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A partially purified membrane protein specific to the brain
Biochimica et Biophysica Acta, 322 (1973) 359-371 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s
BBA
36515
A P A R T I A L L Y P U R I F I E D MEMBRANE P R O T E I N SPECIFIC TO T H E BRAIN
R A M O N LIM AND M A R Y P. G O E D K E N
Division of Neurosurgery and Department of Biochemistry, University of Chicago, Chicago, Ill. 60637
(u.s.A.) (Received March 2oth, 1973)
SUMMARY
A brain-specific protein was partially purified from the particulate fraction of the cerebral cortex by dissociation with lithium diiodosalicylate, phenol treatment and ethanol precipitation. The product was water-soluble and showed several bands on polyacrylamide electrophoresis. Only one of these bands showed antigenic activity. The antigenicity was destroyed by trypsin but not by deoxyribonuclease, ribonuclease, neuraminidase or periodate oxidation. The antigen was not precipitable by vinblastine. The antiserum against this protein did not cross-react with S-Ioo protein, the myelin basic protein, and the liver membrane proteins. The immunological activity of the serum toward the brain protein was not affected by adsorption with membranes from liver, kidney and muscle, but was completely abolished after adsorption with cerebral membranes. The protein antigen was sensitive to acid but resistant to alkali. At physiological pH it was heat-stable. In contrast to most proteins, the protein band took up a pink color rather than blue when stained with Coomassie brilliant blue. The antigen had a molecular weight of 42 ooo and appeared to consist of dissociable subunits.
INTRODUCTION
The information processing function of the brain is dependent on the complexity of its cellular organization, which in turn is reflected in the heterogeneity of its membrane components. In fact, the brain is one of the most membranous organs in the body. Our laboratory 1-4 has been interested in the solubilization, identification and isolation of membrane proteins from the brain, with emphasis on those that are organ specific. It is assumed that chemical knowledge acquired of such proteins might in the long run be relevant to some of the important functions unique to the nervous system. The present report deals with the immunological and chemical characterization of a partially purified brain-specific membrane protein.
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MATERIALS AND METHODS
Isolation of the membrane fraction Pig brains were obtained from a local slaughter house within 3o rain after killing and transported in ice to the laboratory. For each batch of material, 500 g (wet weight) of cerebral cortex, cleared of the meninges and tile bulk of tile white matter, was dissected from the brains. This and all the subsequent steps were conducted at 4 °C. A 25~)o (w/v) homogenate in 0.32 M sucrose was prepared by blending the brain tissue for IO s at 23 ooo rev./min in a Waring blender. The homogenate was centrifuged at 2500 × g for 4 min in a Spineo No. 15 rotor to remove the unbroken cells, cell debris, nuclei, blood vessls and the bulk of the myelin. The supernatant fraction was brought back to a volume of 2 1 with the sucrose solution and then spun at 23 ooo x g for 5 h to collect all the remaining particulate material (henceforth designated as tile "membrane fraction"), which is a mixture of the microsomes, the synaptic membranes, synaptic vesicles, the mitoehondria, some myelin, and the fragmented plasma membranes. The use of the No. 15 rotor enabled us to handle a large volume of sample in each run. The collected membrane mixture was dialyzed against several changes of water and freeze-dried. The usual yield was 4-5 g dry weight. The liver membrane fraction was isolated in a similar manner except that the blending step was extended to I rain. About io g of dried membrane mixture was usually obtained out of 500 g of fresh pig liver.
Partial purification of the membrane antigen The procedure is a modification of tile method of Marchesi and Andrews 5. The membrane fraction from the brain, 4 g dry weight, was suspended in 15o ml of a solution of o.2 M littfium diiodosalicylate and 4 M urea in o.o5 M Tris HC1 at pH 7.5The suspension was sonicated for a total of 3 min with a Branson sonifier (Model W I85D ) using a microtip at 5o W output. Sonication was conducted with 3o-s bursts and I-rain cooling periods between each exposure. Undissolved material was eliminated by centrifugation at 63 ooo × g for I h. The supernatant solution was diluted with 2 vol. of water and mixed with 45o ml of freshly prepared 5o% phenol in water. The mixture was stirred in the cold for 15 rain followed by a centrifugation to separate the two layers. The aqueous phase was concentrated to dryness by alternate dialysis (versus water) and lyophilization. The dried material was washed with 4oo ml of absolute ethanol by stirring it in the cold for 15 min followed by centrifugation. After two more washings the residue was resuspended in 5o ml of water and dialyzed against several changes of water at 4 °C. The material that was insoluble in water at this stage was eliminated by centrifugation. The clear solution was lyophilized. About 15o mg of the dried product (henceforth designated as the "membrane antigen"), was usually obtained. Material isolated from the liver for comparative studies showed a similar yield.
Isolation of the myelin basic protein The myelin basic protein was obtained by a modification of several methods 6-8. I0 g of white matter from pig brain was cut into small pieces, frozen, thawed and ground up with a mortar and pestle. The resulting paste was blended with 190 ml of chloroform-methanol (2 :I, v/v) and centrifuged. The pellet was washed 2 times with
BRAIN SPECIFIC PROTEIN
36I
acetone and extracted twice with 25 ml of o.o5 M HC1 in o.2 M NaC1 (final pH 1.6). The residue was eliminated by eentrifugation. The supernatant fraction was adjusted to p H 7 with N a O H and spun again to remove a small amount of insoluble material. The clear solution was dialyzed against water. Some precipitate that appeared after dialysis was further eliminated. The solution was finally freeze-dried yielding 16 mg of basic protein.
S-zoo protein Bovine S-Ioo protein and the corresponding antiserum were kindly provided by Dr Blake W. Moore of Washington University.
Immunological methods Albino rabbits, about 8 lb body weight, were each inoculated with 5 mg of the brain membrane antigen. The dried material was suspended in I ml of isotonic saline and mixed with I ml of complete Freund's adjuvant (Difco) to form a water in oil emulsion, i ml of the mixture was distributed among the four foot pads, and the remainder injected subcutaneously. A second inoculation was given subcutaneously four weeks later, using 5 mg membrane antigen mixed with incomplete Freund's adjuvant. Antisera were harvested after another two weeks. Control sera obtained prior to immunization did not show any reactivity toward the membrane antigen. The rabbits were neurologically normal throughout the course of immunization. Adsorption of the antisera was conducted by incubating the sera with a membrane fraction at 37 °C for 3o min and then at 4 °C for 48 11; the sera were recovered by centrifugation. Sera incubated in the absence of membrane served as the controls. Immunodiffusion was conducted in Ouchterlony plates made up of 1% Ionagar No. 2 (Colab) in the presence of o.15 M NaC1 and o.o2 M Tris-HC1, p H 7.5 (henceforth designated as "Tris-saline"). Phenol, o.1% was included in all plates as a preservative. Each well contained 4 °/~1 of antiserum or antigen through a single filling. Unless otherwise specified, the antigen concentration was 2 mg dry weight per ml of Tris-saline. Enzyme treatment was conducted by incubating 2 mg of the brain membrane antigen (in I ml of Tris-saline) with i mg of enzyme at 37 °C for 2 h. Bovine pancreatic deoxyribonuclease, bovine pancreatic ribonuclease, Clostridium perfringens neuraminidase and trypsin were products of Sigma Chemical Co. In the case of ribonuclease, the enzyme was preincubated at 8o °C for lO min to eliminate any other possible contaminating enzymes. In the case of trypsin, I mg of egg white trypsin inhibitor was added to terminate the reaction. Controls for deoxyribonuclease, ribonuclease and neuraminidase consisted of incubating the antigen in the absence of enzyme. Control for trypsin consisted of a complete incubation mixture where the inhibitor was added before the enzyme reaction. Control and enzyme-treated samples were directly applied to the antigen wells for immunodiffusion without further treatment. Periodate oxidation was performed by treating the antigen with o.oi M sodium meta-periodate in Tris-saline at 4 °C for 24 11. The excess periodate was subsequently eliminated by dialysis against the same buffer.
