ANALYTICAL
BIOCHEMISTRY
89, 360-371 (1978)
Purification of a Brain-Specific Astroglial by lmmunoaffinity Chromatography DAVID Department
C. RUEGER,’
of Neuropathology. West Roxbury Veteruns
DORIS DAHL,
AND AMICO
Harvard Medic4 School Administration Hospital.
Protein
BIGNAMI
and Spinal Card Injury Boston, Massachusetts
Service.
Received February 13, 1978; accepted April 4, 1978 An immunoaftmity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglobulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immunoaffinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 M acetic acid, 5 M urea, 0.8 M sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mM to 1 mM sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.
Following
the isolation
of myelin
basic protein by Roboz-Einstein by Moore (2) and Moore and McGregor (3) in 1965, several antigens have been shown to be exclusively present in neural tissue (see Refs. 4 and 5 for reviews). Glial fibrillary acidic (GFA) protein (6) is a brain-specific protein selectively localized in astrocytes (7,8). Astrocytes are cells of neuroectodermal origin confined to the central nervous system and containing characteristic bundles of cytoplasmic filaments approximately 7 nm in diameter (9). The close similarity between GFA protein and the major protein contained in isolated brain filaments has been demonstrated recently (10). GFA protein is isolated from human brain and spinal cord by traditional methods such as gel filtration, ion exchange chromatography, and hydroxyapatite adsorption (1 1 - 13). The use of postmortem material for protein characterization studies is limited, however, since the protein is extremely susceptible to in situ proteolysis (12-14). Attempts to obtain nondegraded GFA protein with these methods from phosphate buffer et al. in 1958 (1) and of S-100 protein
’ Send reprint requests to Dr. D. C. Rueger, Spinal Cord Injury Research, West Roxbury Veterans Administration Hospital, Boston, Massachusetts 02132. 0003-2697/78/0892-0360$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
360
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
361
extracts of rapidly frozen bovine brain has resulted in preparations consistently contaminated with tubulin (15). For reasons which are unclear, tubulin contamination does not occur when the protein is isolated in the presence of urea (16); the procedure is, however, time-consuming and only 50% of the isolated protein is in the nondegraded form (16). We now report the purification of bovine GFA protein by immunoaffinity chromatography. The procedure is attractive because of the ease of isolation of highly purified human GFA protein which is used to prepare the antisera (13). Furthermore, the technique is rapid and permits isolation of highly purified protein from phosphate buffer extracts of bovine brain without the problem of tubulin contamination. To our knowledge, this is the first time that immunoaffinity chromatography has been used to purify a brain-specific protein. MATERIALS
AND METHODS
Materials. Beef brains still warm after slaughter were collected on ice. The brain stem and cerebellum were divided from the cerebral hemisphere and discarded. The cerebral hemispheres were sliced into coronal sections at the slaughterhouse and rapidly frozen on dry ice. White matter was roughly dissected from grey matter during freezing. All tissue was stored at -70°C until used. Sepharose 2B and 4B were purchased from Pharmacia. Ultra-pure urea and Tris were obtained from Schwartz/Mann. Sarkosyl NL-97 was purchased from Ciba-Geigy and agarose was purchased from VWR Scientific. Specially pure sodium dodecyl sulfate (SDS) and a crosslinked protein mixture used as molecular weight marker (Product No. 44230 2R) came from Gallard-Schlesinger. Polyacrylamide electrophoresis. Protein samples were prepared in 10 mM sodium phosphate, pH 8.0, containing 1% SDS, 8% sucrose, bromphenol blue, and 1.5% dithiothreitol unless otherwise indicated in the text. The samples were heated for 5 min in a boiling water bath before they were applied to the gels. Sodium phosphate-SDS polyacrylamide electrophoresis was performed according to the procedure of Weber and Osborn (17). Gels contained either 7.5% acrylamide and 0.2% bisacrylamide or 5% acrylamide and 0.13% bisacrylamide. Electrophoresis was performed at 2 mA per gel for 60 min followed by 8 mA per tube for 4 to 5 hr. Tris/glycine-SDS/urea polyacrylamide gel electrophoresis was based on the system described by Bryan (18). The gels contained 8 M urea, 0.1% SDS, 0.025 M Tris, 0.19 M glycine, 5% acrylamide, 0.13% bisacrylamide, 0.05% TEMED, and 0.1% persulfate. The running buffer contained 0.025 M Tris, 0.19 M glycine, 0.1% SDS (pH 8.6). Gels were subjected to electrophoresis at 1.5 mA per gel for about 100 min.
362
RUEGER,
DAHL,
AND
BIGNAMI
Electrophoresis of cyanogen bromide peptides was conducted according to the method of Swank and Munkres (19). The procedure was modified by employing 0.17 M sodium phosphate, pH 6.8, as the gel and running buffer. The gels were electrophoresed at 2 mA per gel for 60 min and then at 3 mA per gel for 18 hr. All gels were stained with Coomassie blue (0.25% in 50% methanol and 10% acetic acid) for 60 min. Destaining of nonurea gels was performed in a solution of 7.5% acetic acid and 7.5% methanol. Ureacontaining gels were destained in a solution of 7.5% acetic acid and 20% methanol. Protein determination. Protein was determined calorimetrically by the method of Lowry et al. (20). Bovine serum albumin was used as the standard. Cyanogen bromide cleavage. Freeze-dried protein was reacted with 1.0 M cyanogen bromide in 70% formic acid for 24 hr in the dark, diluted with 10 vol of distilled water, and freeze-dried. Ouchterlony double diffusion. Immunodiffusion plates were prepared with 1% agarose in 0.05 M Tris-HCl buffer, pH 7.6. Wells were punched in the agarose, six outer wells arranged about a central well, and the latter was filled with antiserum. The outer wells contained crude extracts of brain tissue serially diluted with 0.05 M sodium phosphate, pH 8.0, containing 0.5% Sarkosyl. The plates were incubated at 37°C in a moist box for 24 to 48 hr to permit formation of precipitin lines. Preparation of antiserum. Rabbit anti-GFA protein serum was produced in accordance with previously published methods (13). The antiserum was raised against degraded antigen (40,500 MW) isolated by hydroxyapatite chromatography from 0.05 M sodium phosphate (pH 8.0) extracts of autolysed human spinal cord (13). Bovine brain extracts, bovine GFA protein, and human GFA protein reacted with the antiserum, giving immunodiffusion lines of complete identity. Purijkation of ZgG. Immunoglobulin G was purified according to the procedure described by Livingston (21). In brief, the immunoglobulin fraction was precipitated from serum by adding ammonium sulfate at a final concentration of 50% of saturation. After the precipitate was dissolved in 0.015 M potassium phosphate, pH 6.8, the volume being equal to that of the original serum sample, the immunoglobulin fraction was again made 50% saturated in ammonium sulfate. The precipitate was dissolved in and dialyzed against 0.015 M potassium phosphate, pH 6.8, and applied to a column of DEAE-cellulose (DE-52, Whatman) which had been equilibrated with the same buffer. The column was washed with equilibration buffer until the IgG had passed through and was collected. The IgG was pooled to a concentration of 1 mg/ml and dialyzed against 0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3 (coupling buffer).
