, and then made 8 M in urea and IO% by volume in fl-mercaptoethanol. The solution was concentrated by pressure dialysis to 5 ml and applied to a 50 c m x 2.5 cm Sephadex G-2oo column run in the same solvent system. A 20 ml fraction having a solute distribution coefficient of o.11-o.18 was subjected to electrophoresis as described above with an elution rate of 0.8 ml/min.
Immunization A protein band of mobility o.18 relative to the tracking dye was concentrated to 0.5 mg/ml and dialyzed against normal saline and 0.02 M potassium phosphate buffer (pH 7.o), resulting in precipitation of the protein. The suspension was mixed with equal parts of complete Freund's adjuvant (Difco) and 3 intramuscular injections of 0. 7 ml were made into a sheep. The injections were repeated in 2 weeks and 4 weeks later the sheep was bled.
Immunodiffusion Titer against the antigen was established by double immunodiffusion in Ouchterlony plates containing 1.8 % agarose gels, 0.3 M glycine, 0.02 M potassium phosphate buffer (pH 7.0). The test antigen consisted of membrane proteins extracted with I mM disodium EDTA buffered at pH 7.0 with Tris. Electrophoresis of such extracts revealed that the o.18 band was preferentially extracted by EDTA. Semiquantitation of antigen concentration was achieved by grading the intensity of the precipitin reaction with values I to 5 and noting that each step corresponded to a two-fold dilution of antigen. Preimmunization sera served as controls.
Immunofluorescence Localization of extracted membrane protein to the membrane in intact hepatocytes was investigated by searching for the immunofluorescent 'ring' reaction described by MOLLER4. Suspensions of hepatocytes were prepared in 22 ~o sucrose + 3 mM MgCI~ following liver perfusion with I5~/o sucrose 5. Crude y-globulin was prepared from preimmunization and postimmunization seras by (NH4)2SO 4 precipitation e. Hepatocytes, 30 #1, were incubated with intermittent agitation for 30 min at 4 ° in o.I ml y-globulin diluted 1:27 with buffered saline (this dilution corresponds to 1:5 from the serum concentration of the globulin), washed 2 X by centrifugation in 22~o sucrose, 3 mM MgC12, and incubated in a similar manner with full strength fluorescin conjugated rabbit antisheep y-globulin previously adsorbed with liver powder 7. The conjugated globulin (purchased from Hyland Laboratories, Los Angeles, Calif.) showed a single precipitin line against sheep serum detected down to 1:25 dilution by double immunodiffusion. After washing as before the cells were observed through a fluorescent microscope in the usual manner 7. RESULTS
Purity of isolated protein The protein eluate of the preparative electrophoresis is shown in Fig. I. The bracketed fractions were concentrated and rerun on the analytical gel shown in Fig. 2. A single peak contaminated by a satellite band is seen which, on the basis of a densitometer trace, can account for no more than IO% of the total protein, assuming equal Biochim. Biophys. Acta, 154 (1968) 54o-552
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D.M. NEVILLE, JR
~N
~o 280
350 ElullOn
Volume
{ml}
Fig. I. The protein concentration (LowRY) of the preparative disc electrophoresis eluate is shown. The bracketed volume has been concentrated, its purity checked by analytical eleetrophoresis (Fig. 2) and then injected into a sheep. Fig. 2. Analytical disc electrophoresis (pH 2.7). Samples are (a) 5o/~1 of isolated membrane protein fraction, relative mobility o.18; (b) 5°/21 of unfractionated membrane protein; (c) a mixture of 5 ° #1 (a) + 5 °/zl (b). Note t h a t (c) shows an increase in intensity of the o.18 band.
staining of both bands and the applicability of Beer's law. Fig. 2 shows the relationship between the isolated band and unfractionated membrane protein which have been mixed together and rerun. Fig. 3 shows the single immunodiffusion line obtained when the postimmunization sera is diffused against the EDTA-membrane extract. Membranes extracted with 0. 4 M KC1 contain a variety of proteins as judged by electrophoresis yet also yield a single precipitin line.
Yield of isolated protein Starting with 4o mg of extracted membrane protein 1.2 mg of purified protein of mobility o.18 were obtained. Assuming 40% losses during each concentration step by pressure dialysis and during the electrophoretic run, the isolated protein represents 14 ~o of the applied protein. Since 30-40 ~o of the total membrane protein is not extractable with dilute alkali the isolated protein represents approx. IO% of the total membrane protein. Densitometly of stained analytical electrophoretic patterns indicates that the o. 18 mobility protein accounts for 15 % of the total membrane protein 1. These Biochim. Biophys. Acta, 154 (1968) 54o-552
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Fig. 3. O u c h t e r l o n y plates. T h e c e n t e r wells c o n t a i n sheep a n t i s e r a m a d e a g a i n s t t h e isolated cell m e m b r a n e protein. T h e peripheral wells c o n t a i n E D T A e x t r a c t s of t h e following r a t o r g a n s a n d r a t liver fractions. R i g h t , I, k i d n e y ; 2, spleen; 3, i n t e s t i n a l m u c o s a ; 4, skeletal m u s c l e ; 5, p l a s m a ; 6, liver. Left, I, r o u g h e n d o p l a s m i c r e t i c u l u m ; 2, nuclei; 3, m i t o c h o n d r i a ; 4, Morris h e p a t o m a ; 5, cell m e m b r a n e s (a) ; 6, h e p a t o m a , induced.
figures represent rough estimates since the membranes lose certain proteins and gain others by adsorption during isolation. However it is clear that the isolated protein represents a significant fraction of the total membrane protein.
Estimation of size and molecular weight Analytical electrophoresis oi the G-2oo eluate of membrane protein is shown in Fig. 4. The isolated protein has a relative mobility of o.18 relative to the tracking dye. The solute distribution coefficient, Kd (ref. 8), of this protein was determined by noting the elution volume fraction which displayed the maximum band intensity in the o.18 mobility region of the gel.
%~t//~i~
li~
~ .............
NN;';e i Fig. 4. E x t r a c t e d m e m b r a n e p r o t e i n h a s b e e n c h r o m a t o g r a p h e d on S e p h a d e x G-2oo a n d t h e eluate h a s been a s s a y e d b y a n a l y t i c a l disc electrophoresis a t p H 2. 7 as s h o w n above. E l u t i o n v o l u m e is increasing f r o m left to right. T h e 4 t h gel s h o w s t h e p e a k i n t e n s i t y of t h e o.18 m o b i l i t y b a n d , while t h e c a l i b r a t i n g protein, h e m o g l o b i n , p e a k s b e t w e e n t h e i o t h a n d I i t h gels.