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Electrophoretic methods Two conditions were used for polyacrylamide gel electrophoresis: the original method of Davis 9 without the addition of detergent or other solubilizing agents (henceforth designated as "plain gels") and the sodium dodecyl sulfate technique (henceforth designated as "dodecyl sulfate gels") of Weber and Osborn 1°. In both cases only gels containing 14% acrylamide monomer were prepared and used in the current study; the crossdinker concentration was increased proportionally, thus maintaining the original monomer cross-linker ratios. The gels were cast in 6-ram (internal diameter) tubes and run at 2.5 mA/tube for the plain gel and IO mA/tnbe for the dodecyl sulfate gel, both toward the anode. For the plain gel 2 mg of membrane antigen was dissolved directly in I ml of the electrode buffer (Tris-glycine, pH 8.3) containing 2o~/o sucrose. For the dodecylsulfate gel the same amount of membrane antigen was used per ml of a sample solvent consisting of: o.oi M sodium phosphate at p H 7.o, o.I}/o dodecyl sulfate, IO(~ 2-mercaptoethanol, 4 M urea and o.oo5°/o Bromphenol blue. In both cases IOO/~1 of the sample solution was layered on top of each gel for electrophoresis, giving an equivalence of 2oo/~g membrane antigen per gel. The gels were fixed in 5o')L ethanol containing i o % acetic acid. After leaving in this solution overnight, the gels were stained for 4-6 h with o.25% Coomassie brilliant blue dissolved in the above solution. Destaining was carried out by soaking tile gel in 5% ethanol with 7.5}'o acetic acid for 24 11. The gels were finally stored in 7~/o acetic acid. Enzyme degradation of the membrane antigen was carried out in Tris-glycine buffer at p H 8.3 (for the plain gel), or in o.oi M sodium phosphate at p H 7.0 (for the dodecyl sulfate gel), the buffers used in the corresponding sample solutions. Membrane antigen and enzyme concentrations were 5 mg/ml and I mg/ml, respectively. After incubating at 37 °C for I h, the mixtures were readjusted to contain all the components at their proper concentrations in the complete sample solutions (see above) ; the final membrane antigen concentration being 2 mg/ml. A sample volume of o.I ml was used per gel. Thus, other than the presence of the enzymes, all conditions for electrophoresis were the same as the untreated samples. Protein bands corresponding to the enzymes were detected by electrophoresing each enzyme in separate gels, and the bands subtracted from the protein patterns of the experimental gels. All enzymes used were identical with those described in the immunological methods. Transplantation of acrylamide gel segments was made from plain gels to dodecyl sulfate gels or to other plain gels. The unfixed, unstained gel from which the protein was to be transplanted was sliced crosswise into 16 pieces. Each disc was further split into two equal halves. One half of each pair was frozen and the other was embedded in an agar plate for immunodiffusion. After identifying the segment which contained the antigenically active protein, the corresponding half in storage was thawed, chopped into pieces of approximately I cubic ram, mixed with the sample solvent and placed on top of a gel for electrophoresis. The recipient gel was then treated as usual. Molecular weight determinations were performed in i 4 % dodecyl sulfate gels, using 5 o # g of commercially purified proteins as standards. All standard proteins were products of Sigma Chemical Co.
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RESULTS
Specificity of the brain membrane antigen The b r a i n m e m b r a n e a n t i g e n showed a single p r e c i p i t i n line a g a i n s t its antiser u m (Fig. I). The same serum d i d n o t cross-react w i t h t h e corresponding m a t e r i a l o b t a i n e d from pig liver, nor was a n y r e a c t i o n d e m o n s t r a t e d t o w a r d pig b r a i n cytosol
Fig. i. Immunodiffuslon showing specificity of brain membrane antigen. As, antiserum against brain membrane antigen; BMA, brain membrane antigen; BSP, brain soluble protein (cytosol from a 20% pig brain homogenate in Tris-saline); LMA, liver membrane antigen; MBP, myelin basic protein• a n d m y e l i n basic protein. The organ specificity was f u r t h e r s u b s t a n t i a t e d b y the r e t e n t i o n of the a n t i b o d y a c t i v i t y in the serum after a d s o r p t i o n with liver m e m b r a n e fraction (Fig. 2). A d s o r p t i o n w i t h k i d n e y a n d muscle m e m b r a n e s gave essentially similar results as w i t h liver, whereas a d s o r p t i o n with t h e cerebral m e m b r a n fraction c o m p l e t e l y abolished the a c t i v i t y . Fig. 3 shows t h e i m m u n o l o g i c comparison b e t w e e n this a n t i g e n a n d S - I o o protein. A n t i s e r u m a g a i n s t S-Ioo, in an a m o u n t which r e a c t e d s t r o n g l y with b o t h S-
C
,
Fig. 2. Effect of adsorption on reactivity of antiserum. BMA, brain membrane antigen; As, anti~erum against brain membrane antigen (unadsorbed); As(LMF)I, antiserum against brain membrane antigen (adsorbed with 6 mg liver membrane fraction per ml serum) ; As(LMF)~, antiserum against brain membrane antigen (adsorbed with 12 mg liver membrane fraction per ml serum).
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I I
ii ! i
I
Fig. 3. Absence of cross-reactivity between brain membrane antigen and S-Ioo protein. BMA, brain membrane antigen; As, antiserum against BMA; S-loo, S-ioo protein of Moore (2.5 /~g added); As(S-Ioo), antiserum against S-ioo protein; BSP, brain soluble protein (cytosol from a 2O~o pig brain homogenate in Tris saline). IOO p r o t e i n a n d pig b r a i n cytosol, d i d not form a p r e c i p i t i n line with the cerebral m e m b r a n e antigen, whereas a n t i s e r u m a g a i n s t cerebral m e m b r a n e a n t i g e n r e a c t e d with the homologous antigen b u t n o t w i t h S - I o o a n d cytosol. The fact t h a t pig b r a i n soluble fraction r e a c t e d with a n t i - S - I o o b u t n o t with a n t i - c e r e b r a l - m e m b r a n e would i m p l y t h a t if the s u p p o s e d m e m b r a n e antigen were present in the cytosol, its c o n c e n t r a t i o n m u s t be lower t h a n t h a t of S - I o o which is a b o u t o.Ic~/~, of t o t a l soluble p r o t e i n 1~. Such low c o n c e n t r a t i o n s in the soluble fraction, then, would be v e r y
Fig. 4. Effect of hydrolytic enzymes on antigenicity of brain membrane antigen. 17pper wells, brain membrane antigen (controls); middle wells, antiserum against brain membrane antigen; lower wells, enzyme-treated brain membrane antigen. DNase, deoxyribonuclease-treated; RNase, ribonuclease-treated; NANase, N-acetyl neuraminidase-treated; Tryp, trypsin-treated. Composite photograph of individual agar plates.
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BRAIN SPECIFIC PROTEIN
unlikely to contaminate (during purification) the particulate fraction to an extent as to be detectable by immunodiffusion. Further evidence of the membrane origin of the antigen derives from the fact that omission of lithium diiodosalicylate resulted in negligible yield of the antigen.
Chemical nature of brain membrane antigen The brain membrane antigen was incubated with various degradative enzymes. Fig. 4 shows that the antigenicity was not affected by deoxyribonuclease, ribonuclease and neuraminidase, but was completely abolished by trypsin, indicating the protein nature of the antigen. Periodate oxidation did not inactivate the antigen. The protein was insoluble in ethanol, but was not denatured by this agent even at temperatures above o °C. It was not precipitable by vinblastine, an alkaloid which precipitates many acidic proteins including tubulin. When dissolved in o.15 M NaC1 with 0.02 M Tris-HC1, pH 7.5, the antigenic activity could withstand heating in a boiling water bath for io min. The antigenicity was considerably reduced after bringing the pH down to 3-5 followed by readjustment to neutrality. However, a similar treatment with NaOH to bring the pH up to II did not alter its immunological activity. The brain membrane antigen showed an absorbance ratio (A280 nm/A2~ 0 nm) of
~!iC~iill/ ~i!~i~ili ~i~ Fig. 5. I m m u n o e l e c t r o p h o r e s i s of b r a i n m e m b r a n e antigen. E l e c t r o p h o r e s i s w a s carried o u t in a 1 4 % plain p o l y a c r y l a m i d e gel. T h e u n f i x e d gel w a s split lengthwise. O n e h a l f w a s s t a i n e d w i t h C o o m a s s i e blue a n d t h e o t h e r w a s e m b e d d e d in i % I o n a g a r No. 2 in t h e presence of T r i s - s a l i n e . A n t i s e r u m a g a i n s t b r a i n m e m b r a n e a n t i g e n w a s placed in t h e t r o u g h one d a y later. T h e a c r y l a m i d e gel w a s i n t e n t i o n a l l y b r o k e n a t two sites to p r e v e n t it f r o m curling up. A, result a f t e r i m m u n o diffusion. B, s t a i n i n g p a t t e r n of a c r y l a m i d e gel, s h o w i n g m o b i l i t y v a l u e s of b a n d s w i t h r e s p e c t to t h e f r o n t (f). C, s k e t c h s h o w i n g precipitin line w i t h r e s p e c t to t h e location of p r o t e i n b a n d s in a c r y l a m i d e gel; scale on r i g h t i n d i c a t e s t h e division of t h e gel into 16 e q u a l s e g m e n t s (see Fig. 6).
