129
Biochimica et Biophysica Acta, 558 (1979) 129--141 © Elsevier]North-Holland Biomedical Press
BBA 29093
ISOLATION AND PARTIAL CHARACTERIZATION OF PROTEOGLYCANS FROM SHEEP LUNG PARENCHYMA
PHYLLIS M. SAMPSON a, SERGIO A. JIMENEZ b and REZA I. BASHEY b
a Cardiovascular-Pulmonary Division and b Collagen Research Laboratories, Rheumatology Section, Department of Medicine University of Pennsylvania School of Medicine, Philadelphia PA 19104 (U.S.A.) (Received March 30th, 1979)
Key words: Proteoglycan isolation; Glycosaminoglycan; (Sheep lung parenchyma)
Summary Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE~ellulose in urea and eluted with increasing concentrations of NaC1. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaC1 and further purified by Sepharose CI-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminogiycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single~hain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.
Introduction Collagen and elastin represent more than 90% of the connective tissue matrix of lung parenchyma [1]. These two components have been extensively studied under normal [2--4] and pathological conditions [5,6]. Of the remaining 10%, only the glycosaminoglycans have been studied in detail. It has been shown in
130 several species that all the glycosaminoglycans, except keratan sulfate, occur in lung parenchyma where they comprise less than 1% of the dry weight of the tissue [7]. Isolation of glycosaminoglycans in all cases was accomplished by proteolytic digestion of the tissue, thus excluding the possibility of extraction of intact proteoglycan, the form in which the glycosaminoglycans have been shown to occur in other tissues [8]. To study the role and molecular structure of proteoglycans in lung parenchyma, it is necessary to solubilize the majority of these compounds under conditions which closely preserve their native structure. Since at present there are no studies on the extractability of lung proteoglycans, we have developed a non-enzymic method that results in solubilization of greater than 90% of the uronic acid-containing compounds from sheep lung parenchyma. This report describes these studies and the isolation and partial characterization of a fraction of proteoglycans recovered from one of the extracts. Materials and Methods Adult male sheep were killed by severing the jugular vein and allowing blood to drain from the cut. Lungs were removed immediately and perfused with 4--6 1 0.15 M NaC1 through the pulmonary artery to remove blood. The perfused lungs were placed in an ice-bath, pleura stripped away and the parenchyma cut away from vessels and bronchi. The pieces of parenchyma were frozen immediately in liquid N2 and then shattered and lyophilized. Approx. 1 h was required to prepare parenchyma for lyophilization. Papain, pronase CB(R)B grade (86 400 U/g) and hyaluronidase from Streptomyces hyalurolyticus nov. sp. (3000 U/mg) were obtained from Calbiochem Corp. (LaJolla, CA). Beef pancreas deoxyribonuclease {3280 U/rag) and chondroitinase ABC were purchased from Miles Laboratories (Elkhard, IN)as were the hyaluronic acid, chondroitin sulfates and dermatan sulfate standards. The heparin was laboratory grade obtained from Fisher Scientific Co. (Fairlawn, NJ). Dr. Alfred Linker kindly supplied us with a purified heparan sulfate from beef lung [9]. Guanidine hydrochloride and dithiothreitol were absolute grade products of Research Plus (Denville, NJ). Triton X-100 was purchased from Beckman Instruments (Broomall, PA). All other chemicals used were reagent grade. Pharmacia Fine Chemicals (Uppsala, Sweden) was the source of the Sepharose C1-6B and the blue dextran 2000. For ion-exchange chromatography preswollen microgranular Whatman DE-52 (Whatman, Clifton, NJ) was used.
Uronic acid assay Uronic acid was determined by the method of Bitter and Muir [10] after lyophilized samples were suspended in 0.1 M NaOH and kept at 4°C overnight. Cellulose acetate electrophoresis Electrophoresis of glycosaminoglycans was performed on cellulose acetate strips in a Beckman Microzone Cell (Beckman Instruments, Fullerton, CA) using the following electrophoretic systems: (1) pyridine/1 M acetic acid buffer (pH 3.5) for 15 min at 7 mA [11]; (2) 0.1 M HC1 (pH 1.2) for 2 h at 25 V
131 [12], and (3) 0.2 M calcium acetate (pH 7.2) for 3 h at 7 mA [13]. As markers for electrophoresis, standard glycosaminoglycans and glycosaminoglycans isolated after proteolytic and chemical digestion of lung parenchyma were used. The strips were stained with 0.5% Alcian blue dissolved in 3% acetic acid [12].
Isolation of lung parenchyma glycosaminoglycans The glycosaminoglycans from 2 g lyophilized sheep lung parenchyma were isolated after digestion of the tissue with papain, alkali, and pronase as described previously [ 14,15].