Biochim. Biophys. Acta, 154 (1968) 54o-552
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i). M. NEVILLE, JR
The column was calibrated for the determination of Stokes radii by the method of ACKERS8, using hemoglobin as the calibrating protein. Treatment of hemoglobin with 8 M urea at p H I 1.3 induces dissociation of the heme and uncoiling and dissociation of the a and fl chains which appear as an unresolved doublet of K a --~ 0.37. The sedimentation coefficient for uncoiled hemoglobin in 6 M guanidine hydrochloride has been determined 9. Using this value, a mol. wt. of 15 500 for the globin monomer, and the sedimentation equation and Stokes equation 1° we computed the Stokes radius of uncoiled globin to be 32 A. This value in ACKERS equation yields a pore radius of 15.6 m/,. The isolated protein with K a = o.13 then has a Stokes radius of 58 A. This number must be considered approximate and could be in error b y the difference in uncoiling of hemoglobin in 6 M guanidine hydrochloride vs. 8 M urea io °/o fl-mercaptoethanol. We have determined the K a of bovine serum albumin in 8 M urea run on G-2oo to be o.12. Assuming equal unfolding of both proteins in 8 M urea the molecular weight of the isolated protein will be in the range of that of serum albumin or about 7 ° ooo. The size ~nd molecular weight estimates given above are done under conditions which will dissociate protein subunits which are linked by noncovalent forces. By examining the curvature of the precipitin line we can estimate the size of the native protein with respect to 7-globulin n. Fig. 3 shows that the precipitin line is curved away from the antisera well. This means that the protein antigen diffuses more slowly than 7-globulin and must have a Stokes radius > 5 0 A. This finding is compatible with a highly asymmetric native protein of tool. wt. 7 ° ooo which displays little change in size on treatment with 8 M urea. However if the native protein is globular it must be composed of a number of 7 ° ooo tool. wt. subunits. i
Immunofluorescence (Plate z) A thin intense ring of green fluorescence was present at the periphery of virtually all of the cells incubated initially with postimmunization sera. This type of reaction has been described b y M/3LLER and indicates fluorescent staining of the cell membrane 4. Cells incubated initially with preimmunization sera or with postimmunization sera adsorbed with antigen (300 #g antigen per I ml serum) failed to show the 'ring' reaction. Most of the light coming from the control cells is blue and arises from scattering of the incident light which is normally directed away from the objective b y the dark field condenser. The positive ring reaction taken in conjunctiqn with the appropriate negative controls indicates that the protein antigen is localized on the liver cell surface membrane.
Extraction of protein antigen at neutral pH The procedures adopted for solubilization and fractionation rendered the purified protein insoluble in the absence of urea at neutral pH. We therefore sought extraction procedures which would be compatible with antigen antibody reactions. Dialysis of whole liver homogenates or membranes both prepared in i mM NaHCO 3 against I mM EDTA, p H 7 or 0.3 M KC1, o.15 M potassium phosphate buffer pH 6.5 yielded solutions which gave a single sharp precipitin line on immunodiffusion (Fig. 3). In order to determine if the membrane protein could be a contaminant adsorbed from the homogenizing or washing media we performed serial washings of the total Biochim. 13iophys. Acta, 154 (1968) 540-552
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P l a t e I. P h o t o m i c r o g r a p h s of i m m u n o f l u o r e s c e n c e e x p e r i m e n t s are s h o w n above. T h e localization of t h e purified o r g a n specific p r o t e i n e x t r a c t e d f r o m cell m e m b r a n e p r e p a r a t i o n s is s t u d i e d b y t h e indirect i m m u n o f l u o r e s c e n c e t e c h n i q u e . Liver cell s u s p e n s i o n s h a v e been i l l u m i n a t e d w i t h blue light t h r o u g h a d a r k field c o n d e n s e r a n d p h o t o g r a p h e d t h r o u g h a Zeiss 4o/i.o objective u s i n g K o d a k d a y l i g h t E k t a c h r o m e processed a t ASA 64o. T o p : ceils h a v e been e x p o s e d to sheep p o s t i m m u n i z a t i o n a n t i s e r a followed b y fluorescin c o n j u g a t e d r a b b i t a n t i s h e e p y-globulin. T h e p e r i p h e r y of t h e cells d i s p l a y i n t e n s e green fluorescence characteristic of cell m e m b r a n e localization of t h e a n t i g e n . E x p o s u r e t i m e is o. i see. B o t t o m : control e x p e r i m e n t s p e r f o r m e d on t h e s a m e cell suspension. Prior to s t a i n i n g w i t h c o n j u g a t e d a n t i s h e e p globulin t h e cells a t t h e left were t r e a t e d w i t h p o s t i m m u n i z a t i o n a n t i s e r a a d s o r b e d w i t h a n t i g e n while t h o s e at t h e r i g h t were t r e a t e d w i t h p r e i m m u n i z a t i o n sera. P e r i p h e r a l green fluorescence is n o t seen. T h e low i n t e n s i t y bluish color r e s u l t s f r o m s c a t t e r i n g of t h e i n c i d e n t light b y t h e cells. T h e e x p o s u r e t i m e is 5 ° see. Free nuclei a p p e a r green in b o t h e x p e r i m e n t a l a n d control s t u d i e s i n d i c a t i n g nonspecific b i n d i n g of c o n j u g a t e d globulin. I n t h e control m i c r o g r a p h s free nuclei a p p e a r w h i t e d u e to overexposure.
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ORGAN-SPECIFIC CELL MEMBRANE PROTEIN
liver particulate fraction (17o ooo × g, 15o min). Homogenization in I mM NaHCO 3 failed to solubilize the protein. However a second homogenization in I mM NaHCO, of the centrifuged pellet released large quantities of the antigen. I f membranes or whole liver were homogenized and washed in media containing I mM MgC12 the protein antigen was not released. Extraction with KC1 now failed to release the protein antigen although E D T A was effective. I t appears that KC1 releases antigen from membranes isolated in i mM NaHCO 3 because considerable extraction of divalent cations has occurred during the procedure which weakens the protein membrane complex. Because of these findings all subsequent localization studies were done in the presence of I mM MgC12.
Localization of antigen by liver fractionation Of the homogeneous particulate fractions which can be isolated from liver, only cell membranes yield the protein antigen on extraction with E D T A (see Table I). In addition the specific activity (extractable titer/extractable protein) of cell membranes TABLE I LIVER CELL FRACTION LOCALIZATION OF MEMBRANE PROTEIN P r e s e n c e of t h e i s o l a t e d m e m b r a n e p r o t e i n w a s a s s a y e d b y j u d g i n g t h e i n t e n s i t y of p r e c i p i t i n lines ( a r b i t r a r y v a l u e s 1-5) a f t e r d o u b l e i m m u n o d i f f u s i o n of p o s t i m m u n i z a t i o n g l o b u l i n vs. E D T A e x t r a c t s of l i v e r fractions. P e l l e t e d f r a c t i o n s w e re r e s u s p e n d e d t o gi ve lO-3O % v / v homog e n a t e s a n d d i a l y z e d for 18 h a t 4 ° vs. I mM d i s o d i u m - E D T A - T r i s (pH 7.o), a n d c l e a re d b y cent r i f u g a t i o n p r i o r to i m m u n o d i f f u s i o n a n d LOWRY p r o t e i n d e t e r m i n a t i o n 14.
Fraction
Fraction extract titer
Fraction extract protein (mg/ml)
Cell m e m b r a n e s (a) Cell m e m b r a n e s (b) Nuclei15 Mitochondria S m o o t h e n d o p l a s m i c r e t i c u l u m TM R o u g h e n d o p l a s m i c r e t i c u l u m 17
5 4 o o o o
0.44 1.3 3. i 3.3 2.2 e.2
(a) M e m b r a n e s i s o l a t e d b y t h e second p r o c e d u r e in METHODS in t h e pre s e nc e of i mM MgC12. (b) M e m b r a n e s i s o l a t e d b y t h e first m e t h o d in i mM N a H C O v See METHODS for t h e d e t a i l s of o t h e r f r a c t i o n a t i o n procedures. M i t o c h o n d r i a were p e l l e t e d a t i 0 0o0 × g a ve ra ge , 20 min, a n d w a s h e d b y 4 r e s u s p e n s i o n s a n d p e l l e t i n g in 2 2 % sucrose + I mM MgCI~, a n d floated in a c ont i n u o u s sucrose g r a d i e n t . F l o a t s l i g h t e r t h a n 43 % were d i s c a r d e d due t o c o n t a m i n a t i o n w i t h m e m bran es. All f r a c t i o n s e x c e p t (b) were i s o l a t e d in t h e p r e s e n c e of I mM MgC12.
