305
Biochimica et Biophysica Acta, 578 (1979) 3 0 5 - - 3 1 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 3 8 1 9 2
ASTROGLIAL AND AXONAL PROTEINS IN ISOLATED BRAIN FILAMENTS I. ISOLATION OF THE GLIAL FIBRILLARY ACIDIC PROTEIN AND OF AN IMMUNOLOGICALLY ACTIVE CYANOGEN BROMIDE PEPTIDE FROM BRAIN FILAMENT PREPARATIONS OF BOVINE WHITE MATTER
D. D A H L * a n d A. B I G N A M I
Department o f Neuropathology, Harvard Medical School and Spinal Cord Injury Service, West Rox bury Veterans Administration Hospital, Boston, MA 02132 (U.S.A.) (Received S e p t e m b e r 2 7 t h , 1 9 7 8 )
Key words: Astroglial protein; Axonal protein; Glial fibrillary acidic protein; (Brain filament)
Summary The glial fibrillary acidic protein and an immunologically active cyanogen bromide peptide were purified by immunoaffinity chromatography from 8 M urea extracts of brain filament preparations isolated from bovine white matter according to Norton's procedure. The protein accounted for approximately 30% of the total protein in this preparation and for the largest fraction in the 50 000 molecular weight range. The fraction not absorbed to the immunoSepharose column reacted with neurofilament antisera by double immunodiffusion. On sodium dodecyl sulfate gel electrophoresis the main bands in the non-adsorbed fraction were at 74 000 daltons and above 100 000. Several bands were seen in the 50 000 molecular weight range. It is concluded that glioand neurofilaments co-purify together in Norton's procedure and that neurofilaments are probably heterogeneous in polypeptide composition.
Introduction A major controversy presently exists in the literature as to the polypeptide composition of the mammalian neurofilament. Brain filaments isolated accord-
* Address reprint requests to: Dr. D. Dahl, Spinal Cord Injury Research, Veterans Administxation Hospital, 1400 Veterans of Foreign Wars, Parkway, West Roxbury, Ma 02132.
306 ing to the basic procedure developed in Norton's laboratory [11,26] separate into multiple bands on sodium dodecyl sulfate-acrylamide gel electrophoresis, the major band in the 50 000 mol. wt. range being commonly interpreted as neurofilament protein [8,9,15,25]. On the other hand, studies of neurofilaments isolated from peripheral nerve indicate that they are predominantly formed of a 68 000 dalton polypeptide [22], while studies of the slow component of axonal transport show that three polypeptides with molecular weights of 212 000, 160 000, and 68 000 appear to be associated with a single structure which was tentatively identified as the neurofilament [14]. The purpose of this paper is to report the isolation from brain filaments of the glial fibrillary acidic protein and of an immunologically active cyanogen bromide pep~ide [5] using a procedure recently developed in this laboratory for the purification of the protein from buffer extracts of bovine brain, i.e. immunoaffinity chromatography [ 19]. It will be shown that a major fraction of the approx. 50 000 mol. wt. protein in brain filament preparations is glial fibrillary acidic protein, a brain-specific protein selectively localized in astroglia by immunohistochemical methods [3,12,21]. Methods Brain filaments were isolated from bovine white matter with two modifications of the basic Norton's procedure [25,30]. Immunoaffinity chromatography was performed as described elsewhere [19]. Four different batches of Sepharose 4B immunoadsorbent were used. The columns were underloaded with respect to their capacity which had been determined in preliminary experiments. Limiting amounts of brain filament extracts were applied to the column and non-adsorbed protein was tested f o r the absence of glial fibrillary acidic protein by double immunodiffusion. The brain filament pellet was homogenized at 4°C with 8 M urea in 0.05 M sodium phosphate buffer, pH 8.0. The supernatant after centrifugation at 100 000 X g for 1 h was dialyzed overnight against the column equilibration buffer (2 M urea, 0.8 M sodium chloride, 0.01 M sodium phosphate buffer, pH 7.7). Following high speed centrifugation the 2 M ureasoluble column samples were applied to the Sepharose 4B-immunoabsorbent washed with