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0.60, indicating the presence of a considerable amount of nucleic acid in the product. This was not a surprise since the cerebral membrane fraction included the microsomes, and the fractionation procedure employed (phenol partition and alcohol precipitation) was similar to procedures used for the isolation of RNA. The membrane antigen could be further purified with streptomycinr2, which eliminates the nucleic acids and possibly some acidic proteins. The sample solution was mixed with 0.3 vol. of 5ojo streptomycin sulfate at neutral pH and stirred for 15 min in the cold. The white precipitate, which contained the RNA-streptomycin complex, was removed by centrifugation. The excess streptomycin was subsequently eliminated by dialysis. The antigenic activity was fully retained despite an 800/, reduction in dry weight (a 5-fold purification). The streptomycin-treated products showed an absorbance ratio of 0.83 indicating the possible presence of 6% nucleic acid. However, all the immunological and electrophoretic studies in this report were conducted without streptomycin treatment. Localization
of the antigen
in polyacrylamide
gel
Fig. 3 shows that the cerebral membrane antigen yielded two major bands and three minor bands in the plain gel. Only a minor band (mobility 0.22) formed a precipitin line upon immunodiffusion. Unlike ordinary proteins, this band stained pink rather than blue with Coomassie. Antoher minor protein band (mobility 0.77) also stained pink but did not form a precipitin line. All other protein bands ap-
Fig. 6. Localization of antigen in acrylamide gel. Brain membrane antigen was electrophorescd in 14% plain gel. The unfixed gel was sliced crosswise into 16 equal segments as indicated in Fig. 5. The slices were chopped into smaller pieces and placed in the peripheral wells of an Ouchterlony plate. Immunodiffusion was started a day later by filling the center well with antiserum. Note the presence of a precipitin line only in piece No. 4 which corresponds to the location of the precipitin line in Fig. 5. Pieces of acrylamide’gels were removed from the wells before photography.
BRAIN SPECIFIC PROTEIN
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peared blue a n d were i m m u n o l o g i c a l l y inert, as far as p r e c i p i t a t i n g a n t i b o d y is concerned. The location of the i m m u n o l o g i c a l l y active b a n d was further checked b y slicing the gel crosswise. As seen in Fig. 6, only segment No. 4 reacted with the a n t i s e r u m . T h u s we i d e n t i f y the b a n d with a m o b i l i t y of o.22 as the antigenically active b a n d .
Characteristics of the antigenic band in polyacrylamide gel Protein b a n d s u n d e r various gel conditions are shown in Fig. 7. As in the plain gel, a pink b a n d with a m o b i l i t y of 0.22 ( ' A " band) was observed in the dodecyl sulfate gel. This b a n d was c o n s t a n t l y seen a m o n g different batches of p r o d u c t despite v a r i a b i l i t y in other bands. A protein corresponding to the B b a n d (mobility 0.77 ) was
I
II
III
Fig. 7. Gel patterns of membrane antigen under different electrophoretic conditions. 1, brain membrane antigen in plain gels; duplicate gels are show. II, brain membrane antigen in dodecyl sulfate gels; the two samples were taken from two different batches of product. III, brain membrane antigen (left) compared with liver membrane antigen (right) in dodecyl sulfate gels; the two gels were electrophoresed simultaneously; the brain membrane antigen was from a batch different from those shown in II. Origin at top; front at bottom end (indicated by a line). A and B designate positions of bands with mobilities of o.22 and o.77, respectively. The A band from the brain membrane antigen appears distinctly pinkish in all gels. The B band from the brain is pink only in the plain gel but not in the dodecyl sulfate gel. Liver sample does not show any pink band. Coomassie blue stain. present in the dodecyl sulfate gel, b u t unlike its c o u n t e r p a r t in the plain gel this b a n d was variable a n d s t a i n e d blue with Coomassie. I n contrast to the b r a i n m e m b r a n e antigen, the corresponding m a t e r i a l purified from the liver m e m b r a n e s did n o t reveal a n y distinct b a n d s at positions A a n d B, nor was a n y b a n d observed to be p i n k in color. The absorption spectra of the pink a n d blue b a n d s from cerebral m e m b r a n e are shown in Fig. 8.
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R. LIM, M. P. GOEDKEN
0.5 0.4
z 0.3 0.2 0.1 i
I
,
,
i
i
500
I
,
i
,
i
600
I
700
WAVELENGTH (nm)
Fig. 8. Absorbance s p e c t r a of a pink b a n d ( " A " b a n d in Fig. 7) and a blue b a n d f r o m acrylamide gels stained with Coomassie brilliant blue. Note the presence of a left shoulder w i t h the pink b a n d and a right shoulder with the blue b a n d which p r o b a b l y are responsible for their characteristic colors.
Fig. 9 and IO show the results of enzyme treatment on the band patterns. In both gel systems the band mobilities were not altered after treatment with deoxyribonuclease, ribonuclease and neuraminidase. Tryptic digestion, however, abolished most of the bands, including the A band, yielding smaller peptides close to the front. Thus the results confirm those obtained with immunological studies in suggesting the protein nature of the cerebral membrane antigen.
A~
A~
B~ Cont
"'"'1'
DNase RNase NANase Tryp
i
:!:!:!:i:i:?::? ~:;:i:{:;:;:!:?!
:i:!:!:!:!:!:!:i:
B ~ ::::::::::%::: Cont
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RNase NANase
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Fig. 9- Effect of hydrolytic enzymes on protein p a t t e r n of brain m e m b r a n e antigen in plain gel. Cont, control (sample n o t treated with enzyme); DNase, deoxyribonuclease-treated; RNase, ribonuclease-treated; NANase, N-acetyl n e u r a m i n i d a s e - t r e a t e d ; Tryp, trypsin-treated. Origin at top; f r o n t at b o t t o m end. A and B indicate positions of b a n d s with mobilities of o.22 and o.77, respectively. Fig. io. Effect of hydrolytic enzymes on protein p a t t e r n of brain m e m b r a n e antigen in dodecyl sulfate gel. Origin at top; f r o n t at b o t t o m end. A and B indicate positions of b a n d s with rnobilities of 0.22 and o.77, respectively. Control and e n z y m e - t r e a t e d samples are as designated in Fig. 9-
That the A bands in the plain and dodecyl sulfate gels were identical was borne out by transplantation experiments (Fig. 11) in which a gel segment corresponding to the A band was cut out from an unfixed plain gel and re-electrophoresed in a dodecyl sulfate gel. This resulted in the appearance of a pink band in the location where the A band should be. The similarity in mobility of the antigenic band in the presence or absence of dodecyl sulfate suggests the open chain conformation of the protein and that the protein might be highly acidic. Whether the native conformation of the protein had been modified during the initial membrane solubilization and
369
BRAIN SPECIFIC PROTEIN
A~
~A
B~
~-B I
Z
m/2
Tn-
Fig. II. R e s u l t s of gel t r a n s p l a n t a t i o n . A a n d B indicate positions o f b a n d s w i t h mobilities of 0.22 a n d 0.77, respectively. I, p r o t e i n p a t t e r n of b r a i n m e m b r a n e a n t i g e n in p l a i n gel; II, result of t r a n s p l a n t i n g " A " b a n d f r o m I to a n o t h e r piece of p l a i n gel. I I I a n d IV are t h e t w o different o u t c o m e s of t r a n s p l a n t i n g A b a n d f r o m 1 to dodecyl s u l f a t e gels.