isolated in I mM MgC12 is 3o times greater than the unfractionated homogenate. However, cell membranes isolated in I mM MgC12 only account for lO-150/0 of the total antigen present within the liver homogenate. Therefore the distribution of the antigen with respect to density and sedimentation characteristics was investigated. When the liver homogenate was introduced at the bottom of a continuous sucrose gradient, 54-20%, containing I mM MgCI, and centrifuged for 18 h at 9 ° ooo × g max. all of the detectable antigen was localized between 42 and 430/0 sucrose, corresponding to the isopycnic point of isolated cell membranes. When the 42-43 % sucrose Biochim. Biophys. Acta, 154 (1968) 540-552
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D . M . NEVILLE, JR
fraction was centrifuged at 8000 x g average for IO min all of the antigen was sedimented. This sediment and the IOO ooo × g pellet obtained from its supernatant were subjected to electron microscopy after fixation in glutaraldehyde and staining with osmium tetroxide. The low g pellet was very rich in membranes composed of villous processes. These could be derived from small fragments of blood from cell membranes or bile canaliculi, and they were not as plentiful in the high g pellet. Both pellets were rich in endoplasmic reticulum, rough vesicles predominating in the low g pellet. In view of the fact that the isopycnic point of the particulate antigen coincides with the isopycnic point for cell membranes it is likely that all of the antigen is derived from cell membranes. The membrane isolation procedures select out of necessity only large fragments of membranes (roughly IO # x IO/,), and it appears that most of the cell membrane is disrupted into smaller fragments on cell breakage. We cannot, however, eliminate the possibility that the antigen is also present in a previously undefined vesicular fraction or in a fraction of rough endoplasmic reticulum characterized by a high sedimentation coefficient and an isopycnic point corresTABLE II ORGAN LOCALIZATION
OF LIVER
MEMBRANE
PROTEIN
P r e s e n c e of t h e i s o l a t e d m e m b r a n e p r o t e i n was a s s a y e d b y j u d g i n g i n t e n s i t y of p r e c i p i t i n lines a f t e r d o u b l e i m m u n o d i f f u s i o n of p o s t i m m u n i z a t i o n g l o b u l i n vs. E D T A e x t r a c t s of o r g a n homogenates. 4 0 % h o m o g e n a t e s of each o r g a n were m a d e in 2 2 % sucrose + I mM MgCI~ a n d c e nt ri fu ged a t 17o ooo x g max., i 5 o min. P e l l e t s were r e s u s p e n d e d y i e l d i n g a 5 0 % h o m o g e n a t e a n d d i a l y z e d a t 4 ° for 18 h vs. 500 vols. of I mM d i s o d i u m E D T A - T r i s (pH 7.0), a n d c l e a re d b y cent r i f u g a t i o n prior to i m m u n o d i f f u s i o n a n d LOWRY p r o t e i n d e t e r m i n a t i o n .
Organ
Organ extract titer
Organ extract protein (mg/ml)
R a t liver Rat kidney R a t spleen Rat intestinal mucosa Rat pancreas R a t adipose t i s s u e R a t skeletal muscle Rat erythrocytes Rat embryo liver-mid Rat embryo liver-late G u i n e a pig l i v e r Mouse liver Chicken l i v e r Hepatoma, induced Morris h e p a t o m a Rat plasma
4 o o o o o o o o 3 2 3 o 4 o o
6.7 5.3 6.3 2.3 2.5 1.4 5.7 5.o 3.7 4 .6 6. 4 5.8 4.2 6.8 4.7 --
ponding to 42-43 % sucrose. Most procedures designed to isolate rough endoplasmic reticulum never isolate that fraction having a high sedimentation coefficient. This material either exists as large clumps or is attached to the mitoehondria and nuclei and is discarded in the nuclear-mitochondrial pellet unless very high shear forces are used during homogenization. Biochim. Biophys. Acta, 154 (1968) 54o-552
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Organ specificity The organ specific character of the protein antigen is shown in Table II. A variety of organ homogenates including liver have been extracted with E D T A under identical conditions. Although E D T A extracts protein from all homogenates, precipitin lines are obtained only from liver extracts. The absence of precipitin lines with rat plasma indicates that the protein antigen is not one of the variety of plasma proteins manufactured by the liver. The protein antigen is not highly species specific being detectable in two other rodents but it was not detectable in the chicken. Although the protein antigen is organ specific it was not detectable in midterm embryonic livers although it was present in the latter third of gestation. A highly differentiated second generation hepatoma induced by diacetyl amino fluorene contained the antigen but the more undifferentiated Morris hepatoma failed to elicit a reaction. DISCUSSION
These experiments show that the liver cell membrane contains an organ-specific protein having a minimal or subunit tool. wt. of 7 ° ooo and which comprises roughly IO °/o of the total membrane protein. Three types of experiments document the cell membrane origin of this protein. First, the protein is extractable from cell membranes isolated under conditions which fail to solubilize the protein. This eliminates the possibility that the protein is an adsorbed contaminant from material solubilized during ffactionation. Second, the protein is not detected in extracts of purified nuclei, mitochondria, and endoplasmic reticulum. Third, the protein is localized to the cell membrane of intact hepatocytes b y immunofluorescence. The latter experiments eliminate the possibility that the protein never existed in membrane but was extracted from nonmembrane particles which m a y contaminate the membrane preparations. The organ specific nature of the membrane protein is clearly seen in the results presented in Table II. By organ specific we mean that the quantity of protein in organs other than liver is reduced at least by a factor of ten, this being the limit of the detection system. Liver specific antigens have been previously detected by immunizing with liver particulate fractions and performing adsorptions of antisera with other organs prior to immunodiffusion 1~. However, these antigens have not been isolated or characterized. At present the function of the liver specific membrane protein is unknown. However, the localization studies indicate that this function must be related to liver specific processes rather than general cellular processes necessary for cell viability. The absence of the liver specific protein from midterm embryos and Morris hepatoma (a tissue cultured cell line) as opposed to its presence in late term embryo and the more highly differentiated second generation hepatoma is consistent with a liver specific function. Whether the absence of the protein from the membranes of Morris hepatoma has any relation to the capacity of this cell line for uncontrolled growth in a variety of environments remains to be elucidated. Cellular proteins are generally divided into three functional classes, enzymatic, carrier, and structural proteins. Regardless of the function of the liver specific membrane protein it has unique possibilities to serve as a transmitter of information. A Biochim. Biophys. Acta, 154 (1968) 540-552
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p r o t e i n w h i c h is c h a r a c t e r i s t i c for a g i v e n cell t y p e a n d is localized to t h e surface m e m b r a n e p r o v i d e s a m o l e c u l a r m a r k e r for t h a t p a r t i c u l a r cell t y p e , a m a r k e r w h i c h is accessible to a n y o t h e r cell m a k i n g c e l l u l a r c o n t a c t . S u c h p r o t e i n s we refer to as e i g e n proteins. A n u m b e r o f o b s e r v a t i o n s s u g g e s t t h a t cells are c a p a b l e of r e c o g n i z i n g o t h e r cells, a n d t h a t c e l l u l a r c o n t a c t is i n v o l v e d in t h e r e c o g n i t i o n process 13. S u c h r e c o g n i t i o n w o u l d a p p e a r to be a n e c e s s i t y for o r g a n d i f f e r e n t i a t i o n a n d t h e m a i n t e n a n c e of c e l l u l a r o r g a n i z a t i o n . I t is r e a s o n a b l e t o s u p p o s e t h a t r e c o g n i t i o n occurs at t h e surface m e m b r a n e since t h i s s t r u c t u r e s e p a r a t e s t h e e x t e r n a l f r o m t h e i n t e r n a l e n v i r o n m e n t . W e are i n v e s t i g a t i n g t h e p o s s i b i l i t y t h a t cellular r e c o g n i t i o n is m e d i a t e d b y cell t y p e specific m e m b r a n e p r o t e i n s . I f this is t h e case it s h o u l d be possible to d e m o n s t r a t e t h a t e v e r y d i f f e r e n t cell t y p e has an ' e i g e n ' p r o t e i n , a n d in a d d i t i o n to cell specificity a n d m e m b r a n e l o c a l i z a t i o n t h e s e p r o t e i n s e x e r t p r o f o u n d effects on o r g a n i z e d c e l l u l a r behavior.