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
363
Immunoadsorbent preparation. Both Sepharose 2B and 4B were used for the preparation of IgG-Sepharose. After the packed Sepharose was washed with 40 vol of distilled water on a sintered glass funnel, it was suspended in 3 vol of distilled water, and the suspension was adjusted to pH 11 with 1 N sodium hydroxide. The Sepharose was activated by adding solid cyanogen bromide at a concentration of 0.1 g/ml packed Sepharose. During activation the suspension was rapidly stirred with a magnetic bar and the pH was maintained by adding 2.5 N sodium hydroxide. The reaction was allowed to proceed for either 8 or 30 min and was terminated by washing the CNBr-Sepharose on a sintered glass funnel with 40 vol of ice-cold distilled water. The washed CNBrSepharose was mixed with purified IgG (8 mg of protein/ml of packed Sepharose), and the coupling process was allowed to proceed for 20 hr at 4°C with gentle stirring by a magnetic bar. Following coupling, unbound protein was washed away with coupling buffer, and any remaining active groups were reacted for 2 hr with 1 M glycine in 0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3. Four washing cycles were used to remove noncovalently adsorbed protein, each cycle consisting of a wash at pH 3.6 (0.1 M acetate buffer containing 0.5 M sodium chloride) followed by a wash with coupling buffer. IgGSepharose was stored at 4°C in 0.05 M sodium phosphate, pH 8.0, containing 0.02% sodium azide until used. IgG coupling efficiency was greater than 99% when the reaction utilized CNBr-Sepharose prepared by an 8-min activation period and about 95% when the reaction utilized CNBr-Sepharose prepared by a 30-min activation period. Further reduction in the coupling efficiency resulted in significant IgG leakage from the Sepharose. Coupling efficiency was not altered when Sepharose 2B was substituted for Sepharose 4B. Preparation of crude extracts of brain tissue. Crude extracts were prepared by homogenization of frozen bovine white matter in a Sorvall Omnimixer for a total of 4 min in 30-set bursts. Extractions were performed with various buffers (1:4, w/v), each of which was maintained at pH 8.0 during homogenization by adding 1 N sodium hydroxide. The extracts were cleared by centrifugation at 45,000g for 20 min. RESULTS
Solubility of GFA protein in brain extracts. Previous data from this laboratory (14) have shown that GFA protein is only partly soluble in 0.05 M sodium phosphate (pH 8.0) extracts of bovine brain and that the solubility is greatly increased when the extraction buffer contains 4 M urea. As indicated by immunodiffusion titer, the data in Table 1 show the solubility of GFA protein extracted from tissue with these buffers and other buffers of various ionic strength. The solubility of GFA pro-
364
RUEGER,
AND BIGNAMI
DAHL, TABLE
EFFECT
OF MOLARITY IN
pH
OF SODIUM
&O-BUFFERED
Molarity
PHOSPHATE
EXTRACTS
1 ON THE
OF BOVINE
Immunodiffusion
1 rnM
‘/I 6
10 mM
‘is
50 rnM
‘14
50 mM (with 0.8 M NaCl) 50 mM (with 4 M urea)
‘12 ‘II6
SOLUBILITY BRAIN
titer
WHITE
OF GFA
PROTEIN
MATTER
Total protein Wudml) 6.0 5.6 5.3 4.8
IO
tein increased as the ionic strength of the extraction buffer was lowered. The immunodiffusion titer of 1 mM sodium phosphate (pH 8.0) crude extracts was comparable to that of 4 M urea extracts. When tissue extracted with 1 mM sodium phosphate was subsequently homogenized in 4 M urea containing buffer, only a small immunodiffusion titer (one half or less) was present in the second extract. Insoluble GFA protein remaining in tissue homogenized in 0.05 M sodium phosphate, pH 8.0, could be solubilized when the tissue was subsequently homogenized in the same buffer containing 4 M urea. Crude extracts prepared for affinity chromatography usually contained 0.1 mM EDTA in the extraction buffer. This concentration of EDTA did not change the solubility characteristics of GFA protein in crude extracts. Column chromatography. A typical affinity chromatography is shown in Fig. 1. GFA protein was extracted with 1 mM sodium phosphate, pH 8.0, containing 0.1 mM EDTA (1:4, w/v). The crude extract was diluted with 1 vol of 0.05 M sodium phosphate, 4 M urea, 1.6 M NaCl, 0.1 mM EDTA at pH 8.0 and centrifuged at 45,OOOg for 20 min. Fifteen milliliters of the supernatant was applied to a IO-ml column of IgG-Sepharose 2B which had been equilibrated with 0.025 M sodium phosphate, 2 M urea, 0.8 M NaCl, 0.1 mM EDTA at pH 7.7. The column was washed first with equilibration buffer and then with 0.05 M sodium phosphate, 5 M urea, 0.8 M NaCl, 0.1 mM EDTA at pH 6.0 to remove nonspecifically adsorbed protein. This fraction also contained, however, a relatively minor amount of GFA protein, as indicated by Ouchterlony double diffusion and electrophoretic patterns on SDS-polyacrylamide gels. Purified GFA protein was eluted with 1 M acetic acid, 5 M urea, 0.8 M NaCl at pH 2.5. GFA protein was also purified by affinity chromatography from tissue extracted with the following buffers: (i) 0.05 M sodium phosphate, pH 8.0; (ii) 0.05 M sodium phosphate, pH 8.0, containing 4 M urea; (iii) the buffer in (ii) after an initial extraction of the tissue with buffer (i). In
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA PROTEIN
365
FRACTION NUMBER FIG. 1. Purification of bovine GFA protein by immunoaffinity chromatography. The procedure is as described in the text. IgG-Sepharose was prepared from Sepharose 2B which had been activated with CNBr for 30 min. Chromatography was performed at 4°C with a 0.9 x 16-cm column of immunoadsorbent. Two-milliliter fractions were collected at a Row rate of 20 mlihr. The vertical arrows indicate points at which new buffers were introduced for the elution of nonspecifically bound protein (a) and of GFA protein (b).
these cases, the crude extracts were diluted with 1 vol of the appropriate buffer such that the final solution consisted of 0.05 M sodium phosphate, 2 M urea, 0.8 M NaCl at pH 7.7. After centrifugation, samples were applied to columns of IgG-Sepharose which had been equilibrated with this buffer, and the columns were washed with equilibration buffer. The remainder of the chromatographic procedure was as described above. In some instances the procedure included 0.1 mM EDTA. GFA protein eluted from immunoaffinity columns was usually dialyzed against 1 mM sodium phosphate, pH 8.0, containing 0.1 mM EDTA or 0.05 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA. After dialysis against the latter buffer, a substantial amount of purified GFA protein precipitated. Immunoadsorbent binding capacity. The binding capacity of IgGSepharose was tested by column chromatography for several preparations of the immunoadsorbent. The nonabsorbed protein was titered by Ouchlerlony double diffusion to ensure that an excess of crude extract had been applied to each preparation. Maximum binding capacity was found to be 2.0 to 2.3 mg of GFA protein/l0 ml of immunoadsorbent. This was achieved with IgG-Sepharose prepared from Sepharose 2B or 4B which had been activated with CNBr for a total of 30 min. The capacity of immunoadsorbent prepared from Sepharose activated for 8 min with CNBr was considerably reduced (0.5-0.7 mg per 10 ml of IgGSepharose) .