equilibration buffer and then with buffer containing 5 M urea, 0.8 M NaC1, 0.05 M sodium phosphate, pH 6.0. Protein was eluted with 1 M acetic acid, 5 M urea, 0.8 M NaC1, pH 2.5. The flow rate was 5 ml/h for the first 20 fractions and increased to 20 ml/h afterwards. The cyanogen bromide digests of brain filament preparations were obtained by reacting 8 M urea extracts dialyzed against water and freeze-dried with 1 M cyanogen bromide in 70% formic acid for 24 h at 22°C in the dark. The samples diluted with 10 volume of distilled water were freeze-dried and redissolved in 8 M urea. After dialyzing against the equilibration buffer and centrifugation at 100 000 X g for 1 h the 2 M urea-soluble column samples were applied to the Sepharose-immunoadsorbent. The antiserum to the glial fibrillary acidic protein was raised against degraded antigen (40 500 mol. wt.) isolated by hydroxyapatite chromatography from phosphate buffer extracts of autolysed human spinal cord [6]. The
307 antisera used in this work were obtained from one rabbit kept for 3 years in the laboratory, bled every 2 weeks and boosted w i t h 1 mg of antigen every 3 months. As indicated by immunodiffusion and immunofluorescence the strength of the antisera remained essentially unchanged during the whole period. Neurofilament antiserum was raised in one rabbit injected with urea-soluble chicken brain antigen as previously described [7]. The anti-neurofilament activity first appeared 3 weeks after injection and plateaued at 5 weeks. Maintenace of the activity r e q u i r e d w e e k l y boosters. As previously reported [7], only a weak immunodiffusion line against the glial fibrillary acidic protein could be demonstrated with these antisera Degraded glial fibrillary acidic protein (40 500 mol wt.) was isolated from autolysed h u m a n and bovine spinal cords by hydroxyapatite chromatography [6]. Non-degraded bovine glial fibrillary acidic protein used as a molecular weight standard was isolated by immunoaffinity chromatography [19]. T u b u lin was prepared from calf brain by two cycles of the polymerization procedure [27]. In order to avoid nonspecific precipitation, absorption experiments were conducted with diluted antisera. In fact, preliminary experiments had shown that when non
Biochimica et Biophysica Acta, 578 (1979) 3 0 5 - - 3 1 6 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 3 8 1 9 2
ASTROGLIAL AND AXONAL PROTEINS IN ISOLATED BRAIN FILAMENTS I. ISOLATION OF THE GLIAL FIBRILLARY ACIDIC PROTEIN AND OF AN IMMUNOLOGICALLY ACTIVE CYANOGEN BROMIDE PEPTIDE FROM BRAIN FILAMENT PREPARATIONS OF BOVINE WHITE MATTER
D. D A H L * a n d A. B I G N A M I
Department o f Neuropathology, Harvard Medical School and Spinal Cord Injury Service, West Rox bury Veterans Administration Hospital, Boston, MA 02132 (U.S.A.) (Received S e p t e m b e r 2 7 t h , 1 9 7 8 )
Key words: Astroglial protein; Axonal protein; Glial fibrillary acidic protein; (Brain filament)
Summary The glial fibrillary acidic protein and an immunologically active cyanogen bromide peptide were purified by immunoaffinity chromatography from 8 M urea extracts of brain filament preparations isolated from bovine white matter according to Norton's procedure. The protein accounted for approximately 30% of the total protein in this preparation and for the largest fraction in the 50 000 molecular weight range. The fraction not absorbed to the immunoSepharose column reacted with neurofilament antisera by double immunodiffusion. On sodium dodecyl sulfate gel electrophoresis the main bands in the non-adsorbed fraction were at 74 000 daltons and above 100 000. Several bands were seen in the 50 000 molecular weight range. It is concluded that glioand neurofilaments co-purify together in Norton's procedure and that neurofilaments are probably heterogeneous in polypeptide composition.
Introduction A major controversy presently exists in the literature as to the polypeptide composition of the mammalian neurofilament. Brain filaments isolated accord-
* Address reprint requests to: Dr. D. Dahl, Spinal Cord Injury Research, Veterans Administxation Hospital, 1400 Veterans of Foreign Wars, Parkway, West Roxbury, Ma 02132.