ACKNOWLEDGEMENTS T h e a u t h o r t h a n k s Mr. JAMES BOONE for e x c e l l e n t t e c h n i c a l assistance. L i v e r t u m o r s w e r e gifts f r o m Dr. MELVIN D. REUBER o f t h e N a t i o n a l C a n c e r I n s t i t u t e a n d Dr. EDWARD B. THOMPSON of t h e N a t i o n a l I n s t i t u t e for A r t h r i t i s a n d M e t a b o l i c Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16
17
D. M. NEVILLE, JR., Biochim. Biophys. Acta, 133 (1967) 168. D. M. NEVILLE, JR., J. Biophys. Biochem. Cytol., 8 (196o) 413 . T. JOVlN, A. CHRAMBACHAND M. NAUGHTON, Anal. Biochem., 9 (1964) 351. G. M6LLER, J. Exptl. Med., 114 (1961) 415 . N. G. ANDERSON, Science, 117 (1953) 627. G. GOLDS'rEIN, B. H. SPALDING AND W. B. HUNT, Proc. Soc. Exptl. Biol. Med., I I I (1962) 416. R. C. NAIRN, Fluorescent Protein Tracing, E. and S. Livingstone, Edinburgh and London, 1962. G. K. ACKERS, Biochemistry, 3 (1964) 723 • K. KAWAHARA,A. G. I~IRSHNER AND C. TANEORD, Biochemistry, 4 (1965) 12°3. C. TANFORD, Physical Chemistry of Macromoleeules, John Wiley, New York-London, 1963, Chap. 6. A. J. CROWLE, Immunodiffusion, Academic Press, N.Y., 1961. D. C. DUlVtONDE,Advan. Immunol., 5 (I966) 245. T. HUMPHREYS, in B. D. DAvis AND L. WARREN, The Specificity of Cell Surfaces, PrenticeHall, Englewood Cliffs, N.J., 1967, p. 195. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265 . T. E. CONOVER AND G. SIEBERT, Biochim. Biophys. Acta, 99 (1965) I. J. CHAUVEAU, Y. MOUL]~, C. ROUILLER AND J. SCHNEEBELI, J. Cell Biol., 12 (1962) 17. G. DALLNER, S. ORRENIUS AND A. BERGSTRAND, J. Cell. Biol., 16 (1963) 426.
Bioehim. Biophys. Acta, 154 (1968) 540-552
BIOCHIMICAET BIOPHYSICAACTA
BBA 35173 ISOLATION OF AN ORGAN SPECIFIC P R O T E I N A N T I G E N FROM CELLSURFACE MEMBRANE OF RAT L I V E R
DAVID M. NEVILLE, JR. Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Md. (U.S.A.)
(Received August I4th. 1967)
SUMMARY
I. A single membrane protein accounting for roughly lO% of the total membrane protein has been isolated from alkali extracts of rat liver cell membranes by preparative disc electrophoresis at pH 2.7 in urea. 2. A Stokes radius of 58/~ and a molecular weight of 7 ° ooo has been estimated for the protein by gel filtration in 8 M urea. 3. Divalent cations are involved in the binding of the protein to the membrane matrix. Dialysis of membranes against ! mM EDTA solubilizes the protein at neutral pH. 4. Antisera prepared against the purified protein in sheep yield a single precipitin line after immunodiffusion with EDTA extracts of liver and liver cell membranes. 5. The protein is apparently organ specific not being detectable in rat plasma or E D T A extracts of erythrocytes, kidney, spleen, intestine, pancreas, muscle or adipose tissue. In addition the protein appears to be membrane specific not being detected in extracts of rat liver nuclei, mitochondria, endoplasmic reticulum or cell sap. The protein has been localized to the cell surface membrane of intact hepatocytes b y immunofluorescence. 6. The liver membrane protein could not be detected in extracts of Morris hepatoma but was found to be present in a highly differentiated second generation hepatoma induced by diacetyl amino fluorene.
INTRODUCTION This report describes the isolation and initial characterization of an organ specific protein extracted from cell-surface membranes of rat liver. Previous work from this laboratory has demonstrated that a variety of cell membrane proteins can be solubilized by dilute alkali and effectively fractionated b y disc electrophoresis at p H 2.7 in urea 1. Using these techniques on a preparative scale we now report the isolation of milligram quantities of a single membrane protein. By developing antibodies to the purified protein its localization has been studied. These studies demonstrate that the isolated protein is present in the cell membranes of intact Biochim. Biophys. Acta, 154 (1968) 54o-552
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hepatocytes and that it is organ specific for liver. The significance of these findings is discussed in terms of the role of the cell membrane in the processes of cellular organization and differentiation. MATERIALS AND METHODS
Cell membrane isolation Liver cell plasma membranes were isolated using a modification of the author's original technique 2. The method is described below in detail because attention to these details is necessary to insure reproducibility. In particular the sucrose concentrations are quite critical. These are given here in percent (w/w) at 20 ° and are checked b y an Abbe refractometer. All steps are carried out between 0-4 °. Media refers to o.ooi M NaHCOv (I) Decapitate eight IOO g rats and quickly excise the livers, trim free from connective tissue, place in an iced beaker and mince with scissors. (2) Place IO g of minced liver in large Dounce homogenizer (available from Blaessig Glass Spec. Co., Rochester, N.Y.). Add 25 ml media. Homogenize at 4 ° with 8 vigorous strokes of the loose pestle. Repeat × i. Add the pooled homogenates to 500 ml media (4 °) and stir for 3 rain, then filter first through 2 layers of cheesecloth, then 4 layers. (3) Distribute the filtered homogenate equally between four 25 ° ml glass centrifuge bottles (do not pre-chill the bottles) and spin 15oo × g m a x for IO min. Carefully pour off supernatant and while tube is upside down insert absorbant paper into neck to remove excess supernatant. Now pour off pellets into a large Dounce homogenizer. (4) Repeat steps 2-3 x i. (5) Homogenize pellets with 3 gentle strokes of the loose pestle. (6) Adjust 69 % stock sucrose solution to 69 ± o.I % using refractometer. Place 34 ml into a ioo ml cylinder and cool in ice bucket. Pour in homogenate from step 5. Add H~O to make 60 ml. Mix vigorously with rod until no Schlerin patterns are evident. Check in refractometer. Adjust with H20 or 69 % sucrose until the sucrose-homogenate reads 44.0 i o . I °/o. (7) Pour 20 ml into each of three S-25 tubes. (8) Carefully overlay with IO ml of 42.3 + o . I % sucrose (checked b y refractometer). (9) Balance all three tubes within i 0.05 g by adding 42.3 % sucrose. (io) Load tubes into pre-chilled S-25 rotor (4 °) and spin 25 ooo rev./min (9° ooo X g max) for 2 h, brake on. Handle tubes carefully to preserve the density interface. Make certain caps are well greased to keep tubes at I atm. (See Step 13 now.) (ii) Remove float with spatula. Add 8 ml media. Spin to pack sediment 25 ooo X g m a x for IO rain. (12) Add 4 ml media and resuspend pellet by squirting I x through No. 22 needle. (13) Into the bottom of 2 siliconized S-25 tubes place a 'cushion' of 4.1 ml of 50 + 1% sucrose. Over each form a linear sucrose gradient from 37-3%. (14) Overlay gradients with 2 ml of resuspended homogenate and spin 2000 rev./min (550 x g max) for I h with brake off. (15) Using a syringe and long blunt No. 20 needle remove material at cushion Biochim. Biophys. Acta, 154 (1968) 540-552