Extraction of lung parenchyma proteoglycans with guanidine hydrochloride A series of four samples of 1.5 g each of lyophilized lung parenchyma was suspended in 30 ml each of 0.2 M acetate buffer (pH 6.0), containing 2, 3, 4 or 5 M guanidine hydrochloride. 5 mM benzamidine hydrochloride, 5 mM iodoacetamide, 0.1 M 6-aminocaproic acid and 0.01 M disodium EDTA were incorporated to inhibit proteolysis [16]. The suspensions were stirred for 20 h at room temperature and centrifuged at 27 000 Xg for 1 h. The supernatants were dialyzed 40 h against cold, running tap water followed by 6 h against distilled water at 4°C and lyophilized. The residues were washed with distilled water until chloride and acetate free, dried and assayed for uronic acid content.
Sequential extraction with guanidine hydrochloride, dithiothreitol and Triton X-1 O0 After pilot experiments established that 4 M guanidine hydrochloride extraction was the most efficient, 20 g tissue was extracted three times with 4 M guanidine hydrochloride. The residue was extracted twice under N2 with 0.1 M dithiothreitol in 4 M guanidine hydrochloride buffer containing protease inhibitors for 20 h at room temperature. The extracts were dialyzed exhaustively at 4°C against distilled water containing the inhibitors followed by 6 h against distilled water and they were then lyophilized. The dithiothreitol residue was re-extracted twice with a solution of 1% Triton X-100 in 0.05 M Tris-HC1, (pH 7.5), 0.15 M NaC1 and the protease inhibitots. The extracts were separated from the Triton X-100 by dialysis and acetone precipitation and lyophilized for analyses. The residue remaining after these extractions was extensively washed with distilled water, rinsed with acetone and ether and dried in a dessicator.
Enzymic digestion of extracts and residues To ascertain the presence of glycosaminoglycans in the extracts and residues, they were treated with papain as described previously [15]. The digests were precipitated with 5% trichloroacetic acid and the supernatants neutralized, dialyzed and lyophilized. A portion of the lyophilized samples was redissolved in 0.1 M acetate buffer (pH 5.1) and incubated with ribonuclease (250 U/10 mg) at 37°C for 6 h. The solution was adjusted to 0.2 mM MgSO4, deoxyribonuclease (250 U/10 mg) was added and the solution reincubated under toluene for 18 h at 37°C. The samples were then treated as the papain
Biochimica et Biophysica Acta, 558 (1979) 129--141 © Elsevier]North-Holland Biomedical Press
BBA 29093
ISOLATION AND PARTIAL CHARACTERIZATION OF PROTEOGLYCANS FROM SHEEP LUNG PARENCHYMA
PHYLLIS M. SAMPSON a, SERGIO A. JIMENEZ b and REZA I. BASHEY b
a Cardiovascular-Pulmonary Division and b Collagen Research Laboratories, Rheumatology Section, Department of Medicine University of Pennsylvania School of Medicine, Philadelphia PA 19104 (U.S.A.) (Received March 30th, 1979)
Key words: Proteoglycan isolation; Glycosaminoglycan; (Sheep lung parenchyma)
Summary Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE~ellulose in urea and eluted with increasing concentrations of NaC1. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaC1 and further purified by Sepharose CI-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminogiycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single~hain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.
Introduction Collagen and elastin represent more than 90% of the connective tissue matrix of lung parenchyma [1]. These two components have been extensively studied under normal [2--4] and pathological conditions [5,6]. Of the remaining 10%, only the glycosaminoglycans have been studied in detail. It has been shown in
130 several species that all the glycosaminoglycans, except keratan sulfate, occur in lung parenchyma where they comprise less than 1% of the dry weight of the tissue [7]. Isolation of glycosaminoglycans in all cases was accomplished by proteolytic digestion of the tissue, thus excluding the possibility of extraction of intact proteoglycan, the form in which the glycosaminoglycans have been shown to occur in other tissues [8]. To study the role and molecular structure of proteoglycans in lung parenchyma, it is necessary to solubilize the majority of these compounds under conditions which closely preserve their native structure. Since at present there are no studies on the extractability of lung proteoglycans, we have developed a non-enzymic method that results in solubilization of greater than 90% of the uronic acid-containing compounds from sheep lung parenchyma. This report describes these studies and the isolation and partial characterization of a fraction of proteoglycans recovered from one of the extracts. Materials and Methods Adult male sheep were killed by severing the jugular vein and allowing blood to drain from the cut. Lungs were removed immediately and perfused with 4--6 1 0.15 M NaC1 through the pulmonary artery to remove blood. The perfused lungs were placed in an ice-bath, pleura stripped away and the parenchyma cut away from vessels and bronchi. The pieces of parenchyma were frozen immediately in liquid N2 and then shattered and lyophilized. Approx. 1 h was required to prepare parenchyma for lyophilization. Papain, pronase CB(R)B grade (86 400 U/g) and hyaluronidase from Streptomyces hyalurolyticus nov. sp. (3000 U/mg) were obtained from Calbiochem Corp. (LaJolla, CA). Beef pancreas deoxyribonuclease {3280 U/rag) and chondroitinase ABC were purchased from Miles Laboratories (Elkhard, IN)as were the hyaluronic acid, chondroitin sulfates and dermatan sulfate standards. The heparin was laboratory grade obtained from Fisher Scientific Co. (Fairlawn, NJ). Dr. Alfred Linker kindly supplied us with a purified heparan sulfate from beef lung [9]. Guanidine hydrochloride and dithiothreitol were absolute grade products of Research Plus (Denville, NJ). Triton X-100 was purchased from Beckman Instruments (Broomall, PA). All other chemicals used were reagent grade. Pharmacia Fine Chemicals (Uppsala, Sweden) was the source of the Sepharose C1-6B and the blue dextran 2000. For ion-exchange chromatography preswollen microgranular Whatman DE-52 (Whatman, Clifton, NJ) was used.