subsequent purification steps is not known. Nevertheless, we feel safe to say that the antigenic determining site appears to be unaltered since the antiserum prepared against the purified protein reacted with the intact membrane fraction as well. We observed that the transplantation of the A band from a plain gel to a dodecyl sulfate gel occasionally resulted in the appearance of the B band, instead of the A band, in the recipient gel (Fig. II). In this instance the nascent B band stained blue. Thus it appears that some relationship existed between the A and B bands. Considerable effort had been exerted without success to predictably direct the transplanted band to either the A or B position. Such efforts included the addition or omission of 2-mercaptoethanol, dithiothreitol and iodoacetamide at various concentrations and in various sequences, as well as incubating the sample mixture at 37 °C. The possibility that an endogenous protease might have been activated by sodium dodecyl sulfate is unlikely since increasing the sodium dodecyl sulfate concentration in the sample mixture from o. I to i °/o and heating the sample in a boiling water bath did not prevent the formation of B band in the dodecyl sulfate gel. Further evidence 9080-
1
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MOBILITY
Fig. 12. Molecular w e i g h t d e t e r m i n a t i o n w i t h 14 % dodecyl s u l f a t e gel. Mobility v a l u e s are w i t h r e s p e c t to t h e t r a c k i n g d y e ( B r o m p h e n o l blue). T h e s t a n d a r d p r o t e i n s are: I, b o v i n e s e r u m alb u m i n ; 2, c a t a l a s e ; 3, o v a l b u m i n ; 4, c a r b o x y p e p t i d a s e A; 5, t r y p s i n ; 6, m y o g l o b i n ; 7, ribon u c l e a s e ; 8, c y t o c h r o m e c; 9, insulin. T h e use of e - m e r c a p t o e t h a n o l w a s o m i t t e d for insulin. A r r o w s i n d i c a t e t h e locations o f A a n d B b a n d s of b r a i n m e m b r a n e antigen.
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R. LIM, M. P. G O E D K E N
against protein degradation is provided by the reverse conversion of B band to A band, a phenomenon observed when transplanting the B band from a plain gel to another plain gel. It seems as if the protein antigen consisted of dissociable subunits, although the mechanism of association is not known. Molecular weight determination (Fig. 12) shows that the A and B bands have molecular weights of about 42 ooo and 7ooo, respectively, suggesting that the membrane antigen was composed of 6 subunits. Whether these subunits were homomonomers or heteromonomers of similar molecular size remains to be determined. DISCUSSION
One is confronted with a paradox when attempting to isolate individual membrane proteins from the brain. The dissociation of proteins from the particulate material usually necessitates the use of a detergent or other solubilizing agents. Such procedures often lead to an irreversible denaturation of the proteins with complete loss of their biological activity, a result which defeats an important purpose of studying these proteins. Lithium diiodosalicylate, a chaotropic agent, has been reported by Marchesi 5 to be useful for the isolation of an immunologically active glycoprotein from the erythrocyte membrane. Unlike Triton X-Ioo and sodium dodecyl sulfate, lithium diiodosalicylate is easily removable by dialysis. Furthernlore, Triton X - I o o appears to solubilize proteins by micelle formation rather than through dissociation. The effectiveness of sodium dodecyl sulfate as a dissociative agent for brain proteins has also been questioned 13. The use of lithium diiodosalicylate in connection with urea has enabled us to obtain an immunologically active organspecific protein from the cerebral membranes. Although the protein is by no means pure, its final isolation should not be too difficult because the protein is now water soluble and can be handled like other soluble proteins. The presence of immunological activity in this protein opens up several possibilities for future research. First, affinity chromatography m a y be used for the complete purification of the protein, taking advantage of the fact that it is the only antigenically active component in the partially purified fraction. Secondly, the distribution of this protein in various subcellular fractions can be measured with a quantitative complement-fixation test4, a4, or its exact morphological origin localized with immunoelectron microscopy. Finally, the antibody can be employed as a chemical probe for elucidating the physiological role of the protein in cultured brain cells. Several lines of evidence indicate the organ specificity of this protein: (I) the absence of reactivity of the antiserum toward liver membrane antigen similarly prepared; (2) the retention of immunological activity of the serum toward the brain membrane antigen even after adsorption with liver, kidney or muscle total membrane fraction ; (3) the protein in acrylamide gel appeared as a pink band when stained with Coomassie, whereas all the bands obtained from the corresponding liver fraction were blue. Although the original procedure of Marchesi a was designed for the isolation of glycoproteins, there is no evidence that our cerebral membrane antigen is a glycoprotein. Furthermore, none of the protein bands in the gel was periodic acid-Schiffpositive. Inasmuch as a reliable chemical analysis of the protein is not feasible in its current state of purity, the presence of a carbohydrate moiety in a manner not
BRAIN SPECIFIC PROTEIN
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critical for a n t i g e n i c i t y a n d e l e c t r o p h o r e t i c m o b i l i t y r e m a i n s to be proven. The a n t i g e n p r e p a r a t i o n was c o n t a m i n a t e d w i t h nucleic acid, even after s t r e p t o m y c i n t r e a t m e n t . However, t h e nucleic acid d i d n o t a p p e a r to interfere w i t h the i m m u n o l o g i c a l a n d e l e c t r o p h o r e t i c studies. On the o t h e r hand, the possibility t h a t t h e p r o t e i n a n t i g e n m i g h t c o n t a i n some t i g h t l y b o u n d nucleotides deserves serious consideration. A t a n y rate, the p i n k color e x h i b i t e d b y the p r o t e i n with Coomassie stain suggests molecular peculiarities, a n d favors the likelihood of a c o n j u g a t e d protein r a t h e r t h a n a simple protein. A n u m b e r of brain-specific m e m b r a n e p r o t e i n s have been r e p o r t e d (for review, see Shooter a n d Einstein15). A m o n g the well s t u d i e d are the m y e l i n proteins such as the p r o t e o l i p i d protein, the basic encephalitogenic p r o t e i n a n d the acidic p r o t e o l i p i d p r o t e i n of Wolfgram. O t h e r p r o t e i n s t h a t are p r e d o m i n a n t l y found in the brain, t h o u g h n o t s t r i c t l y organ-specific, are p r o t e i n s u b u n i t s of the cytoskeletons, such as filarin 16 a n d t u b u l i n l L R e c e n t l y an acidic p r o t e i n has been o b t a i n e d from the glial fibrillary c o m p o n e n t TM.A l t h o u g h the last three p r o t e i n s form the s t r u c t u r a l elements for i n t r a c e l l u l a r s u p p o r t , t h e y are n o t s t r i c t l y m e m b r a n e p r o t e i n s since t h e y are r e a d i l y e x t r a c t a b l e b y o r d i n a r y buffers. The p r o t e i n r e p o r t e d in this c o m m u n i c a t i o n a p p e a r s to be different from a n y of the b r a i n proteins p r e v i o u s l y s t u d i e d a n d m a y be w o r t h p u r s u i n g from b o t h chemical a n d biological p o i n t s of view. ACKNOWLEDGEMENTS This w o r k was p a r t i a l l y s u p p o r t e d b y U.S. P u b l i c H e a l t h Service G r a n t No. NS-09228 (to R.L.). W e t h a n k Dr F e r e n c J. K e z d y for o b t a i n i n g the a b s o r p t i o n spectra.
REFERENCES 1 Lim, R. and Tadayyon, E. (197o) Anal. Biochem. 34, 9 15 2 Lim, R. (1971) Trans. Am. Soc. Neurochem. 2, 93 3 Lim, R. (1971) Abstracts, 3rd Meet. Int. Soc. Neurochem., Budapest, p. 216 4 Lim, R. and Hsu, L. W. (1971) Biochim. Biophys. Acta 249, 569-582 5 Marchesi, V. T. and Andrews, E. P. (1971) Science 174, i247-i248 6 Bencina, B. and Carnegie, P. R. (1969) F E B S Lett. 4, 9-12 70shiro, Y. and Eylar, E. H. (197o) Arch. Biochem. Biophys. 138, 392-396 8 Greenfield, S., Norton, W. T. and Morell, P. (1971) J. Neurochem. 18, 2II9--2128 9 Davis, B. J. (1964) Ann. N . Y . Acad. Sci. 121, 4o4-427 io Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44o6 4412 I i Moore, B. W. (1969) in Handbook o[ Neurochemislry (Lajtha, A., ed.), Vol. i, pp. 93-99, Plenum, New York 12 Lim, R. and Cohen, S. S. (1966) J. Biol. Chem. 241, 43o4-4315 13 Katzman, R. L. (1972) Biochim. Biophys. Acta 266, 269-272 14 Moore, B. W. and Perez, V. J. (1966) J. Immunol. 96, lOOO-lOO5 15 Shooter, E. M. and Einstein, E. R. (1971) A n n u . Rev. Biochem. 4o, 635-652 16 Huneeus, F. C. and Davison, P. F. (197o) J. Mol. Biol. 52, 415-428 17 Davison, P. F. and Huneeus, F. C. (197o) J. Mol. Biol. 52, 429-439 18 Bignami, A., Eng, L. F., Dahl, D. and Uyeda, C. T. (1972) Brain Res. 43, 429-435
BBA
36515
A P A R T I A L L Y P U R I F I E D MEMBRANE P R O T E I N SPECIFIC TO T H E BRAIN
R A M O N LIM AND M A R Y P. G O E D K E N
Division of Neurosurgery and Department of Biochemistry, University of Chicago, Chicago, Ill. 60637
(u.s.A.) (Received March 2oth, 1973)
SUMMARY
A brain-specific protein was partially purified from the particulate fraction of the cerebral cortex by dissociation with lithium diiodosalicylate, phenol treatment and ethanol precipitation. The product was water-soluble and showed several bands on polyacrylamide electrophoresis. Only one of these bands showed antigenic activity. The antigenicity was destroyed by trypsin but not by deoxyribonuclease, ribonuclease, neuraminidase or periodate oxidation. The antigen was not precipitable by vinblastine. The antiserum against this protein did not cross-react with S-Ioo protein, the myelin basic protein, and the liver membrane proteins. The immunological activity of the serum toward the brain protein was not affected by adsorption with membranes from liver, kidney and muscle, but was completely abolished after adsorption with cerebral membranes. The protein antigen was sensitive to acid but resistant to alkali. At physiological pH it was heat-stable. In contrast to most proteins, the protein band took up a pink color rather than blue when stained with Coomassie brilliant blue. The antigen had a molecular weight of 42 ooo and appeared to consist of dissociable subunits.