366
RUEGER,
DAHL,
AND
BIGNAMI
The y-globulin fraction of anti-GFA protein serum, prepared by ammonium sulfate precipitation at 40% of saturation, could not be used to prepare immunoadsorbent. The maximum capacity of several different batches of anti-GFA protein globulin fraction-Sepharose 4B was less than 0.1 mg of GFA protein per 10 ml of immunoadsorbent. The effect of flow rate of the column procedure on the recovery of GFA protein was studied. When flow rates of 8, 20, and 40 mYhr were employed, the yields of GFA protein were not significantly changed. After chromatography, immunoadsorbent columns were regenerated by washing with 0.05 M sodium phosphate, pH 8.0. The columns were used repeatedly, although a small decrease in binding capacity occurred with each column run. Yield of GFA protein per gram of tissue. To determine the yield of GFA protein, affinity chromatography was performed with a limiting amount of crude extract. Protein that did not adsorb to IgG-Sepharose under these conditions was tested for the absence of GFA protein by Ouchterlony double diffusion. A 1 mM sodium phosphate (pH 8.0) extract of bovine white matter was found to yield about 1.5 mg of GFA protein/g of tissue. Tissue extracted with 0.05 M sodium phosphate, pH 8.0, yielded about 0.5 mg/g of tissue, the low yield confirming immunodiffusion data (Table 1) that a low amount of GFA protein was solubilized in this buffer. Purity of GFA protein. Figure 2A shows the electrophoretic pattern of purified GFA protein on Tris/glycine-SDS/urea polyacrylamide (5%) gels. GFA protein migrated as a single band and did not contain tubulin as a contaminant. Tubulin and GFA protein have similar mobilities on phosphate-SDS polyacrylamide electrophoresis (15,22). Cyanogen bromide peptide maps were used to identify further the protein isolated by affinity chromatography as GFA protein. Figure 2B shows a digested preparation electrophoresed on the polyacrylamide gel system described by Swank and Munkres (19) for low molecular weight peptides. The digest was similar to that of bovine brain GFA protein isolated from 4 M urea extracts by DEAE Bio-Gel A and hydroxyapatite chromatography (15) and to the digest of human GFA protein isolated from buffer extracts by hydroxyapatite chromatography (12). Figure 3 shows the electrophoretic pattern on phosphate-SDS polyacrylamide (7.5%) gels. The protein migrated as one major band when samples prepared for electrophoresis contained dithiothreitol. Minor components migrating ahead of the major band were seen when gets were heavily loaded. These components apparently represent degraded GFA protein species, as described previously from this laboratory (12,14). The molecular weight of nondegraded GFA protein (52,000 when the determination is performed with 0.1 M phosphate-SDS gels containing 7.5% acrylamide) has been studied using several different polyacrylamide gel systems in a companion paper (22). The molecular weight is in agree-
IMMUNOAFFINITY
12
CHROMATOGRAPHY
OF GFA PROTEIN
367
12
2. (A) Comparison of GFA protein (Gel 1) isolated by immunoafktity chromatography with tubulin prepared from calf brain by two cycles of the polymerization procedure (30) on Trislglycine-SDS/urea gel electrophoresis. Separation of the proteins was unsatisfactory on phosphate-SDS gel electrophoresis. (B) Cyanogen bromide peptides of GFA protein isolated by immunoaffinity chromatography on SDS/urea gel electrophoresis. Gel 1 is an incomplete digest of myoglobin used as the standard (19). From top to bottom: myoglobin (17,ooO). peptides I and II (14,900), peptide I (8270), peptide II (6420), and peptide III (2550). Gel 2 is a digest of GFA protein. The bands above the peptide in the myoglobin range are due to aggregation and not to incomplete cleavage since they were not seen on SDS gel electrophoresis. FIG.
ment with that determined for the nondegraded portion of GFA protein isolated from 4 M urea extracts of bovine brain by DEAE-Bio-Gel A and hydroxyapatite chromatography (23). The gels shown in Figs. 2 and 3 show GFA protein preparations that were purified from tissue extracted without 4 M urea. Comparable results, however, were achieved with material extracted in 4 M urea. Effect of EDTA. An effect of EDTA in the purification procedure was observed when purified GFA protein was prepared for polyacrylamide electrophoresis in the absence of reducing agents. Figure 3A shows a preparation of GFA protein purified without EDTA and electrophoresed in the presence and absence of dithiothreitol. The protein migrated as two major bands without reductant; the lowest molecular weight
368
RUEGER, DAHL,
12
AND BIGNAMI
12
FIG. 3. Effect of dithiothreitol on the electrophoretic mobility in phosphate-SDS polyacrylamide gels of GFA protein isolated from bovine brain in the presence or absence of EDTA. (A) Without EDTA. Gel 1 is a nonreduced sample; Gel 2 is a reduced sample. (B) With EDTA. Gel 1 is a nonreduced sample; Gel 2 is a reduced sample. Electrophoresis in A and B was performed with gels containing 7.5% acrylamide. (C) Molecular weight determination of the upper band in nonreduced samples; the gels contained 5% acrylamide. Gel 1 is crosslinked protein used as standard (31.32): a (159,000); b (106,000); and c (53,000). Gel 2 is nonreduced GFA protein; Gel 3 is a mixture of crosslinked marker protein with nonreduced GFA protein.