Uronic acid assay Uronic acid was determined by the method of Bitter and Muir [10] after lyophilized samples were suspended in 0.1 M NaOH and kept at 4°C overnight. Cellulose acetate electrophoresis Electrophoresis of glycosaminoglycans was performed on cellulose acetate strips in a Beckman Microzone Cell (Beckman Instruments, Fullerton, CA) using the following electrophoretic systems: (1) pyridine/1 M acetic acid buffer (pH 3.5) for 15 min at 7 mA [11]; (2) 0.1 M HC1 (pH 1.2) for 2 h at 25 V
131 [12], and (3) 0.2 M calcium acetate (pH 7.2) for 3 h at 7 mA [13]. As markers for electrophoresis, standard glycosaminoglycans and glycosaminoglycans isolated after proteolytic and chemical digestion of lung parenchyma were used. The strips were stained with 0.5% Alcian blue dissolved in 3% acetic acid [12].
Isolation of lung parenchyma glycosaminoglycans The glycosaminoglycans from 2 g lyophilized sheep lung parenchyma were isolated after digestion of the tissue with papain, alkali, and pronase as described previously [ 14,15].
Extraction of lung parenchyma proteoglycans with guanidine hydrochloride A series of four samples of 1.5 g each of lyophilized lung parenchyma was suspended in 30 ml each of 0.2 M acetate buffer (pH 6.0), containing 2, 3, 4 or 5 M guanidine hydrochloride. 5 mM benzamidine hydrochloride, 5 mM iodoacetamide, 0.1 M 6-aminocaproic acid and 0.01 M disodium EDTA were incorporated to inhibit proteolysis [16]. The suspensions were stirred for 20 h at room temperature and centrifuged at 27 000 Xg for 1 h. The supernatants were dialyzed 40 h against cold, running tap water followed by 6 h against distilled water at 4°C and lyophilized. The residues were washed with distilled water until chloride and acetate free, dried and assayed for uronic acid content.
Sequential extraction with guanidine hydrochloride, dithiothreitol and Triton X-1 O0 After pilot experiments established that 4 M guanidine hydrochloride extraction was the most efficient, 20 g tissue was extracted three times with 4 M guanidine hydrochloride. The residue was extracted twice under N2 with 0.1 M dithiothreitol in 4 M guanidine hydrochloride buffer containing protease inhibitors for 20 h at room temperature. The extracts were dialyzed exhaustively at 4°C against distilled water containing the inhibitors followed by 6 h against distilled water and they were then lyophilized. The dithiothreitol residue was re-extracted twice with a solution of 1% Triton X-100 in 0.05 M Tris-HC1, (pH 7.5), 0.15 M NaC1 and the protease inhibitots. The extracts were separated from the Triton X-100 by dialysis and acetone precipitation and lyophilized for analyses. The residue remaining after these extractions was extensively washed with distilled water, rinsed with acetone and ether and dried in a dessicator.
Enzymic digestion of extracts and residues To ascertain the presence of glycosaminoglycans in the extracts and residues, they were treated with papain as described previously [15]. The digests were precipitated with 5% trichloroacetic acid and the supernatants neutralized, dialyzed and lyophilized. A portion of the lyophilized samples was redissolved in 0.1 M acetate buffer (pH 5.1) and incubated with ribonuclease (250 U/10 mg) at 37°C for 6 h. The solution was adjusted to 0.2 mM MgSO4, deoxyribonuclease (250 U/10 mg) was added and the solution reincubated under toluene for 18 h at 37°C. The samples were then treated as the papain