INTRODUCTION
The information processing function of the brain is dependent on the complexity of its cellular organization, which in turn is reflected in the heterogeneity of its membrane components. In fact, the brain is one of the most membranous organs in the body. Our laboratory 1-4 has been interested in the solubilization, identification and isolation of membrane proteins from the brain, with emphasis on those that are organ specific. It is assumed that chemical knowledge acquired of such proteins might in the long run be relevant to some of the important functions unique to the nervous system. The present report deals with the immunological and chemical characterization of a partially purified brain-specific membrane protein.
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MATERIALS AND METHODS
Isolation of the membrane fraction Pig brains were obtained from a local slaughter house within 3o rain after killing and transported in ice to the laboratory. For each batch of material, 500 g (wet weight) of cerebral cortex, cleared of the meninges and tile bulk of tile white matter, was dissected from the brains. This and all the subsequent steps were conducted at 4 °C. A 25~)o (w/v) homogenate in 0.32 M sucrose was prepared by blending the brain tissue for IO s at 23 ooo rev./min in a Waring blender. The homogenate was centrifuged at 2500 × g for 4 min in a Spineo No. 15 rotor to remove the unbroken cells, cell debris, nuclei, blood vessls and the bulk of the myelin. The supernatant fraction was brought back to a volume of 2 1 with the sucrose solution and then spun at 23 ooo x g for 5 h to collect all the remaining particulate material (henceforth designated as tile "membrane fraction"), which is a mixture of the microsomes, the synaptic membranes, synaptic vesicles, the mitoehondria, some myelin, and the fragmented plasma membranes. The use of the No. 15 rotor enabled us to handle a large volume of sample in each run. The collected membrane mixture was dialyzed against several changes of water and freeze-dried. The usual yield was 4-5 g dry weight. The liver membrane fraction was isolated in a similar manner except that the blending step was extended to I rain. About io g of dried membrane mixture was usually obtained out of 500 g of fresh pig liver.
Partial purification of the membrane antigen The procedure is a modification of tile method of Marchesi and Andrews 5. The membrane fraction from the brain, 4 g dry weight, was suspended in 15o ml of a solution of o.2 M littfium diiodosalicylate and 4 M urea in o.o5 M Tris HC1 at pH 7.5The suspension was sonicated for a total of 3 min with a Branson sonifier (Model W I85D ) using a microtip at 5o W output. Sonication was conducted with 3o-s bursts and I-rain cooling periods between each exposure. Undissolved material was eliminated by centrifugation at 63 ooo × g for I h. The supernatant solution was diluted with 2 vol. of water and mixed with 45o ml of freshly prepared 5o% phenol in water. The mixture was stirred in the cold for 15 rain followed by a centrifugation to separate the two layers. The aqueous phase was concentrated to dryness by alternate dialysis (versus water) and lyophilization. The dried material was washed with 4oo ml of absolute ethanol by stirring it in the cold for 15 min followed by centrifugation. After two more washings the residue was resuspended in 5o ml of water and dialyzed against several changes of water at 4 °C. The material that was insoluble in water at this stage was eliminated by centrifugation. The clear solution was lyophilized. About 15o mg of the dried product (henceforth designated as the "membrane antigen"), was usually obtained. Material isolated from the liver for comparative studies showed a similar yield.
Isolation of the myelin basic protein The myelin basic protein was obtained by a modification of several methods 6-8. I0 g of white matter from pig brain was cut into small pieces, frozen, thawed and ground up with a mortar and pestle. The resulting paste was blended with 190 ml of chloroform-methanol (2 :I, v/v) and centrifuged. The pellet was washed 2 times with
BRAIN SPECIFIC PROTEIN
36I
acetone and extracted twice with 25 ml of o.o5 M HC1 in o.2 M NaC1 (final pH 1.6). The residue was eliminated by eentrifugation. The supernatant fraction was adjusted to p H 7 with N a O H and spun again to remove a small amount of insoluble material. The clear solution was dialyzed against water. Some precipitate that appeared after dialysis was further eliminated. The solution was finally freeze-dried yielding 16 mg of basic protein.
S-zoo protein Bovine S-Ioo protein and the corresponding antiserum were kindly provided by Dr Blake W. Moore of Washington University.
Immunological methods Albino rabbits, about 8 lb body weight, were each inoculated with 5 mg of the brain membrane antigen. The dried material was suspended in I ml of isotonic saline and mixed with I ml of complete Freund's adjuvant (Difco) to form a water in oil emulsion, i ml of the mixture was distributed among the four foot pads, and the remainder injected subcutaneously. A second inoculation was given subcutaneously four weeks later, using 5 mg membrane antigen mixed with incomplete Freund's adjuvant. Antisera were harvested after another two weeks. Control sera obtained prior to immunization did not show any reactivity toward the membrane antigen. The rabbits were neurologically normal throughout the course of immunization. Adsorption of the antisera was conducted by incubating the sera with a membrane fraction at 37 °C for 3o min and then at 4 °C for 48 11; the sera were recovered by centrifugation. Sera incubated in the absence of membrane served as the controls. Immunodiffusion was conducted in Ouchterlony plates made up of 1% Ionagar No. 2 (Colab) in the presence of o.15 M NaC1 and o.o2 M Tris-HC1, p H 7.5 (henceforth designated as "Tris-saline"). Phenol, o.1% was included in all plates as a preservative. Each well contained 4 °/~1 of antiserum or antigen through a single filling. Unless otherwise specified, the antigen concentration was 2 mg dry weight per ml of Tris-saline. Enzyme treatment was conducted by incubating 2 mg of the brain membrane antigen (in I ml of Tris-saline) with i mg of enzyme at 37 °C for 2 h. Bovine pancreatic deoxyribonuclease, bovine pancreatic ribonuclease, Clostridium perfringens neuraminidase and trypsin were products of Sigma Chemical Co. In the case of ribonuclease, the enzyme was preincubated at 8o °C for lO min to eliminate any other possible contaminating enzymes. In the case of trypsin, I mg of egg white trypsin inhibitor was added to terminate the reaction. Controls for deoxyribonuclease, ribonuclease and neuraminidase consisted of incubating the antigen in the absence of enzyme. Control for trypsin consisted of a complete incubation mixture where the inhibitor was added before the enzyme reaction. Control and enzyme-treated samples were directly applied to the antigen wells for immunodiffusion without further treatment. Periodate oxidation was performed by treating the antigen with o.oi M sodium meta-periodate in Tris-saline at 4 °C for 24 11. The excess periodate was subsequently eliminated by dialysis against the same buffer.