migrated with a mobility identical to that of the reduced protein. Figure 3B shows a preparation of GFA protein purified in the presence of 0.1 mM EDTA and electrophoresed in the presence and absence of dithiothreitol. Under these conditions, the protein migrated almost entirely in the reduced form when the reducing agent was not included. Figure 3C shows that the high molecular weight band observed in preparations electrophoresed in the absence of dithiothreitol corresponded to an apparent dimeric form of the protein. With regard to the mobility of proteins in the absence of reducing agent, it should be noted that the crosslinked protein used as the standard in Fig. 3C migrated with identical mobilities in the presence or absence of dithiothreitol. A preparation of GFA protein that electrophoresed primarily in the
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
369
monomeric state could be converted to one that electrophoresed primarily in the dimeric state by dialysis against 0.05 M sodium phosphate, pH 8.0. Prolonged dialysis (4-5 days) produced higher molecular aggregates, some of which did not penetrate 7.5% polyacrylamide gels. In all cases, however, GFA protein migrated in the monomeric state in the presence of reducing agent. In experiments employing GFA protein dialyzed against a buffer in which the protein was only partially soluble, soluble and insoluble fractions (separated by centrifugation at 45,OOOg for 30 min) showed no differences in monomer-dimer distributions. In this regard, the presence of 0.1 mM EDTA in the dialysis buffer did not alter the solubility of GFA protein. DISCUSSION
The yield of GFA protein isolated by immunoaffinity chromatography from 0.05 M sodium phosphate buffer extracts of bovine white matter (0.5 mg/g of wet tissue) may be compared with the content of GFA protein in rat cerebral white matter extracted with the same buffer (0.25 mg/g of wet tissue), as determined by a two-site immunoradiometric assay (24). The lower concentration in rat brain could be due to differences in the techniques used or may reflect the decreased need for supportive fibrous neuroglia in the smaller brain, as suggested by the fivefold increase of GFA protein in human brain compared with rat brain by the same technique (24). This marked difference, however, may also reflect the increased solubility of GFA protein in 0.05 M sodium phosphate buffer following tissue autolysis (14). The enhanced solubility of GFA protein at low ionic strength resulting in a marked increase of the yield (1.5 mg/g of wet tissue extracted with 1 mM sodium phosphate buffer against 0.5 mg in 0.05 M buffer) is of interest in view of a recent report on the disruption of mammalian neurofilaments from peripheral nerve by decreasing the salinity of incubational media (25). Glial filaments and neurofilaments are morphologically and biochemically related (9,26). It is also interesting to note that the solubility of GFA protein is quite low in 0.05 M sodium phosphate extracting buffer containing 0.8 M NaCl. Whereas the mammalian neurofilament is relatively stable in hypertonic solutions (25), invertebrate neurofilaments readily dissolve in molar salt concentrations (27,28). The effect of reducing agents on the electrophoretic mobility of GFA protein is consistent with a recent publication from this laboratory reporting the presence of interchain disulfide bridges in GFA protein isolated from 4 M urea extracts of bovine brain by DEAE-Bio-Gel A and hydroxyapatite chromatography (23). Primarily monomeric GFA protein, however, was prepared by immunoaffinity chromatography when the proce-
370
RUEGER, DAHL, AND BIGNAMI
dure included EDTA in the buffers. In the absence of EDTA, GFA protein was prepared as a mixture of the monomer and an apparent dimeric species. Prolonged dialysis of the protein produced higher molecular weight aggregates, some of which did not penetrate into 7.5% polyacrylamide gels in the absence of reducing agents. It thus appears that the mode of isolation dramatically affects the degree of covalent aggregation, and it is interesting to note that a component claimed to be an oxidation product and migrating as a trimer of the monomeric form has been recently reported in neurofilament protein isolated from the axonal fraction of bovine white matter (29). Immunoaffinity chromatography has been utilized for the purification of a variety of proteins, but we are not aware of reports on the isolation of tissue-specific antigens by this procedure. Considering the relevance of these proteins to the problem of differentiation, we believe that the method may find wider application. With respect to GFA protein, the isolation of the water soluble fraction in the nondegraded form may prove to be essential for the understanding of the structural organization of glial filaments. ACKNOWLEDGMENTS This work was supported by USPHS Grant No. NS 13034 and by the Veterans Administration. We wish to thank William Frech for technical assistance.
REFERENCES 1. Roboz-Einstein,
E., Henderson,
N., and Kies, M. W. (1958) J. Neurochem.
2,
254-260.
Moore, B. W. (1965) Biochem. Biophys. Res. Commun. 19, 739-744. Moore, B. W., and McGregor, D. (1965) J. Biol. Chem. 240, 1647-1653. 4. Rauch, H. C., and Einstein, E. R. (1974) in Reviews of Neuroscience (Ehrenpreis, S., and Kopin, I. J., eds.), Vol. 1, pp. 283-343, Raven, New York. 5. Thompson, E. J. (1976) in Biochemistry and Neurological Disease (Davison, A. N., ed.), pp. 278-316, Blackwell, Oxford. 6. Eng, L. F., Vanderhaeghen, J. J., Bignami, A., and Gerstl, B. (1971) Brain Res. 28, 2. 3.
351-354.
Bignami, A., and Dahl, D. (1974) J. Comp. Neurol. 153, 27-38. Schachner, M., Hedley-Whyte, E. T., Hsu, D. W., Schoonmaker, G., and Bignami, A. (1977) J. Cell Biol. 75, 67-73. 9. Wuerker, R. B. (1970) Tissue and Cell 2, l-9. 10. Yen, S.-H., Dahl, D., Schachner, M., and Schelanski, M. L. (1976) Proc. Nat. Acnd. 7. 8.
Sci.
USA
73, 529-533.
11. Dahl, D., and Bignami, A. (1973) Brain Res. 57, 343-360. 12. Dahl, D., and Bignami, A. (1975) Biochim. Biophys. Acfa 386, 41-51. 13. Dahl, D., and Bignami, A. (1976) Brain Res. 116, 150-157. 14. Dahl, D. (1976) Biochim. Biophys. Acta 420, 142-154. 15. Bignami, A., Dahl, D., and Rueger, D. C. (1977) in Mechanisms, Regulation and Special Functions of Protein Synthesis in the Brain (Roberts, S., Lajtha, A., and Gispen, W. H., eds.), pp. 153-160, Elsevier/North Holland.
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA PROTEIN
371
16. Chan, P. H., Huston, J. S., Moo-Penn, W., Dahl, D., and Bignami, A. (1976) in Proceedings of the Second Annual Maine Biomedical Science Symposium Vol. 2, pp. 496-524, University of Maine Press, Orono, Maine. 17. Weber, K., and Osbom, M. (1969) J. Bid/. Chem. 244, 4406-4412. 18. Bryan, J. (1974) Fed. Proc. 33, 152-157. 19. Swank, R. T., and Munkres, K. D. (1971) Anal. Biochem. 39, 462-477. 20. Lowry, 0. H.. Rosebrough. N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 21. Livingston, D. M. (1974) Methods Enzymol. 34, 723-731. 22. Rueger, D. C., Dahl, D., and Bignami, A. (1978) Brain Research, in press. 23. Huston, J. S., and Bignami, A. (1977) Biochim. Biophys. Acfa 493, 97-103. 24. Eng, L. F., Lee, Y. L., and Miles, L. E. M. (1976) Anal. Biochem. 71, 243-259. 25. Schlaepfer, W. W. (1978) J. Cell Biol. 76, 50-56. 26. Dahl, D., and Bignami, A. (1976) FEBS Let?. 66, 281-284. 27. Huneeus, F. C., and Davison, P. F. (1970) J. Mol. Biol. 52, 415-428. 28. Gilbert, D. S., Newby, B. J., and Anderton, B. H. (1975) Nature (London) 256, 586589. 29. Davison, P. F., and Hong, B.-S. (1977) Brain Res. 134, 287-295. 30. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70, 765-768. 31. Molecular Weight Markers for SDS Polyacrylamide Gel Electrophoresis, GallardSchlesinger Chemical Manufacturing Corp., Carle Place, N. Y. 32. Steele, Jr., J. C. H., and Nielsen, T. B. (1978) Anal. Biochem. 84, 218-224.