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interface. Check purity in phase scope. Count ratio of vesicles per membrane which should not exceed I. In order to increase the quantity of membranes produced the volume of the flotation rather than the tissue concentration must be increased. I f a three place 60 ml swinging bucket is available (Spinco S-25.2) steps 1- 4 m a y be repeated and the volumes of homogenate and sucrose in step 6 doubled. 4 ° ml are poured into each tube and overlayed with 20 ml of 42. 3 -6o.1% sucrose. Spin at 25 ooo rev./min (lO7 ooo × g max) for 15o min. Three gradients should now be used in step 13. When examined b y phase microscopy these membranes appeared similar to the initial preparation except for their larger size. Electron micrographs of this preparation compared to the initial method showed better preservation of the villous processes of the bile canaliculi and an occasional patch of rough endoplasmic reticulum. These differences reflect the substitution of a single zonal centrifugation for repeated washings b y ordinary centrifugation. Cell membranes were also isolated using a media of 22% sucrose plus I mM MgCIv After homogenizing as before the membranes were collected in a 13 ooo × g max, IO rain, pellet. This pellet was washed by recentrifuging 2 x , taken up in 43~/o sucrose + I mM MgC12and floated at 9 ° ooo x g max for 2 h. The float was resuspended and applied to a 37-15 ~o sucrose gradient, containing I mM MgC12, overlaying a 50% sucrose cushion. Centrifugation and collection of membranes was carried out as described in the first procedure. Membranes appeared identical to those harvested in I mM NaHCO 8 when examined by phase microscopy. However this procedure is not entirely reproducible. On some occasions large quantities of vesicles float at 43~/o sucrose, and these have high sedimentation rates and cannot be separated from the membranes by zonal centrifugation. It is our opinion that the density of the large vesicle population changes with the nutritional and physical states of the animal and we have not been successful in controlling these variables.
Preparative electrophoresis Preparative polyacrylamide gel disc electrophoresis was performed as described by JovlN, CHRAMBACHAND •AUGHTON 3 using the Buchler 'Poly-Prep' instrument. The p H 2.7 gel system 1 was used with the following modifications. Upper and lower buffers were made 5 M in urea. The membrane holder was filled with lower buffer. The elution buffer was identical to the lower gel buffer and 9 M in urea. All buffers were degassed prior to use. The lower and upper gels were photopolymerized with 0.05 ~o ammonium persulfate and riboflavin, 0.o0025% lower gel, and 0.0005 ~o upper gel. The high catalyst concentration is necessary because the polyethylene gel forming insert inhibits polymerization leaving a soft uneven gel surface. In part this can be overcome by purging the apparatus with N~ prior to casting the gel. The lower gel solution is overlayed with water saturated with pure oxygen to prevent polymerization of the curved meniscus of the lower gel solution. Dissymmetry of banding was noticed with this gel system until the upper electrode was made symmetrical b y wrapping platinum wire around the central cooling tube just below the upper buffer level. Using a I½ cm lower gel, a I cm upper gel and a flow rate of 3-4 1/min through each cooling jacket at 25°; 35 mA could be drawn without causing band dissymetry. A 2 cm 3 pellet of cell membranes obtained from 16o g of liver was extracted with Biochim. Biophys. Acta, 154 (1968) 540-552
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2o ml of o.o5 M K2CO3 as previously described 1, and then made 8 M in urea and IO% by volume in fl-mercaptoethanol. The solution was concentrated by pressure dialysis to 5 ml and applied to a 50 c m x 2.5 cm Sephadex G-2oo column run in the same solvent system. A 20 ml fraction having a solute distribution coefficient of o.11-o.18 was subjected to electrophoresis as described above with an elution rate of 0.8 ml/min.
Immunization A protein band of mobility o.18 relative to the tracking dye was concentrated to 0.5 mg/ml and dialyzed against normal saline and 0.02 M potassium phosphate buffer (pH 7.o), resulting in precipitation of the protein. The suspension was mixed with equal parts of complete Freund's adjuvant (Difco) and 3 intramuscular injections of 0. 7 ml were made into a sheep. The injections were repeated in 2 weeks and 4 weeks later the sheep was bled.
Immunodiffusion Titer against the antigen was established by double immunodiffusion in Ouchterlony plates containing 1.8 % agarose gels, 0.3 M glycine, 0.02 M potassium phosphate buffer (pH 7.0). The test antigen consisted of membrane proteins extracted with I mM disodium EDTA buffered at pH 7.0 with Tris. Electrophoresis of such extracts revealed that the o.18 band was preferentially extracted by EDTA. Semiquantitation of antigen concentration was achieved by grading the intensity of the precipitin reaction with values I to 5 and noting that each step corresponded to a two-fold dilution of antigen. Preimmunization sera served as controls.
Immunofluorescence Localization of extracted membrane protein to the membrane in intact hepatocytes was investigated by searching for the immunofluorescent 'ring' reaction described by MOLLER4. Suspensions of hepatocytes were prepared in 22 ~o sucrose + 3 mM MgCI~ following liver perfusion with I5~/o sucrose 5. Crude y-globulin was prepared from preimmunization and postimmunization seras by (NH4)2SO 4 precipitation e. Hepatocytes, 30 #1, were incubated with intermittent agitation for 30 min at 4 ° in o.I ml y-globulin diluted 1:27 with buffered saline (this dilution corresponds to 1:5 from the serum concentration of the globulin), washed 2 X by centrifugation in 22~o sucrose, 3 mM MgC12, and incubated in a similar manner with full strength fluorescin conjugated rabbit antisheep y-globulin previously adsorbed with liver powder 7. The conjugated globulin (purchased from Hyland Laboratories, Los Angeles, Calif.) showed a single precipitin line against sheep serum detected down to 1:25 dilution by double immunodiffusion. After washing as before the cells were observed through a fluorescent microscope in the usual manner 7. RESULTS
Purity of isolated protein The protein eluate of the preparative electrophoresis is shown in Fig. I. The bracketed fractions were concentrated and rerun on the analytical gel shown in Fig. 2. A single peak contaminated by a satellite band is seen which, on the basis of a densitometer trace, can account for no more than IO% of the total protein, assuming equal Biochim. Biophys. Acta, 154 (1968) 54o-552
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D.M. NEVILLE, JR
~N
~o 280
350 ElullOn
Volume
{ml}
Fig. I. The protein concentration (LowRY) of the preparative disc electrophoresis eluate is shown. The bracketed volume has been concentrated, its purity checked by analytical eleetrophoresis (Fig. 2) and then injected into a sheep. Fig. 2. Analytical disc electrophoresis (pH 2.7). Samples are (a) 5o/~1 of isolated membrane protein fraction, relative mobility o.18; (b) 5°/21 of unfractionated membrane protein; (c) a mixture of 5 ° #1 (a) + 5 °/zl (b). Note t h a t (c) shows an increase in intensity of the o.18 band.
staining of both bands and the applicability of Beer's law. Fig. 2 shows the relationship between the isolated band and unfractionated membrane protein which have been mixed together and rerun. Fig. 3 shows the single immunodiffusion line obtained when the postimmunization sera is diffused against the EDTA-membrane extract. Membranes extracted with 0. 4 M KC1 contain a variety of proteins as judged by electrophoresis yet also yield a single precipitin line.