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Electrophoretic methods Two conditions were used for polyacrylamide gel electrophoresis: the original method of Davis 9 without the addition of detergent or other solubilizing agents (henceforth designated as "plain gels") and the sodium dodecyl sulfate technique (henceforth designated as "dodecyl sulfate gels") of Weber and Osborn 1°. In both cases only gels containing 14% acrylamide monomer were prepared and used in the current study; the crossdinker concentration was increased proportionally, thus maintaining the original monomer cross-linker ratios. The gels were cast in 6-ram (internal diameter) tubes and run at 2.5 mA/tube for the plain gel and IO mA/tnbe for the dodecyl sulfate gel, both toward the anode. For the plain gel 2 mg of membrane antigen was dissolved directly in I ml of the electrode buffer (Tris-glycine, pH 8.3) containing 2o~/o sucrose. For the dodecylsulfate gel the same amount of membrane antigen was used per ml of a sample solvent consisting of: o.oi M sodium phosphate at p H 7.o, o.I}/o dodecyl sulfate, IO(~ 2-mercaptoethanol, 4 M urea and o.oo5°/o Bromphenol blue. In both cases IOO/~1 of the sample solution was layered on top of each gel for electrophoresis, giving an equivalence of 2oo/~g membrane antigen per gel. The gels were fixed in 5o')L ethanol containing i o % acetic acid. After leaving in this solution overnight, the gels were stained for 4-6 h with o.25% Coomassie brilliant blue dissolved in the above solution. Destaining was carried out by soaking tile gel in 5% ethanol with 7.5}'o acetic acid for 24 11. The gels were finally stored in 7~/o acetic acid. Enzyme degradation of the membrane antigen was carried out in Tris-glycine buffer at p H 8.3 (for the plain gel), or in o.oi M sodium phosphate at p H 7.0 (for the dodecyl sulfate gel), the buffers used in the corresponding sample solutions. Membrane antigen and enzyme concentrations were 5 mg/ml and I mg/ml, respectively. After incubating at 37 °C for I h, the mixtures were readjusted to contain all the components at their proper concentrations in the complete sample solutions (see above) ; the final membrane antigen concentration being 2 mg/ml. A sample volume of o.I ml was used per gel. Thus, other than the presence of the enzymes, all conditions for electrophoresis were the same as the untreated samples. Protein bands corresponding to the enzymes were detected by electrophoresing each enzyme in separate gels, and the bands subtracted from the protein patterns of the experimental gels. All enzymes used were identical with those described in the immunological methods. Transplantation of acrylamide gel segments was made from plain gels to dodecyl sulfate gels or to other plain gels. The unfixed, unstained gel from which the protein was to be transplanted was sliced crosswise into 16 pieces. Each disc was further split into two equal halves. One half of each pair was frozen and the other was embedded in an agar plate for immunodiffusion. After identifying the segment which contained the antigenically active protein, the corresponding half in storage was thawed, chopped into pieces of approximately I cubic ram, mixed with the sample solvent and placed on top of a gel for electrophoresis. The recipient gel was then treated as usual. Molecular weight determinations were performed in i 4 % dodecyl sulfate gels, using 5 o # g of commercially purified proteins as standards. All standard proteins were products of Sigma Chemical Co.
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RESULTS
Specificity of the brain membrane antigen The b r a i n m e m b r a n e a n t i g e n showed a single p r e c i p i t i n line a g a i n s t its antiser u m (Fig. I). The same serum d i d n o t cross-react w i t h t h e corresponding m a t e r i a l o b t a i n e d from pig liver, nor was a n y r e a c t i o n d e m o n s t r a t e d t o w a r d pig b r a i n cytosol
Fig. i. Immunodiffuslon showing specificity of brain membrane antigen. As, antiserum against brain membrane antigen; BMA, brain membrane antigen; BSP, brain soluble protein (cytosol from a 20% pig brain homogenate in Tris-saline); LMA, liver membrane antigen; MBP, myelin basic protein• a n d m y e l i n basic protein. The organ specificity was f u r t h e r s u b s t a n t i a t e d b y the r e t e n t i o n of the a n t i b o d y a c t i v i t y in the serum after a d s o r p t i o n with liver m e m b r a n e fraction (Fig. 2). A d s o r p t i o n w i t h k i d n e y a n d muscle m e m b r a n e s gave essentially similar results as w i t h liver, whereas a d s o r p t i o n with t h e cerebral m e m b r a n fraction c o m p l e t e l y abolished the a c t i v i t y . Fig. 3 shows t h e i m m u n o l o g i c comparison b e t w e e n this a n t i g e n a n d S - I o o protein. A n t i s e r u m a g a i n s t S-Ioo, in an a m o u n t which r e a c t e d s t r o n g l y with b o t h S-
C
,
Fig. 2. Effect of adsorption on reactivity of antiserum. BMA, brain membrane antigen; As, anti~erum against brain membrane antigen (unadsorbed); As(LMF)I, antiserum against brain membrane antigen (adsorbed with 6 mg liver membrane fraction per ml serum) ; As(LMF)~, antiserum against brain membrane antigen (adsorbed with 12 mg liver membrane fraction per ml serum).
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R. LIM, M. P. GOEDKEN
I I
ii ! i
I
Fig. 3. Absence of cross-reactivity between brain membrane antigen and S-Ioo protein. BMA, brain membrane antigen; As, antiserum against BMA; S-loo, S-ioo protein of Moore (2.5 /~g added); As(S-Ioo), antiserum against S-ioo protein; BSP, brain soluble protein (cytosol from a 2O~o pig brain homogenate in Tris saline). IOO p r o t e i n a n d pig b r a i n cytosol, d i d not form a p r e c i p i t i n line with the cerebral m e m b r a n e antigen, whereas a n t i s e r u m a g a i n s t cerebral m e m b r a n e a n t i g e n r e a c t e d with the homologous antigen b u t n o t w i t h S - I o o a n d cytosol. The fact t h a t pig b r a i n soluble fraction r e a c t e d with a n t i - S - I o o b u t n o t with a n t i - c e r e b r a l - m e m b r a n e would i m p l y t h a t if the s u p p o s e d m e m b r a n e antigen were present in the cytosol, its c o n c e n t r a t i o n m u s t be lower t h a n t h a t of S - I o o which is a b o u t o.Ic~/~, of t o t a l soluble p r o t e i n 1~. Such low c o n c e n t r a t i o n s in the soluble fraction, then, would be v e r y
Fig. 4. Effect of hydrolytic enzymes on antigenicity of brain membrane antigen. 17pper wells, brain membrane antigen (controls); middle wells, antiserum against brain membrane antigen; lower wells, enzyme-treated brain membrane antigen. DNase, deoxyribonuclease-treated; RNase, ribonuclease-treated; NANase, N-acetyl neuraminidase-treated; Tryp, trypsin-treated. Composite photograph of individual agar plates.
365
BRAIN SPECIFIC PROTEIN
unlikely to contaminate (during purification) the particulate fraction to an extent as to be detectable by immunodiffusion. Further evidence of the membrane origin of the antigen derives from the fact that omission of lithium diiodosalicylate resulted in negligible yield of the antigen.
Chemical nature of brain membrane antigen The brain membrane antigen was incubated with various degradative enzymes. Fig. 4 shows that the antigenicity was not affected by deoxyribonuclease, ribonuclease and neuraminidase, but was completely abolished by trypsin, indicating the protein nature of the antigen. Periodate oxidation did not inactivate the antigen. The protein was insoluble in ethanol, but was not denatured by this agent even at temperatures above o °C. It was not precipitable by vinblastine, an alkaloid which precipitates many acidic proteins including tubulin. When dissolved in o.15 M NaC1 with 0.02 M Tris-HC1, pH 7.5, the antigenic activity could withstand heating in a boiling water bath for io min. The antigenicity was considerably reduced after bringing the pH down to 3-5 followed by readjustment to neutrality. However, a similar treatment with NaOH to bring the pH up to II did not alter its immunological activity. The brain membrane antigen showed an absorbance ratio (A280 nm/A2~ 0 nm) of
~!iC~iill/ ~i!~i~ili ~i~ Fig. 5. I m m u n o e l e c t r o p h o r e s i s of b r a i n m e m b r a n e antigen. E l e c t r o p h o r e s i s w a s carried o u t in a 1 4 % plain p o l y a c r y l a m i d e gel. T h e u n f i x e d gel w a s split lengthwise. O n e h a l f w a s s t a i n e d w i t h C o o m a s s i e blue a n d t h e o t h e r w a s e m b e d d e d in i % I o n a g a r No. 2 in t h e presence of T r i s - s a l i n e . A n t i s e r u m a g a i n s t b r a i n m e m b r a n e a n t i g e n w a s placed in t h e t r o u g h one d a y later. T h e a c r y l a m i d e gel w a s i n t e n t i o n a l l y b r o k e n a t two sites to p r e v e n t it f r o m curling up. A, result a f t e r i m m u n o diffusion. B, s t a i n i n g p a t t e r n of a c r y l a m i d e gel, s h o w i n g m o b i l i t y v a l u e s of b a n d s w i t h r e s p e c t to t h e f r o n t (f). C, s k e t c h s h o w i n g precipitin line w i t h r e s p e c t to t h e location of p r o t e i n b a n d s in a c r y l a m i d e gel; scale on r i g h t i n d i c a t e s t h e division of t h e gel into 16 e q u a l s e g m e n t s (see Fig. 6).