BIOCHEMISTRY
89, 360-371 (1978)
Purification of a Brain-Specific Astroglial by lmmunoaffinity Chromatography DAVID Department
C. RUEGER,’
of Neuropathology. West Roxbury Veteruns
DORIS DAHL,
AND AMICO
Harvard Medic4 School Administration Hospital.
Protein
BIGNAMI
and Spinal Card Injury Boston, Massachusetts
Service.
Received February 13, 1978; accepted April 4, 1978 An immunoaftmity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglobulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immunoaffinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 M acetic acid, 5 M urea, 0.8 M sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mM to 1 mM sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.
Following
the isolation
of myelin
basic protein by Roboz-Einstein by Moore (2) and Moore and McGregor (3) in 1965, several antigens have been shown to be exclusively present in neural tissue (see Refs. 4 and 5 for reviews). Glial fibrillary acidic (GFA) protein (6) is a brain-specific protein selectively localized in astrocytes (7,8). Astrocytes are cells of neuroectodermal origin confined to the central nervous system and containing characteristic bundles of cytoplasmic filaments approximately 7 nm in diameter (9). The close similarity between GFA protein and the major protein contained in isolated brain filaments has been demonstrated recently (10). GFA protein is isolated from human brain and spinal cord by traditional methods such as gel filtration, ion exchange chromatography, and hydroxyapatite adsorption (1 1 - 13). The use of postmortem material for protein characterization studies is limited, however, since the protein is extremely susceptible to in situ proteolysis (12-14). Attempts to obtain nondegraded GFA protein with these methods from phosphate buffer et al. in 1958 (1) and of S-100 protein
’ Send reprint requests to Dr. D. C. Rueger, Spinal Cord Injury Research, West Roxbury Veterans Administration Hospital, Boston, Massachusetts 02132. 0003-2697/78/0892-0360$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
360
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
361
extracts of rapidly frozen bovine brain has resulted in preparations consistently contaminated with tubulin (15). For reasons which are unclear, tubulin contamination does not occur when the protein is isolated in the presence of urea (16); the procedure is, however, time-consuming and only 50% of the isolated protein is in the nondegraded form (16). We now report the purification of bovine GFA protein by immunoaffinity chromatography. The procedure is attractive because of the ease of isolation of highly purified human GFA protein which is used to prepare the antisera (13). Furthermore, the technique is rapid and permits isolation of highly purified protein from phosphate buffer extracts of bovine brain without the problem of tubulin contamination. To our knowledge, this is the first time that immunoaffinity chromatography has been used to purify a brain-specific protein. MATERIALS
AND METHODS
Materials. Beef brains still warm after slaughter were collected on ice. The brain stem and cerebellum were divided from the cerebral hemisphere and discarded. The cerebral hemispheres were sliced into coronal sections at the slaughterhouse and rapidly frozen on dry ice. White matter was roughly dissected from grey matter during freezing. All tissue was stored at -70°C until used. Sepharose 2B and 4B were purchased from Pharmacia. Ultra-pure urea and Tris were obtained from Schwartz/Mann. Sarkosyl NL-97 was purchased from Ciba-Geigy and agarose was purchased from VWR Scientific. Specially pure sodium dodecyl sulfate (SDS) and a crosslinked protein mixture used as molecular weight marker (Product No. 44230 2R) came from Gallard-Schlesinger. Polyacrylamide electrophoresis. Protein samples were prepared in 10 mM sodium phosphate, pH 8.0, containing 1% SDS, 8% sucrose, bromphenol blue, and 1.5% dithiothreitol unless otherwise indicated in the text. The samples were heated for 5 min in a boiling water bath before they were applied to the gels. Sodium phosphate-SDS polyacrylamide electrophoresis was performed according to the procedure of Weber and Osborn (17). Gels contained either 7.5% acrylamide and 0.2% bisacrylamide or 5% acrylamide and 0.13% bisacrylamide. Electrophoresis was performed at 2 mA per gel for 60 min followed by 8 mA per tube for 4 to 5 hr. Tris/glycine-SDS/urea polyacrylamide gel electrophoresis was based on the system described by Bryan (18). The gels contained 8 M urea, 0.1% SDS, 0.025 M Tris, 0.19 M glycine, 5% acrylamide, 0.13% bisacrylamide, 0.05% TEMED, and 0.1% persulfate. The running buffer contained 0.025 M Tris, 0.19 M glycine, 0.1% SDS (pH 8.6). Gels were subjected to electrophoresis at 1.5 mA per gel for about 100 min.
362
RUEGER,
DAHL,
AND
BIGNAMI
Electrophoresis of cyanogen bromide peptides was conducted according to the method of Swank and Munkres (19). The procedure was modified by employing 0.17 M sodium phosphate, pH 6.8, as the gel and running buffer. The gels were electrophoresed at 2 mA per gel for 60 min and then at 3 mA per gel for 18 hr. All gels were stained with Coomassie blue (0.25% in 50% methanol and 10% acetic acid) for 60 min. Destaining of nonurea gels was performed in a solution of 7.5% acetic acid and 7.5% methanol. Ureacontaining gels were destained in a solution of 7.5% acetic acid and 20% methanol. Protein determination. Protein was determined calorimetrically by the method of Lowry et al. (20). Bovine serum albumin was used as the standard. Cyanogen bromide cleavage. Freeze-dried protein was reacted with 1.0 M cyanogen bromide in 70% formic acid for 24 hr in the dark, diluted with 10 vol of distilled water, and freeze-dried. Ouchterlony double diffusion. Immunodiffusion plates were prepared with 1% agarose in 0.05 M Tris-HCl buffer, pH 7.6. Wells were punched in the agarose, six outer wells arranged about a central well, and the latter was filled with antiserum. The outer wells contained crude extracts of brain tissue serially diluted with 0.05 M sodium phosphate, pH 8.0, containing 0.5% Sarkosyl. The plates were incubated at 37°C in a moist box for 24 to 48 hr to permit formation of precipitin lines. Preparation of antiserum. Rabbit anti-GFA protein serum was produced in accordance with previously published methods (13). The antiserum was raised against degraded antigen (40,500 MW) isolated by hydroxyapatite chromatography from 0.05 M sodium phosphate (pH 8.0) extracts of autolysed human spinal cord (13). Bovine brain extracts, bovine GFA protein, and human GFA protein reacted with the antiserum, giving immunodiffusion lines of complete identity. Purijkation of ZgG. Immunoglobulin G was purified according to the procedure described by Livingston (21). In brief, the immunoglobulin fraction was precipitated from serum by adding ammonium sulfate at a final concentration of 50% of saturation. After the precipitate was dissolved in 0.015 M potassium phosphate, pH 6.8, the volume being equal to that of the original serum sample, the immunoglobulin fraction was again made 50% saturated in ammonium sulfate. The precipitate was dissolved in and dialyzed against 0.015 M potassium phosphate, pH 6.8, and applied to a column of DEAE-cellulose (DE-52, Whatman) which had been equilibrated with the same buffer. The column was washed with equilibration buffer until the IgG had passed through and was collected. The IgG was pooled to a concentration of 1 mg/ml and dialyzed against 0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3 (coupling buffer).