Yield of isolated protein Starting with 4o mg of extracted membrane protein 1.2 mg of purified protein of mobility o.18 were obtained. Assuming 40% losses during each concentration step by pressure dialysis and during the electrophoretic run, the isolated protein represents 14 ~o of the applied protein. Since 30-40 ~o of the total membrane protein is not extractable with dilute alkali the isolated protein represents approx. IO% of the total membrane protein. Densitometly of stained analytical electrophoretic patterns indicates that the o. 18 mobility protein accounts for 15 % of the total membrane protein 1. These Biochim. Biophys. Acta, 154 (1968) 54o-552
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Fig. 3. O u c h t e r l o n y plates. T h e c e n t e r wells c o n t a i n sheep a n t i s e r a m a d e a g a i n s t t h e isolated cell m e m b r a n e protein. T h e peripheral wells c o n t a i n E D T A e x t r a c t s of t h e following r a t o r g a n s a n d r a t liver fractions. R i g h t , I, k i d n e y ; 2, spleen; 3, i n t e s t i n a l m u c o s a ; 4, skeletal m u s c l e ; 5, p l a s m a ; 6, liver. Left, I, r o u g h e n d o p l a s m i c r e t i c u l u m ; 2, nuclei; 3, m i t o c h o n d r i a ; 4, Morris h e p a t o m a ; 5, cell m e m b r a n e s (a) ; 6, h e p a t o m a , induced.
figures represent rough estimates since the membranes lose certain proteins and gain others by adsorption during isolation. However it is clear that the isolated protein represents a significant fraction of the total membrane protein.
Estimation of size and molecular weight Analytical electrophoresis oi the G-2oo eluate of membrane protein is shown in Fig. 4. The isolated protein has a relative mobility of o.18 relative to the tracking dye. The solute distribution coefficient, Kd (ref. 8), of this protein was determined by noting the elution volume fraction which displayed the maximum band intensity in the o.18 mobility region of the gel.
%~t//~i~
li~
~ .............
NN;';e i Fig. 4. E x t r a c t e d m e m b r a n e p r o t e i n h a s b e e n c h r o m a t o g r a p h e d on S e p h a d e x G-2oo a n d t h e eluate h a s been a s s a y e d b y a n a l y t i c a l disc electrophoresis a t p H 2. 7 as s h o w n above. E l u t i o n v o l u m e is increasing f r o m left to right. T h e 4 t h gel s h o w s t h e p e a k i n t e n s i t y of t h e o.18 m o b i l i t y b a n d , while t h e c a l i b r a t i n g protein, h e m o g l o b i n , p e a k s b e t w e e n t h e i o t h a n d I i t h gels.
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i). M. NEVILLE, JR
The column was calibrated for the determination of Stokes radii by the method of ACKERS8, using hemoglobin as the calibrating protein. Treatment of hemoglobin with 8 M urea at p H I 1.3 induces dissociation of the heme and uncoiling and dissociation of the a and fl chains which appear as an unresolved doublet of K a --~ 0.37. The sedimentation coefficient for uncoiled hemoglobin in 6 M guanidine hydrochloride has been determined 9. Using this value, a mol. wt. of 15 500 for the globin monomer, and the sedimentation equation and Stokes equation 1° we computed the Stokes radius of uncoiled globin to be 32 A. This value in ACKERS equation yields a pore radius of 15.6 m/,. The isolated protein with K a = o.13 then has a Stokes radius of 58 A. This number must be considered approximate and could be in error b y the difference in uncoiling of hemoglobin in 6 M guanidine hydrochloride vs. 8 M urea io °/o fl-mercaptoethanol. We have determined the K a of bovine serum albumin in 8 M urea run on G-2oo to be o.12. Assuming equal unfolding of both proteins in 8 M urea the molecular weight of the isolated protein will be in the range of that of serum albumin or about 7 ° ooo. The size ~nd molecular weight estimates given above are done under conditions which will dissociate protein subunits which are linked by noncovalent forces. By examining the curvature of the precipitin line we can estimate the size of the native protein with respect to 7-globulin n. Fig. 3 shows that the precipitin line is curved away from the antisera well. This means that the protein antigen diffuses more slowly than 7-globulin and must have a Stokes radius > 5 0 A. This finding is compatible with a highly asymmetric native protein of tool. wt. 7 ° ooo which displays little change in size on treatment with 8 M urea. However if the native protein is globular it must be composed of a number of 7 ° ooo tool. wt. subunits. i
Immunofluorescence (Plate z) A thin intense ring of green fluorescence was present at the periphery of virtually all of the cells incubated initially with postimmunization sera. This type of reaction has been described b y M/3LLER and indicates fluorescent staining of the cell membrane 4. Cells incubated initially with preimmunization sera or with postimmunization sera adsorbed with antigen (300 #g antigen per I ml serum) failed to show the 'ring' reaction. Most of the light coming from the control cells is blue and arises from scattering of the incident light which is normally directed away from the objective b y the dark field condenser. The positive ring reaction taken in conjunctiqn with the appropriate negative controls indicates that the protein antigen is localized on the liver cell surface membrane.
Extraction of protein antigen at neutral pH The procedures adopted for solubilization and fractionation rendered the purified protein insoluble in the absence of urea at neutral pH. We therefore sought extraction procedures which would be compatible with antigen antibody reactions. Dialysis of whole liver homogenates or membranes both prepared in i mM NaHCO 3 against I mM EDTA, p H 7 or 0.3 M KC1, o.15 M potassium phosphate buffer pH 6.5 yielded solutions which gave a single sharp precipitin line on immunodiffusion (Fig. 3). In order to determine if the membrane protein could be a contaminant adsorbed from the homogenizing or washing media we performed serial washings of the total Biochim. 13iophys. Acta, 154 (1968) 540-552
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P l a t e I. P h o t o m i c r o g r a p h s of i m m u n o f l u o r e s c e n c e e x p e r i m e n t s are s h o w n above. T h e localization of t h e purified o r g a n specific p r o t e i n e x t r a c t e d f r o m cell m e m b r a n e p r e p a r a t i o n s is s t u d i e d b y t h e indirect i m m u n o f l u o r e s c e n c e t e c h n i q u e . Liver cell s u s p e n s i o n s h a v e been i l l u m i n a t e d w i t h blue light t h r o u g h a d a r k field c o n d e n s e r a n d p h o t o g r a p h e d t h r o u g h a Zeiss 4o/i.o objective u s i n g K o d a k d a y l i g h t E k t a c h r o m e processed a t ASA 64o. T o p : ceils h a v e been e x p o s e d to sheep p o s t i m m u n i z a t i o n a n t i s e r a followed b y fluorescin c o n j u g a t e d r a b b i t a n t i s h e e p y-globulin. T h e p e r i p h e r y of t h e cells d i s p l a y i n t e n s e green fluorescence characteristic of cell m e m b r a n e localization of t h e a n t i g e n . E x p o s u r e t i m e is o. i see. B o t t o m : control e x p e r i m e n t s p e r f o r m e d on t h e s a m e cell suspension. Prior to s t a i n i n g w i t h c o n j u g a t e d a n t i s h e e p globulin t h e cells a t t h e left were t r e a t e d w i t h p o s t i m m u n i z a t i o n a n t i s e r a a d s o r b e d w i t h a n t i g e n while t h o s e at t h e r i g h t were t r e a t e d w i t h p r e i m m u n i z a t i o n sera. P e r i p h e r a l green fluorescence is n o t seen. T h e low i n t e n s i t y bluish color r e s u l t s f r o m s c a t t e r i n g of t h e i n c i d e n t light b y t h e cells. T h e e x p o s u r e t i m e is 5 ° see. Free nuclei a p p e a r green in b o t h e x p e r i m e n t a l a n d control s t u d i e s i n d i c a t i n g nonspecific b i n d i n g of c o n j u g a t e d globulin. I n t h e control m i c r o g r a p h s free nuclei a p p e a r w h i t e d u e to overexposure.