R. LIM, M. I’. GOEDKEN
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0.60, indicating the presence of a considerable amount of nucleic acid in the product. This was not a surprise since the cerebral membrane fraction included the microsomes, and the fractionation procedure employed (phenol partition and alcohol precipitation) was similar to procedures used for the isolation of RNA. The membrane antigen could be further purified with streptomycinr2, which eliminates the nucleic acids and possibly some acidic proteins. The sample solution was mixed with 0.3 vol. of 5ojo streptomycin sulfate at neutral pH and stirred for 15 min in the cold. The white precipitate, which contained the RNA-streptomycin complex, was removed by centrifugation. The excess streptomycin was subsequently eliminated by dialysis. The antigenic activity was fully retained despite an 800/, reduction in dry weight (a 5-fold purification). The streptomycin-treated products showed an absorbance ratio of 0.83 indicating the possible presence of 6% nucleic acid. However, all the immunological and electrophoretic studies in this report were conducted without streptomycin treatment. Localization
of the antigen
in polyacrylamide
gel
Fig. 3 shows that the cerebral membrane antigen yielded two major bands and three minor bands in the plain gel. Only a minor band (mobility 0.22) formed a precipitin line upon immunodiffusion. Unlike ordinary proteins, this band stained pink rather than blue with Coomassie. Antoher minor protein band (mobility 0.77) also stained pink but did not form a precipitin line. All other protein bands ap-
Fig. 6. Localization of antigen in acrylamide gel. Brain membrane antigen was electrophorescd in 14% plain gel. The unfixed gel was sliced crosswise into 16 equal segments as indicated in Fig. 5. The slices were chopped into smaller pieces and placed in the peripheral wells of an Ouchterlony plate. Immunodiffusion was started a day later by filling the center well with antiserum. Note the presence of a precipitin line only in piece No. 4 which corresponds to the location of the precipitin line in Fig. 5. Pieces of acrylamide’gels were removed from the wells before photography.
BRAIN SPECIFIC PROTEIN
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peared blue a n d were i m m u n o l o g i c a l l y inert, as far as p r e c i p i t a t i n g a n t i b o d y is concerned. The location of the i m m u n o l o g i c a l l y active b a n d was further checked b y slicing the gel crosswise. As seen in Fig. 6, only segment No. 4 reacted with the a n t i s e r u m . T h u s we i d e n t i f y the b a n d with a m o b i l i t y of o.22 as the antigenically active b a n d .
Characteristics of the antigenic band in polyacrylamide gel Protein b a n d s u n d e r various gel conditions are shown in Fig. 7. As in the plain gel, a pink b a n d with a m o b i l i t y of 0.22 ( ' A " band) was observed in the dodecyl sulfate gel. This b a n d was c o n s t a n t l y seen a m o n g different batches of p r o d u c t despite v a r i a b i l i t y in other bands. A protein corresponding to the B b a n d (mobility 0.77 ) was
I
II
III
Fig. 7. Gel patterns of membrane antigen under different electrophoretic conditions. 1, brain membrane antigen in plain gels; duplicate gels are show. II, brain membrane antigen in dodecyl sulfate gels; the two samples were taken from two different batches of product. III, brain membrane antigen (left) compared with liver membrane antigen (right) in dodecyl sulfate gels; the two gels were electrophoresed simultaneously; the brain membrane antigen was from a batch different from those shown in II. Origin at top; front at bottom end (indicated by a line). A and B designate positions of bands with mobilities of o.22 and o.77, respectively. The A band from the brain membrane antigen appears distinctly pinkish in all gels. The B band from the brain is pink only in the plain gel but not in the dodecyl sulfate gel. Liver sample does not show any pink band. Coomassie blue stain. present in the dodecyl sulfate gel, b u t unlike its c o u n t e r p a r t in the plain gel this b a n d was variable a n d s t a i n e d blue with Coomassie. I n contrast to the b r a i n m e m b r a n e antigen, the corresponding m a t e r i a l purified from the liver m e m b r a n e s did n o t reveal a n y distinct b a n d s at positions A a n d B, nor was a n y b a n d observed to be p i n k in color. The absorption spectra of the pink a n d blue b a n d s from cerebral m e m b r a n e are shown in Fig. 8.
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R. LIM, M. P. GOEDKEN
0.5 0.4
z 0.3 0.2 0.1 i
I
,
,
i
i
500
I
,
i
,
i
600
I
700
WAVELENGTH (nm)
Fig. 8. Absorbance s p e c t r a of a pink b a n d ( " A " b a n d in Fig. 7) and a blue b a n d f r o m acrylamide gels stained with Coomassie brilliant blue. Note the presence of a left shoulder w i t h the pink b a n d and a right shoulder with the blue b a n d which p r o b a b l y are responsible for their characteristic colors.
Fig. 9 and IO show the results of enzyme treatment on the band patterns. In both gel systems the band mobilities were not altered after treatment with deoxyribonuclease, ribonuclease and neuraminidase. Tryptic digestion, however, abolished most of the bands, including the A band, yielding smaller peptides close to the front. Thus the results confirm those obtained with immunological studies in suggesting the protein nature of the cerebral membrane antigen.
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Fig. 9- Effect of hydrolytic enzymes on protein p a t t e r n of brain m e m b r a n e antigen in plain gel. Cont, control (sample n o t treated with enzyme); DNase, deoxyribonuclease-treated; RNase, ribonuclease-treated; NANase, N-acetyl n e u r a m i n i d a s e - t r e a t e d ; Tryp, trypsin-treated. Origin at top; f r o n t at b o t t o m end. A and B indicate positions of b a n d s with mobilities of o.22 and o.77, respectively. Fig. io. Effect of hydrolytic enzymes on protein p a t t e r n of brain m e m b r a n e antigen in dodecyl sulfate gel. Origin at top; f r o n t at b o t t o m end. A and B indicate positions of b a n d s with rnobilities of 0.22 and o.77, respectively. Control and e n z y m e - t r e a t e d samples are as designated in Fig. 9-
That the A bands in the plain and dodecyl sulfate gels were identical was borne out by transplantation experiments (Fig. 11) in which a gel segment corresponding to the A band was cut out from an unfixed plain gel and re-electrophoresed in a dodecyl sulfate gel. This resulted in the appearance of a pink band in the location where the A band should be. The similarity in mobility of the antigenic band in the presence or absence of dodecyl sulfate suggests the open chain conformation of the protein and that the protein might be highly acidic. Whether the native conformation of the protein had been modified during the initial membrane solubilization and
369
BRAIN SPECIFIC PROTEIN
A~
~A
B~
~-B I
Z
m/2
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Fig. II. R e s u l t s of gel t r a n s p l a n t a t i o n . A a n d B indicate positions o f b a n d s w i t h mobilities of 0.22 a n d 0.77, respectively. I, p r o t e i n p a t t e r n of b r a i n m e m b r a n e a n t i g e n in p l a i n gel; II, result of t r a n s p l a n t i n g " A " b a n d f r o m I to a n o t h e r piece of p l a i n gel. I I I a n d IV are t h e t w o different o u t c o m e s of t r a n s p l a n t i n g A b a n d f r o m 1 to dodecyl s u l f a t e gels.
subsequent purification steps is not known. Nevertheless, we feel safe to say that the antigenic determining site appears to be unaltered since the antiserum prepared against the purified protein reacted with the intact membrane fraction as well. We observed that the transplantation of the A band from a plain gel to a dodecyl sulfate gel occasionally resulted in the appearance of the B band, instead of the A band, in the recipient gel (Fig. II). In this instance the nascent B band stained blue. Thus it appears that some relationship existed between the A and B bands. Considerable effort had been exerted without success to predictably direct the transplanted band to either the A or B position. Such efforts included the addition or omission of 2-mercaptoethanol, dithiothreitol and iodoacetamide at various concentrations and in various sequences, as well as incubating the sample mixture at 37 °C. The possibility that an endogenous protease might have been activated by sodium dodecyl sulfate is unlikely since increasing the sodium dodecyl sulfate concentration in the sample mixture from o. I to i °/o and heating the sample in a boiling water bath did not prevent the formation of B band in the dodecyl sulfate gel. Further evidence 9080-
1
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2
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4
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I
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I
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MOBILITY
Fig. 12. Molecular w e i g h t d e t e r m i n a t i o n w i t h 14 % dodecyl s u l f a t e gel. Mobility v a l u e s are w i t h r e s p e c t to t h e t r a c k i n g d y e ( B r o m p h e n o l blue). T h e s t a n d a r d p r o t e i n s are: I, b o v i n e s e r u m alb u m i n ; 2, c a t a l a s e ; 3, o v a l b u m i n ; 4, c a r b o x y p e p t i d a s e A; 5, t r y p s i n ; 6, m y o g l o b i n ; 7, ribon u c l e a s e ; 8, c y t o c h r o m e c; 9, insulin. T h e use of e - m e r c a p t o e t h a n o l w a s o m i t t e d for insulin. A r r o w s i n d i c a t e t h e locations o f A a n d B b a n d s of b r a i n m e m b r a n e antigen.