306 ing to the basic procedure developed in Norton's laboratory [11,26] separate into multiple bands on sodium dodecyl sulfate-acrylamide gel electrophoresis, the major band in the 50 000 mol. wt. range being commonly interpreted as neurofilament protein [8,9,15,25]. On the other hand, studies of neurofilaments isolated from peripheral nerve indicate that they are predominantly formed of a 68 000 dalton polypeptide [22], while studies of the slow component of axonal transport show that three polypeptides with molecular weights of 212 000, 160 000, and 68 000 appear to be associated with a single structure which was tentatively identified as the neurofilament [14]. The purpose of this paper is to report the isolation from brain filaments of the glial fibrillary acidic protein and of an immunologically active cyanogen bromide pep~ide [5] using a procedure recently developed in this laboratory for the purification of the protein from buffer extracts of bovine brain, i.e. immunoaffinity chromatography [ 19]. It will be shown that a major fraction of the approx. 50 000 mol. wt. protein in brain filament preparations is glial fibrillary acidic protein, a brain-specific protein selectively localized in astroglia by immunohistochemical methods [3,12,21]. Methods Brain filaments were isolated from bovine white matter with two modifications of the basic Norton's procedure [25,30]. Immunoaffinity chromatography was performed as described elsewhere [19]. Four different batches of Sepharose 4B immunoadsorbent were used. The columns were underloaded with respect to their capacity which had been determined in preliminary experiments. Limiting amounts of brain filament extracts were applied to the column and non-adsorbed protein was tested f o r the absence of glial fibrillary acidic protein by double immunodiffusion. The brain filament pellet was homogenized at 4°C with 8 M urea in 0.05 M sodium phosphate buffer, pH 8.0. The supernatant after centrifugation at 100 000 X g for 1 h was dialyzed overnight against the column equilibration buffer (2 M urea, 0.8 M sodium chloride, 0.01 M sodium phosphate buffer, pH 7.7). Following high speed centrifugation the 2 M ureasoluble column samples were applied to the Sepharose 4B-immunoabsorbent washed with equilibration buffer and then with buffer containing 5 M urea, 0.8 M NaC1, 0.05 M sodium phosphate, pH 6.0. Protein was eluted with 1 M acetic acid, 5 M urea, 0.8 M NaC1, pH 2.5. The flow rate was 5 ml/h for the first 20 fractions and increased to 20 ml/h afterwards. The cyanogen bromide digests of brain filament preparations were obtained by reacting 8 M urea extracts dialyzed against water and freeze-dried with 1 M cyanogen bromide in 70% formic acid for 24 h at 22°C in the dark. The samples diluted with 10 volume of distilled water were freeze-dried and redissolved in 8 M urea. After dialyzing against the equilibration buffer and centrifugation at 100 000 X g for 1 h the 2 M urea-soluble column samples were applied to the Sepharose-immunoadsorbent. The antiserum to the glial fibrillary acidic protein was raised against degraded antigen (40 500 mol. wt.) isolated by hydroxyapatite chromatography from phosphate buffer extracts of autolysed human spinal cord [6]. The
307 antisera used in this work were obtained from one rabbit kept for 3 years in the laboratory, bled every 2 weeks and boosted w i t h 1 mg of antigen every 3 months. As indicated by immunodiffusion and immunofluorescence the strength of the antisera remained essentially unchanged during the whole period. Neurofilament antiserum was raised in one rabbit injected with urea-soluble chicken brain antigen as previously described [7]. The anti-neurofilament activity first appeared 3 weeks after injection and plateaued at 5 weeks. Maintenace of the activity r e q u i r e d w e e k l y boosters. As previously reported [7], only a weak immunodiffusion line against the glial fibrillary acidic protein could be demonstrated with these antisera Degraded glial fibrillary acidic protein (40 500 mol wt.) was isolated from autolysed h u m a n and bovine spinal cords by hydroxyapatite chromatography [6]. Non-degraded bovine glial fibrillary acidic protein used as a molecular weight standard was isolated by immunoaffinity chromatography [19]. T u b u lin was prepared from calf brain by two cycles of the polymerization procedure [27]. In order to avoid nonspecific precipitation, absorption experiments were conducted with diluted antisera. In fact, preliminary experiments had shown that when non