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ORGAN-SPECIFIC CELL MEMBRANE PROTEIN
liver particulate fraction (17o ooo × g, 15o min). Homogenization in I mM NaHCO 3 failed to solubilize the protein. However a second homogenization in I mM NaHCO, of the centrifuged pellet released large quantities of the antigen. I f membranes or whole liver were homogenized and washed in media containing I mM MgC12 the protein antigen was not released. Extraction with KC1 now failed to release the protein antigen although E D T A was effective. I t appears that KC1 releases antigen from membranes isolated in i mM NaHCO 3 because considerable extraction of divalent cations has occurred during the procedure which weakens the protein membrane complex. Because of these findings all subsequent localization studies were done in the presence of I mM MgC12.
Localization of antigen by liver fractionation Of the homogeneous particulate fractions which can be isolated from liver, only cell membranes yield the protein antigen on extraction with E D T A (see Table I). In addition the specific activity (extractable titer/extractable protein) of cell membranes TABLE I LIVER CELL FRACTION LOCALIZATION OF MEMBRANE PROTEIN P r e s e n c e of t h e i s o l a t e d m e m b r a n e p r o t e i n w a s a s s a y e d b y j u d g i n g t h e i n t e n s i t y of p r e c i p i t i n lines ( a r b i t r a r y v a l u e s 1-5) a f t e r d o u b l e i m m u n o d i f f u s i o n of p o s t i m m u n i z a t i o n g l o b u l i n vs. E D T A e x t r a c t s of l i v e r fractions. P e l l e t e d f r a c t i o n s w e re r e s u s p e n d e d t o gi ve lO-3O % v / v homog e n a t e s a n d d i a l y z e d for 18 h a t 4 ° vs. I mM d i s o d i u m - E D T A - T r i s (pH 7.o), a n d c l e a re d b y cent r i f u g a t i o n p r i o r to i m m u n o d i f f u s i o n a n d LOWRY p r o t e i n d e t e r m i n a t i o n 14.
Fraction
Fraction extract titer
Fraction extract protein (mg/ml)
Cell m e m b r a n e s (a) Cell m e m b r a n e s (b) Nuclei15 Mitochondria S m o o t h e n d o p l a s m i c r e t i c u l u m TM R o u g h e n d o p l a s m i c r e t i c u l u m 17
5 4 o o o o
0.44 1.3 3. i 3.3 2.2 e.2
(a) M e m b r a n e s i s o l a t e d b y t h e second p r o c e d u r e in METHODS in t h e pre s e nc e of i mM MgC12. (b) M e m b r a n e s i s o l a t e d b y t h e first m e t h o d in i mM N a H C O v See METHODS for t h e d e t a i l s of o t h e r f r a c t i o n a t i o n procedures. M i t o c h o n d r i a were p e l l e t e d a t i 0 0o0 × g a ve ra ge , 20 min, a n d w a s h e d b y 4 r e s u s p e n s i o n s a n d p e l l e t i n g in 2 2 % sucrose + I mM MgCI~, a n d floated in a c ont i n u o u s sucrose g r a d i e n t . F l o a t s l i g h t e r t h a n 43 % were d i s c a r d e d due t o c o n t a m i n a t i o n w i t h m e m bran es. All f r a c t i o n s e x c e p t (b) were i s o l a t e d in t h e p r e s e n c e of I mM MgC12.
isolated in I mM MgC12 is 3o times greater than the unfractionated homogenate. However, cell membranes isolated in I mM MgC12 only account for lO-150/0 of the total antigen present within the liver homogenate. Therefore the distribution of the antigen with respect to density and sedimentation characteristics was investigated. When the liver homogenate was introduced at the bottom of a continuous sucrose gradient, 54-20%, containing I mM MgCI, and centrifuged for 18 h at 9 ° ooo × g max. all of the detectable antigen was localized between 42 and 430/0 sucrose, corresponding to the isopycnic point of isolated cell membranes. When the 42-43 % sucrose Biochim. Biophys. Acta, 154 (1968) 540-552
55 °
D . M . NEVILLE, JR
fraction was centrifuged at 8000 x g average for IO min all of the antigen was sedimented. This sediment and the IOO ooo × g pellet obtained from its supernatant were subjected to electron microscopy after fixation in glutaraldehyde and staining with osmium tetroxide. The low g pellet was very rich in membranes composed of villous processes. These could be derived from small fragments of blood from cell membranes or bile canaliculi, and they were not as plentiful in the high g pellet. Both pellets were rich in endoplasmic reticulum, rough vesicles predominating in the low g pellet. In view of the fact that the isopycnic point of the particulate antigen coincides with the isopycnic point for cell membranes it is likely that all of the antigen is derived from cell membranes. The membrane isolation procedures select out of necessity only large fragments of membranes (roughly IO # x IO/,), and it appears that most of the cell membrane is disrupted into smaller fragments on cell breakage. We cannot, however, eliminate the possibility that the antigen is also present in a previously undefined vesicular fraction or in a fraction of rough endoplasmic reticulum characterized by a high sedimentation coefficient and an isopycnic point corresTABLE II ORGAN LOCALIZATION
OF LIVER
MEMBRANE
PROTEIN
P r e s e n c e of t h e i s o l a t e d m e m b r a n e p r o t e i n was a s s a y e d b y j u d g i n g i n t e n s i t y of p r e c i p i t i n lines a f t e r d o u b l e i m m u n o d i f f u s i o n of p o s t i m m u n i z a t i o n g l o b u l i n vs. E D T A e x t r a c t s of o r g a n homogenates. 4 0 % h o m o g e n a t e s of each o r g a n were m a d e in 2 2 % sucrose + I mM MgCI~ a n d c e nt ri fu ged a t 17o ooo x g max., i 5 o min. P e l l e t s were r e s u s p e n d e d y i e l d i n g a 5 0 % h o m o g e n a t e a n d d i a l y z e d a t 4 ° for 18 h vs. 500 vols. of I mM d i s o d i u m E D T A - T r i s (pH 7.0), a n d c l e a re d b y cent r i f u g a t i o n prior to i m m u n o d i f f u s i o n a n d LOWRY p r o t e i n d e t e r m i n a t i o n .