37 °
R. LIM, M. P. G O E D K E N
against protein degradation is provided by the reverse conversion of B band to A band, a phenomenon observed when transplanting the B band from a plain gel to another plain gel. It seems as if the protein antigen consisted of dissociable subunits, although the mechanism of association is not known. Molecular weight determination (Fig. 12) shows that the A and B bands have molecular weights of about 42 ooo and 7ooo, respectively, suggesting that the membrane antigen was composed of 6 subunits. Whether these subunits were homomonomers or heteromonomers of similar molecular size remains to be determined. DISCUSSION
One is confronted with a paradox when attempting to isolate individual membrane proteins from the brain. The dissociation of proteins from the particulate material usually necessitates the use of a detergent or other solubilizing agents. Such procedures often lead to an irreversible denaturation of the proteins with complete loss of their biological activity, a result which defeats an important purpose of studying these proteins. Lithium diiodosalicylate, a chaotropic agent, has been reported by Marchesi 5 to be useful for the isolation of an immunologically active glycoprotein from the erythrocyte membrane. Unlike Triton X-Ioo and sodium dodecyl sulfate, lithium diiodosalicylate is easily removable by dialysis. Furthernlore, Triton X - I o o appears to solubilize proteins by micelle formation rather than through dissociation. The effectiveness of sodium dodecyl sulfate as a dissociative agent for brain proteins has also been questioned 13. The use of lithium diiodosalicylate in connection with urea has enabled us to obtain an immunologically active organspecific protein from the cerebral membranes. Although the protein is by no means pure, its final isolation should not be too difficult because the protein is now water soluble and can be handled like other soluble proteins. The presence of immunological activity in this protein opens up several possibilities for future research. First, affinity chromatography m a y be used for the complete purification of the protein, taking advantage of the fact that it is the only antigenically active component in the partially purified fraction. Secondly, the distribution of this protein in various subcellular fractions can be measured with a quantitative complement-fixation test4, a4, or its exact morphological origin localized with immunoelectron microscopy. Finally, the antibody can be employed as a chemical probe for elucidating the physiological role of the protein in cultured brain cells. Several lines of evidence indicate the organ specificity of this protein: (I) the absence of reactivity of the antiserum toward liver membrane antigen similarly prepared; (2) the retention of immunological activity of the serum toward the brain membrane antigen even after adsorption with liver, kidney or muscle total membrane fraction ; (3) the protein in acrylamide gel appeared as a pink band when stained with Coomassie, whereas all the bands obtained from the corresponding liver fraction were blue. Although the original procedure of Marchesi a was designed for the isolation of glycoproteins, there is no evidence that our cerebral membrane antigen is a glycoprotein. Furthermore, none of the protein bands in the gel was periodic acid-Schiffpositive. Inasmuch as a reliable chemical analysis of the protein is not feasible in its current state of purity, the presence of a carbohydrate moiety in a manner not
BRAIN SPECIFIC PROTEIN
371
critical for a n t i g e n i c i t y a n d e l e c t r o p h o r e t i c m o b i l i t y r e m a i n s to be proven. The a n t i g e n p r e p a r a t i o n was c o n t a m i n a t e d w i t h nucleic acid, even after s t r e p t o m y c i n t r e a t m e n t . However, t h e nucleic acid d i d n o t a p p e a r to interfere w i t h the i m m u n o l o g i c a l a n d e l e c t r o p h o r e t i c studies. On the o t h e r hand, the possibility t h a t t h e p r o t e i n a n t i g e n m i g h t c o n t a i n some t i g h t l y b o u n d nucleotides deserves serious consideration. A t a n y rate, the p i n k color e x h i b i t e d b y the p r o t e i n with Coomassie stain suggests molecular peculiarities, a n d favors the likelihood of a c o n j u g a t e d protein r a t h e r t h a n a simple protein. A n u m b e r of brain-specific m e m b r a n e p r o t e i n s have been r e p o r t e d (for review, see Shooter a n d Einstein15). A m o n g the well s t u d i e d are the m y e l i n proteins such as the p r o t e o l i p i d protein, the basic encephalitogenic p r o t e i n a n d the acidic p r o t e o l i p i d p r o t e i n of Wolfgram. O t h e r p r o t e i n s t h a t are p r e d o m i n a n t l y found in the brain, t h o u g h n o t s t r i c t l y organ-specific, are p r o t e i n s u b u n i t s of the cytoskeletons, such as filarin 16 a n d t u b u l i n l L R e c e n t l y an acidic p r o t e i n has been o b t a i n e d from the glial fibrillary c o m p o n e n t TM.A l t h o u g h the last three p r o t e i n s form the s t r u c t u r a l elements for i n t r a c e l l u l a r s u p p o r t , t h e y are n o t s t r i c t l y m e m b r a n e p r o t e i n s since t h e y are r e a d i l y e x t r a c t a b l e b y o r d i n a r y buffers. The p r o t e i n r e p o r t e d in this c o m m u n i c a t i o n a p p e a r s to be different from a n y of the b r a i n proteins p r e v i o u s l y s t u d i e d a n d m a y be w o r t h p u r s u i n g from b o t h chemical a n d biological p o i n t s of view. ACKNOWLEDGEMENTS This w o r k was p a r t i a l l y s u p p o r t e d b y U.S. P u b l i c H e a l t h Service G r a n t No. NS-09228 (to R.L.). W e t h a n k Dr F e r e n c J. K e z d y for o b t a i n i n g the a b s o r p t i o n spectra.
REFERENCES 1 Lim, R. and Tadayyon, E. (197o) Anal. Biochem. 34, 9 15 2 Lim, R. (1971) Trans. Am. Soc. Neurochem. 2, 93 3 Lim, R. (1971) Abstracts, 3rd Meet. Int. Soc. Neurochem., Budapest, p. 216 4 Lim, R. and Hsu, L. W. (1971) Biochim. Biophys. Acta 249, 569-582 5 Marchesi, V. T. and Andrews, E. P. (1971) Science 174, i247-i248 6 Bencina, B. and Carnegie, P. R. (1969) F E B S Lett. 4, 9-12 70shiro, Y. and Eylar, E. H. (197o) Arch. Biochem. Biophys. 138, 392-396 8 Greenfield, S., Norton, W. T. and Morell, P. (1971) J. Neurochem. 18, 2II9--2128 9 Davis, B. J. (1964) Ann. N . Y . Acad. Sci. 121, 4o4-427 io Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 44o6 4412 I i Moore, B. W. (1969) in Handbook o[ Neurochemislry (Lajtha, A., ed.), Vol. i, pp. 93-99, Plenum, New York 12 Lim, R. and Cohen, S. S. (1966) J. Biol. Chem. 241, 43o4-4315 13 Katzman, R. L. (1972) Biochim. Biophys. Acta 266, 269-272 14 Moore, B. W. and Perez, V. J. (1966) J. Immunol. 96, lOOO-lOO5 15 Shooter, E. M. and Einstein, E. R. (1971) A n n u . Rev. Biochem. 4o, 635-652 16 Huneeus, F. C. and Davison, P. F. (197o) J. Mol. Biol. 52, 415-428 17 Davison, P. F. and Huneeus, F. C. (197o) J. Mol. Biol. 52, 429-439 18 Bignami, A., Eng, L. F., Dahl, D. and Uyeda, C. T. (1972) Brain Res. 43, 429-435