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
363
Immunoadsorbent preparation. Both Sepharose 2B and 4B were used for the preparation of IgG-Sepharose. After the packed Sepharose was washed with 40 vol of distilled water on a sintered glass funnel, it was suspended in 3 vol of distilled water, and the suspension was adjusted to pH 11 with 1 N sodium hydroxide. The Sepharose was activated by adding solid cyanogen bromide at a concentration of 0.1 g/ml packed Sepharose. During activation the suspension was rapidly stirred with a magnetic bar and the pH was maintained by adding 2.5 N sodium hydroxide. The reaction was allowed to proceed for either 8 or 30 min and was terminated by washing the CNBr-Sepharose on a sintered glass funnel with 40 vol of ice-cold distilled water. The washed CNBrSepharose was mixed with purified IgG (8 mg of protein/ml of packed Sepharose), and the coupling process was allowed to proceed for 20 hr at 4°C with gentle stirring by a magnetic bar. Following coupling, unbound protein was washed away with coupling buffer, and any remaining active groups were reacted for 2 hr with 1 M glycine in 0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.3. Four washing cycles were used to remove noncovalently adsorbed protein, each cycle consisting of a wash at pH 3.6 (0.1 M acetate buffer containing 0.5 M sodium chloride) followed by a wash with coupling buffer. IgGSepharose was stored at 4°C in 0.05 M sodium phosphate, pH 8.0, containing 0.02% sodium azide until used. IgG coupling efficiency was greater than 99% when the reaction utilized CNBr-Sepharose prepared by an 8-min activation period and about 95% when the reaction utilized CNBr-Sepharose prepared by a 30-min activation period. Further reduction in the coupling efficiency resulted in significant IgG leakage from the Sepharose. Coupling efficiency was not altered when Sepharose 2B was substituted for Sepharose 4B. Preparation of crude extracts of brain tissue. Crude extracts were prepared by homogenization of frozen bovine white matter in a Sorvall Omnimixer for a total of 4 min in 30-set bursts. Extractions were performed with various buffers (1:4, w/v), each of which was maintained at pH 8.0 during homogenization by adding 1 N sodium hydroxide. The extracts were cleared by centrifugation at 45,000g for 20 min. RESULTS
Solubility of GFA protein in brain extracts. Previous data from this laboratory (14) have shown that GFA protein is only partly soluble in 0.05 M sodium phosphate (pH 8.0) extracts of bovine brain and that the solubility is greatly increased when the extraction buffer contains 4 M urea. As indicated by immunodiffusion titer, the data in Table 1 show the solubility of GFA protein extracted from tissue with these buffers and other buffers of various ionic strength. The solubility of GFA pro-
364
RUEGER,
AND BIGNAMI
DAHL, TABLE
EFFECT
OF MOLARITY IN
pH
OF SODIUM
&O-BUFFERED
Molarity
PHOSPHATE
EXTRACTS
1 ON THE
OF BOVINE
Immunodiffusion
1 rnM
‘/I 6
10 mM
‘is
50 rnM
‘14
50 mM (with 0.8 M NaCl) 50 mM (with 4 M urea)
‘12 ‘II6
SOLUBILITY BRAIN
titer
WHITE
OF GFA
PROTEIN
MATTER
Total protein Wudml) 6.0 5.6 5.3 4.8
IO
tein increased as the ionic strength of the extraction buffer was lowered. The immunodiffusion titer of 1 mM sodium phosphate (pH 8.0) crude extracts was comparable to that of 4 M urea extracts. When tissue extracted with 1 mM sodium phosphate was subsequently homogenized in 4 M urea containing buffer, only a small immunodiffusion titer (one half or less) was present in the second extract. Insoluble GFA protein remaining in tissue homogenized in 0.05 M sodium phosphate, pH 8.0, could be solubilized when the tissue was subsequently homogenized in the same buffer containing 4 M urea. Crude extracts prepared for affinity chromatography usually contained 0.1 mM EDTA in the extraction buffer. This concentration of EDTA did not change the solubility characteristics of GFA protein in crude extracts. Column chromatography. A typical affinity chromatography is shown in Fig. 1. GFA protein was extracted with 1 mM sodium phosphate, pH 8.0, containing 0.1 mM EDTA (1:4, w/v). The crude extract was diluted with 1 vol of 0.05 M sodium phosphate, 4 M urea, 1.6 M NaCl, 0.1 mM EDTA at pH 8.0 and centrifuged at 45,OOOg for 20 min. Fifteen milliliters of the supernatant was applied to a IO-ml column of IgG-Sepharose 2B which had been equilibrated with 0.025 M sodium phosphate, 2 M urea, 0.8 M NaCl, 0.1 mM EDTA at pH 7.7. The column was washed first with equilibration buffer and then with 0.05 M sodium phosphate, 5 M urea, 0.8 M NaCl, 0.1 mM EDTA at pH 6.0 to remove nonspecifically adsorbed protein. This fraction also contained, however, a relatively minor amount of GFA protein, as indicated by Ouchterlony double diffusion and electrophoretic patterns on SDS-polyacrylamide gels. Purified GFA protein was eluted with 1 M acetic acid, 5 M urea, 0.8 M NaCl at pH 2.5. GFA protein was also purified by affinity chromatography from tissue extracted with the following buffers: (i) 0.05 M sodium phosphate, pH 8.0; (ii) 0.05 M sodium phosphate, pH 8.0, containing 4 M urea; (iii) the buffer in (ii) after an initial extraction of the tissue with buffer (i). In
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA PROTEIN
365
FRACTION NUMBER FIG. 1. Purification of bovine GFA protein by immunoaffinity chromatography. The procedure is as described in the text. IgG-Sepharose was prepared from Sepharose 2B which had been activated with CNBr for 30 min. Chromatography was performed at 4°C with a 0.9 x 16-cm column of immunoadsorbent. Two-milliliter fractions were collected at a Row rate of 20 mlihr. The vertical arrows indicate points at which new buffers were introduced for the elution of nonspecifically bound protein (a) and of GFA protein (b).
these cases, the crude extracts were diluted with 1 vol of the appropriate buffer such that the final solution consisted of 0.05 M sodium phosphate, 2 M urea, 0.8 M NaCl at pH 7.7. After centrifugation, samples were applied to columns of IgG-Sepharose which had been equilibrated with this buffer, and the columns were washed with equilibration buffer. The remainder of the chromatographic procedure was as described above. In some instances the procedure included 0.1 mM EDTA. GFA protein eluted from immunoaffinity columns was usually dialyzed against 1 mM sodium phosphate, pH 8.0, containing 0.1 mM EDTA or 0.05 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA. After dialysis against the latter buffer, a substantial amount of purified GFA protein precipitated. Immunoadsorbent binding capacity. The binding capacity of IgGSepharose was tested by column chromatography for several preparations of the immunoadsorbent. The nonabsorbed protein was titered by Ouchlerlony double diffusion to ensure that an excess of crude extract had been applied to each preparation. Maximum binding capacity was found to be 2.0 to 2.3 mg of GFA protein/l0 ml of immunoadsorbent. This was achieved with IgG-Sepharose prepared from Sepharose 2B or 4B which had been activated with CNBr for a total of 30 min. The capacity of immunoadsorbent prepared from Sepharose activated for 8 min with CNBr was considerably reduced (0.5-0.7 mg per 10 ml of IgGSepharose) .