Organ
Organ extract titer
Organ extract protein (mg/ml)
R a t liver Rat kidney R a t spleen Rat intestinal mucosa Rat pancreas R a t adipose t i s s u e R a t skeletal muscle Rat erythrocytes Rat embryo liver-mid Rat embryo liver-late G u i n e a pig l i v e r Mouse liver Chicken l i v e r Hepatoma, induced Morris h e p a t o m a Rat plasma
4 o o o o o o o o 3 2 3 o 4 o o
6.7 5.3 6.3 2.3 2.5 1.4 5.7 5.o 3.7 4 .6 6. 4 5.8 4.2 6.8 4.7 --
ponding to 42-43 % sucrose. Most procedures designed to isolate rough endoplasmic reticulum never isolate that fraction having a high sedimentation coefficient. This material either exists as large clumps or is attached to the mitoehondria and nuclei and is discarded in the nuclear-mitochondrial pellet unless very high shear forces are used during homogenization. Biochim. Biophys. Acta, 154 (1968) 54o-552
ORGAN-SPECIFIC CELL MEMBRANE PROTEIN
551
Organ specificity The organ specific character of the protein antigen is shown in Table II. A variety of organ homogenates including liver have been extracted with E D T A under identical conditions. Although E D T A extracts protein from all homogenates, precipitin lines are obtained only from liver extracts. The absence of precipitin lines with rat plasma indicates that the protein antigen is not one of the variety of plasma proteins manufactured by the liver. The protein antigen is not highly species specific being detectable in two other rodents but it was not detectable in the chicken. Although the protein antigen is organ specific it was not detectable in midterm embryonic livers although it was present in the latter third of gestation. A highly differentiated second generation hepatoma induced by diacetyl amino fluorene contained the antigen but the more undifferentiated Morris hepatoma failed to elicit a reaction. DISCUSSION
These experiments show that the liver cell membrane contains an organ-specific protein having a minimal or subunit tool. wt. of 7 ° ooo and which comprises roughly IO °/o of the total membrane protein. Three types of experiments document the cell membrane origin of this protein. First, the protein is extractable from cell membranes isolated under conditions which fail to solubilize the protein. This eliminates the possibility that the protein is an adsorbed contaminant from material solubilized during ffactionation. Second, the protein is not detected in extracts of purified nuclei, mitochondria, and endoplasmic reticulum. Third, the protein is localized to the cell membrane of intact hepatocytes b y immunofluorescence. The latter experiments eliminate the possibility that the protein never existed in membrane but was extracted from nonmembrane particles which m a y contaminate the membrane preparations. The organ specific nature of the membrane protein is clearly seen in the results presented in Table II. By organ specific we mean that the quantity of protein in organs other than liver is reduced at least by a factor of ten, this being the limit of the detection system. Liver specific antigens have been previously detected by immunizing with liver particulate fractions and performing adsorptions of antisera with other organs prior to immunodiffusion 1~. However, these antigens have not been isolated or characterized. At present the function of the liver specific membrane protein is unknown. However, the localization studies indicate that this function must be related to liver specific processes rather than general cellular processes necessary for cell viability. The absence of the liver specific protein from midterm embryos and Morris hepatoma (a tissue cultured cell line) as opposed to its presence in late term embryo and the more highly differentiated second generation hepatoma is consistent with a liver specific function. Whether the absence of the protein from the membranes of Morris hepatoma has any relation to the capacity of this cell line for uncontrolled growth in a variety of environments remains to be elucidated. Cellular proteins are generally divided into three functional classes, enzymatic, carrier, and structural proteins. Regardless of the function of the liver specific membrane protein it has unique possibilities to serve as a transmitter of information. A Biochim. Biophys. Acta, 154 (1968) 540-552
552
D . M . NEVILLE, JR
p r o t e i n w h i c h is c h a r a c t e r i s t i c for a g i v e n cell t y p e a n d is localized to t h e surface m e m b r a n e p r o v i d e s a m o l e c u l a r m a r k e r for t h a t p a r t i c u l a r cell t y p e , a m a r k e r w h i c h is accessible to a n y o t h e r cell m a k i n g c e l l u l a r c o n t a c t . S u c h p r o t e i n s we refer to as e i g e n proteins. A n u m b e r o f o b s e r v a t i o n s s u g g e s t t h a t cells are c a p a b l e of r e c o g n i z i n g o t h e r cells, a n d t h a t c e l l u l a r c o n t a c t is i n v o l v e d in t h e r e c o g n i t i o n process 13. S u c h r e c o g n i t i o n w o u l d a p p e a r to be a n e c e s s i t y for o r g a n d i f f e r e n t i a t i o n a n d t h e m a i n t e n a n c e of c e l l u l a r o r g a n i z a t i o n . I t is r e a s o n a b l e t o s u p p o s e t h a t r e c o g n i t i o n occurs at t h e surface m e m b r a n e since t h i s s t r u c t u r e s e p a r a t e s t h e e x t e r n a l f r o m t h e i n t e r n a l e n v i r o n m e n t . W e are i n v e s t i g a t i n g t h e p o s s i b i l i t y t h a t cellular r e c o g n i t i o n is m e d i a t e d b y cell t y p e specific m e m b r a n e p r o t e i n s . I f this is t h e case it s h o u l d be possible to d e m o n s t r a t e t h a t e v e r y d i f f e r e n t cell t y p e has an ' e i g e n ' p r o t e i n , a n d in a d d i t i o n to cell specificity a n d m e m b r a n e l o c a l i z a t i o n t h e s e p r o t e i n s e x e r t p r o f o u n d effects on o r g a n i z e d c e l l u l a r behavior.
ACKNOWLEDGEMENTS T h e a u t h o r t h a n k s Mr. JAMES BOONE for e x c e l l e n t t e c h n i c a l assistance. L i v e r t u m o r s w e r e gifts f r o m Dr. MELVIN D. REUBER o f t h e N a t i o n a l C a n c e r I n s t i t u t e a n d Dr. EDWARD B. THOMPSON of t h e N a t i o n a l I n s t i t u t e for A r t h r i t i s a n d M e t a b o l i c Diseases. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16
17
D. M. NEVILLE, JR., Biochim. Biophys. Acta, 133 (1967) 168. D. M. NEVILLE, JR., J. Biophys. Biochem. Cytol., 8 (196o) 413 . T. JOVlN, A. CHRAMBACHAND M. NAUGHTON, Anal. Biochem., 9 (1964) 351. G. M6LLER, J. Exptl. Med., 114 (1961) 415 . N. G. ANDERSON, Science, 117 (1953) 627. G. GOLDS'rEIN, B. H. SPALDING AND W. B. HUNT, Proc. Soc. Exptl. Biol. Med., I I I (1962) 416. R. C. NAIRN, Fluorescent Protein Tracing, E. and S. Livingstone, Edinburgh and London, 1962. G. K. ACKERS, Biochemistry, 3 (1964) 723 • K. KAWAHARA,A. G. I~IRSHNER AND C. TANEORD, Biochemistry, 4 (1965) 12°3. C. TANFORD, Physical Chemistry of Macromoleeules, John Wiley, New York-London, 1963, Chap. 6. A. J. CROWLE, Immunodiffusion, Academic Press, N.Y., 1961. D. C. DUlVtONDE,Advan. Immunol., 5 (I966) 245. T. HUMPHREYS, in B. D. DAvis AND L. WARREN, The Specificity of Cell Surfaces, PrenticeHall, Englewood Cliffs, N.J., 1967, p. 195. O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265 . T. E. CONOVER AND G. SIEBERT, Biochim. Biophys. Acta, 99 (1965) I. J. CHAUVEAU, Y. MOUL]~, C. ROUILLER AND J. SCHNEEBELI, J. Cell Biol., 12 (1962) 17. G. DALLNER, S. ORRENIUS AND A. BERGSTRAND, J. Cell. Biol., 16 (1963) 426.
Bioehim. Biophys. Acta, 154 (1968) 540-552