366
RUEGER,
DAHL,
AND
BIGNAMI
The y-globulin fraction of anti-GFA protein serum, prepared by ammonium sulfate precipitation at 40% of saturation, could not be used to prepare immunoadsorbent. The maximum capacity of several different batches of anti-GFA protein globulin fraction-Sepharose 4B was less than 0.1 mg of GFA protein per 10 ml of immunoadsorbent. The effect of flow rate of the column procedure on the recovery of GFA protein was studied. When flow rates of 8, 20, and 40 mYhr were employed, the yields of GFA protein were not significantly changed. After chromatography, immunoadsorbent columns were regenerated by washing with 0.05 M sodium phosphate, pH 8.0. The columns were used repeatedly, although a small decrease in binding capacity occurred with each column run. Yield of GFA protein per gram of tissue. To determine the yield of GFA protein, affinity chromatography was performed with a limiting amount of crude extract. Protein that did not adsorb to IgG-Sepharose under these conditions was tested for the absence of GFA protein by Ouchterlony double diffusion. A 1 mM sodium phosphate (pH 8.0) extract of bovine white matter was found to yield about 1.5 mg of GFA protein/g of tissue. Tissue extracted with 0.05 M sodium phosphate, pH 8.0, yielded about 0.5 mg/g of tissue, the low yield confirming immunodiffusion data (Table 1) that a low amount of GFA protein was solubilized in this buffer. Purity of GFA protein. Figure 2A shows the electrophoretic pattern of purified GFA protein on Tris/glycine-SDS/urea polyacrylamide (5%) gels. GFA protein migrated as a single band and did not contain tubulin as a contaminant. Tubulin and GFA protein have similar mobilities on phosphate-SDS polyacrylamide electrophoresis (15,22). Cyanogen bromide peptide maps were used to identify further the protein isolated by affinity chromatography as GFA protein. Figure 2B shows a digested preparation electrophoresed on the polyacrylamide gel system described by Swank and Munkres (19) for low molecular weight peptides. The digest was similar to that of bovine brain GFA protein isolated from 4 M urea extracts by DEAE Bio-Gel A and hydroxyapatite chromatography (15) and to the digest of human GFA protein isolated from buffer extracts by hydroxyapatite chromatography (12). Figure 3 shows the electrophoretic pattern on phosphate-SDS polyacrylamide (7.5%) gels. The protein migrated as one major band when samples prepared for electrophoresis contained dithiothreitol. Minor components migrating ahead of the major band were seen when gets were heavily loaded. These components apparently represent degraded GFA protein species, as described previously from this laboratory (12,14). The molecular weight of nondegraded GFA protein (52,000 when the determination is performed with 0.1 M phosphate-SDS gels containing 7.5% acrylamide) has been studied using several different polyacrylamide gel systems in a companion paper (22). The molecular weight is in agree-
IMMUNOAFFINITY
12
CHROMATOGRAPHY
OF GFA PROTEIN
367
12
2. (A) Comparison of GFA protein (Gel 1) isolated by immunoafktity chromatography with tubulin prepared from calf brain by two cycles of the polymerization procedure (30) on Trislglycine-SDS/urea gel electrophoresis. Separation of the proteins was unsatisfactory on phosphate-SDS gel electrophoresis. (B) Cyanogen bromide peptides of GFA protein isolated by immunoaffinity chromatography on SDS/urea gel electrophoresis. Gel 1 is an incomplete digest of myoglobin used as the standard (19). From top to bottom: myoglobin (17,ooO). peptides I and II (14,900), peptide I (8270), peptide II (6420), and peptide III (2550). Gel 2 is a digest of GFA protein. The bands above the peptide in the myoglobin range are due to aggregation and not to incomplete cleavage since they were not seen on SDS gel electrophoresis. FIG.
ment with that determined for the nondegraded portion of GFA protein isolated from 4 M urea extracts of bovine brain by DEAE-Bio-Gel A and hydroxyapatite chromatography (23). The gels shown in Figs. 2 and 3 show GFA protein preparations that were purified from tissue extracted without 4 M urea. Comparable results, however, were achieved with material extracted in 4 M urea. Effect of EDTA. An effect of EDTA in the purification procedure was observed when purified GFA protein was prepared for polyacrylamide electrophoresis in the absence of reducing agents. Figure 3A shows a preparation of GFA protein purified without EDTA and electrophoresed in the presence and absence of dithiothreitol. The protein migrated as two major bands without reductant; the lowest molecular weight
368
RUEGER, DAHL,
12
AND BIGNAMI
12
FIG. 3. Effect of dithiothreitol on the electrophoretic mobility in phosphate-SDS polyacrylamide gels of GFA protein isolated from bovine brain in the presence or absence of EDTA. (A) Without EDTA. Gel 1 is a nonreduced sample; Gel 2 is a reduced sample. (B) With EDTA. Gel 1 is a nonreduced sample; Gel 2 is a reduced sample. Electrophoresis in A and B was performed with gels containing 7.5% acrylamide. (C) Molecular weight determination of the upper band in nonreduced samples; the gels contained 5% acrylamide. Gel 1 is crosslinked protein used as standard (31.32): a (159,000); b (106,000); and c (53,000). Gel 2 is nonreduced GFA protein; Gel 3 is a mixture of crosslinked marker protein with nonreduced GFA protein.
migrated with a mobility identical to that of the reduced protein. Figure 3B shows a preparation of GFA protein purified in the presence of 0.1 mM EDTA and electrophoresed in the presence and absence of dithiothreitol. Under these conditions, the protein migrated almost entirely in the reduced form when the reducing agent was not included. Figure 3C shows that the high molecular weight band observed in preparations electrophoresed in the absence of dithiothreitol corresponded to an apparent dimeric form of the protein. With regard to the mobility of proteins in the absence of reducing agent, it should be noted that the crosslinked protein used as the standard in Fig. 3C migrated with identical mobilities in the presence or absence of dithiothreitol. A preparation of GFA protein that electrophoresed primarily in the
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA
PROTEIN
369
monomeric state could be converted to one that electrophoresed primarily in the dimeric state by dialysis against 0.05 M sodium phosphate, pH 8.0. Prolonged dialysis (4-5 days) produced higher molecular aggregates, some of which did not penetrate 7.5% polyacrylamide gels. In all cases, however, GFA protein migrated in the monomeric state in the presence of reducing agent. In experiments employing GFA protein dialyzed against a buffer in which the protein was only partially soluble, soluble and insoluble fractions (separated by centrifugation at 45,OOOg for 30 min) showed no differences in monomer-dimer distributions. In this regard, the presence of 0.1 mM EDTA in the dialysis buffer did not alter the solubility of GFA protein. DISCUSSION
The yield of GFA protein isolated by immunoaffinity chromatography from 0.05 M sodium phosphate buffer extracts of bovine white matter (0.5 mg/g of wet tissue) may be compared with the content of GFA protein in rat cerebral white matter extracted with the same buffer (0.25 mg/g of wet tissue), as determined by a two-site immunoradiometric assay (24). The lower concentration in rat brain could be due to differences in the techniques used or may reflect the decreased need for supportive fibrous neuroglia in the smaller brain, as suggested by the fivefold increase of GFA protein in human brain compared with rat brain by the same technique (24). This marked difference, however, may also reflect the increased solubility of GFA protein in 0.05 M sodium phosphate buffer following tissue autolysis (14). The enhanced solubility of GFA protein at low ionic strength resulting in a marked increase of the yield (1.5 mg/g of wet tissue extracted with 1 mM sodium phosphate buffer against 0.5 mg in 0.05 M buffer) is of interest in view of a recent report on the disruption of mammalian neurofilaments from peripheral nerve by decreasing the salinity of incubational media (25). Glial filaments and neurofilaments are morphologically and biochemically related (9,26). It is also interesting to note that the solubility of GFA protein is quite low in 0.05 M sodium phosphate extracting buffer containing 0.8 M NaCl. Whereas the mammalian neurofilament is relatively stable in hypertonic solutions (25), invertebrate neurofilaments readily dissolve in molar salt concentrations (27,28). The effect of reducing agents on the electrophoretic mobility of GFA protein is consistent with a recent publication from this laboratory reporting the presence of interchain disulfide bridges in GFA protein isolated from 4 M urea extracts of bovine brain by DEAE-Bio-Gel A and hydroxyapatite chromatography (23). Primarily monomeric GFA protein, however, was prepared by immunoaffinity chromatography when the proce-
370
RUEGER, DAHL, AND BIGNAMI
dure included EDTA in the buffers. In the absence of EDTA, GFA protein was prepared as a mixture of the monomer and an apparent dimeric species. Prolonged dialysis of the protein produced higher molecular weight aggregates, some of which did not penetrate into 7.5% polyacrylamide gels in the absence of reducing agents. It thus appears that the mode of isolation dramatically affects the degree of covalent aggregation, and it is interesting to note that a component claimed to be an oxidation product and migrating as a trimer of the monomeric form has been recently reported in neurofilament protein isolated from the axonal fraction of bovine white matter (29). Immunoaffinity chromatography has been utilized for the purification of a variety of proteins, but we are not aware of reports on the isolation of tissue-specific antigens by this procedure. Considering the relevance of these proteins to the problem of differentiation, we believe that the method may find wider application. With respect to GFA protein, the isolation of the water soluble fraction in the nondegraded form may prove to be essential for the understanding of the structural organization of glial filaments. ACKNOWLEDGMENTS This work was supported by USPHS Grant No. NS 13034 and by the Veterans Administration. We wish to thank William Frech for technical assistance.
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Moore, B. W. (1965) Biochem. Biophys. Res. Commun. 19, 739-744. Moore, B. W., and McGregor, D. (1965) J. Biol. Chem. 240, 1647-1653. 4. Rauch, H. C., and Einstein, E. R. (1974) in Reviews of Neuroscience (Ehrenpreis, S., and Kopin, I. J., eds.), Vol. 1, pp. 283-343, Raven, New York. 5. Thompson, E. J. (1976) in Biochemistry and Neurological Disease (Davison, A. N., ed.), pp. 278-316, Blackwell, Oxford. 6. Eng, L. F., Vanderhaeghen, J. J., Bignami, A., and Gerstl, B. (1971) Brain Res. 28, 2. 3.
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11. Dahl, D., and Bignami, A. (1973) Brain Res. 57, 343-360. 12. Dahl, D., and Bignami, A. (1975) Biochim. Biophys. Acfa 386, 41-51. 13. Dahl, D., and Bignami, A. (1976) Brain Res. 116, 150-157. 14. Dahl, D. (1976) Biochim. Biophys. Acta 420, 142-154. 15. Bignami, A., Dahl, D., and Rueger, D. C. (1977) in Mechanisms, Regulation and Special Functions of Protein Synthesis in the Brain (Roberts, S., Lajtha, A., and Gispen, W. H., eds.), pp. 153-160, Elsevier/North Holland.
IMMUNOAFFINITY
CHROMATOGRAPHY
OF GFA PROTEIN
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16. Chan, P. H., Huston, J. S., Moo-Penn, W., Dahl, D., and Bignami, A. (1976) in Proceedings of the Second Annual Maine Biomedical Science Symposium Vol. 2, pp. 496-524, University of Maine Press, Orono, Maine. 17. Weber, K., and Osbom, M. (1969) J. Bid/. Chem. 244, 4406-4412. 18. Bryan, J. (1974) Fed. Proc. 33, 152-157. 19. Swank, R. T., and Munkres, K. D. (1971) Anal. Biochem. 39, 462-477. 20. Lowry, 0. H.. Rosebrough. N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 21. Livingston, D. M. (1974) Methods Enzymol. 34, 723-731. 22. Rueger, D. C., Dahl, D., and Bignami, A. (1978) Brain Research, in press. 23. Huston, J. S., and Bignami, A. (1977) Biochim. Biophys. Acfa 493, 97-103. 24. Eng, L. F., Lee, Y. L., and Miles, L. E. M. (1976) Anal. Biochem. 71, 243-259. 25. Schlaepfer, W. W. (1978) J. Cell Biol. 76, 50-56. 26. Dahl, D., and Bignami, A. (1976) FEBS Let?. 66, 281-284. 27. Huneeus, F. C., and Davison, P. F. (1970) J. Mol. Biol. 52, 415-428. 28. Gilbert, D. S., Newby, B. J., and Anderton, B. H. (1975) Nature (London) 256, 586589. 29. Davison, P. F., and Hong, B.-S. (1977) Brain Res. 134, 287-295. 30. Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Nat. Acad. Sci. USA 70, 765-768. 31. Molecular Weight Markers for SDS Polyacrylamide Gel Electrophoresis, GallardSchlesinger Chemical Manufacturing Corp., Carle Place, N. Y. 32. Steele, Jr., J. C. H., and Nielsen, T. B. (1978) Anal. Biochem. 84, 218-224.
