60

Biochimica et Biophysica Acta, 1117(1992) 60-70 © 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00

BBAGEN 23684

Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma Mikio Arai

1, Toshikazu

Yada, Sakaru Suzuki and Koji Kimata

Institute for Molecular Science of Medicine, Aichi Medical Uniuersity, Nagakute, Aichi (Japan)

(Received 12 November 1991) (Revised manuscript received 3 February 1992)

Key words: Chondroitin sulfate; Proteoglycan;Swarm rat chondrosarcoma;Type IX collagen Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was further separated by anion exchange chromatography into an unbound and a bound fraction in an A230 ratio of about 5 : 1. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the bound fraction appeared as a broad band, whose molecular mass ranged from 250 to 270 kDa. Digestion with chondroitinase ABC reduced the apparent molecular mass of the bound fraction to about 250 kDa, a value comparable to the molecular mass of the unbound fraction. Tryptic peptide maps of the protein moieties of unbound and bound forms showed that their molecular structures were basically identical. A monoclonal antibody specific for LMW, one of the pepsin-resistant fragments of the rat sarcoma type IX, reacted with both the unbound and bound fractions. Together the results indicate that the unbound and bound fractions represent a type IX collagen devoid of the chondroitin sulfate chain and its proteoglycan form with covalently bound chondroitin sulfate, respectively. The extent of glycosaminoglycan attachment to type IX collagen molecules in rat chondrosarcoma (about 16%) is quite different from the extents described in chick embryo cartilage (about 80%), chick vitreous humour (100%) and bovine cartilage ( < 5%). Further studies on the neoplastic tissue will offer additional information regarding the biological basis and biological consequences of the glycosaminoglycan attachment to type IX collagen molecules.

Introduction Recent electron microscopic studies on mixed fibrils of type II and type IX collagens from embryonic chick cartilage have shown that type IX collagen molecules are localized in a periodic manner along the surface of collagen fibrils, with its N-terminal domain projecting out from the fibrils and presumably interacting with the surrounding matrix [1-5]; see also the reviews by Shimokomaki et al. [6] and Gordon and Olsen [7]. The type IX collagen is also a proteoglycan (PG-Lt; see Noro et al. [8]; Vaughan et al. [9]), bearing a chond r o i t i = / d e r m a t a n sulfate chain covalently bound to the NC3 domain of a2(IX) chain [10,11]. This glycosaminoglycan chain is another potential site through which the molecule could interact with matrix components of the cartilage.

Correspondence to: K. Kimata, Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan. Present address: Tokyo Research Institute, Seikagaku Corporation, Tokyo 207, Japan. Abbreviations: HMW and LMW, high- and low-molecular weight disulfide-linked fragments, respectively, isolated from peptic digests of type IX collagen.

Comparative studies on mixed fibrils of type II and type IX collagen from adult chicken vitreous humour have shown that the fibrils are different from those in cartilage in that they are completely invested by a coat of chondroitinase-sensitive glycosaminoglycan, which probably represents chondroitin sulfate side chains of type IX collagen-proteoglycan molecules [12]. It has, indeed, been shown [13,14] that the avian vitreous contains a type IX collagen-proteoglycan characterized by the occurrence of a short form (M r 61000) of a l ( I X ) polypeptide lacking the NC4 domain, by the attachment of extraordinarily large chondroitin sulfate chain (M r 350000), and by the absence of a type IX collagen devoid of the chondroitin sulfate chain. The results, coupled with the finding that the avian vitreous contains little or no hyaluronic acid [13], suggest that the high-molecular weight chondroitin sulfate chains extending for a considerable distance from the fibrils, may provide a vitreous matrix for physicochemical support to maintain internal eye structure and function. Because of the known or suspected involvement of type IX collagen-proteoglycan in the generation of tissue-specific fibrillar patterns of mixed collagen fibrils, further characterization of type IX collagen molecules associated with cartilagenous tissues of dif-

61 ferent histological features would clearly be desirable. We describe here the isolation and characterization of intact type IX collagen from the Swarm rat chondrosarcoma, a transplantable, neoplastic tissue producing a large amount of soft gelatinous matrix containing cartilage-type proteoglycan [15], type II collagen [16], a parent molecule of type M collagen [17], which is now designated type IX collagen [18], and type IX collagen fragments from limited pepsin digests [19]. Our results indicate that a significant proportion of the type IX collagen of rat chondrosarcoma occurs as a proteoglycan and that the ratio of the proteoglycan form to a glycosaminoglycan-lacking form is quite different from the ratios described in other avian and mammalian tissues. Materials and Methods

L-[3H]Proline (1 mCi/ml) and sodium [125I]iodide (carrier-free) were purchased from Amersham Japan (Tokyo, Japan); [35S]sulfate (carrier-free) was from Japan Radioisotope Association (Tokyo, Japan); chondroitin ABC lyase (EC 4.2.2.4), chondroitin AC lyase (EC 4.2.2.5) and bacterial collagenase (EC 3.4.24.3) were from Seikagaku (Tokyo, Japan); Actinase E was from Kakenseiyaku (Tokyo, Japan); pepsin (EC 3.4.23.1) and complete and incomplete Freund's adjuvant were from Difco Laboratories (Detroit, MS, USA); trypsin (EC 3.4.21.4) and 3-aminopropionitrile were from Sigma Chemical (St. Louis, MO, USA); chemicals for tissue culture were from Nissui Seiyaku (Tokyo, Japan); foetal calf serum was from Commonwealth Serum Laboratories (Melbourne, Australia); reagents for enzyme-linked immunosorbent assay were from Miles-Yeda (Rehovot, Israel); mouse monoclonal isotyping kit was from Zymed Laboratories (South San Francisco, CA, USA); Bio-Gel A5m was from Bio-Rad Laboratories Japan (Tokyo, Japan); DEAE-cellulose was from Whatman (Maidstone Kent, UK); LiCrosorbNH 2 was from Merck (Darmstadt, FRG); TSK-G2500, TSK-G3000 and TSK-G4000 were from Tosoh (Tokyo, Japan); silica gel-coated thin-layer plates (20 × 20 cm, 0.25 mm thickness) were from Merck (Darmstadt, FRG); X-ray films were from Fuji Photo Film (Kanagawa, Japan). Collagen standards, /3(II), al(II) and a3(IX) were prepared from chick embryo cartilages, as described in Yasui et al. [20] and Vaughan et al. [9]. Chondroitin sulfate standards with light scattering molecular weights of 39 100, 18 900 and 8050 were gifts from Dr. K. Horie, Tokyo Research Institute, Seikagaku (Tokyo, Japan). The chondrosarcoma was maintained by serial transplantation in female Sprague-Dawley rat [16] without feeding lathyrogen. Peptic fragments (HMW and LMW) of rat chondrosarcoma were prepared by a modification of the

method of Reese and Mayne [21] for chick cartilage HMW and LMW. The tumours, about 100 g (wet weight), were homogenized at 4°C in 300 ml of H 2 0 with a polytron homogenizer and the homogenate was centrifuged at 27000 × g for 20 min. The pellet was extracted twice (4°C, 24 h) with 500 ml each of 4 M guanidine HC1/0.05 M Tris-HCl (pH 8.0), containing 10 mM EDTA, 1 mM N-ethylmaleimide and 1 mM phenylmethanesulphonyl fluoride (hereafter the mixture of the three reagents will be referred to as "protease inhibitors"). The final residue was washed three times with H 2 0 and suspended in 250 ml of 0.2 M NaC1/0.5 M acetic acid containing 250 mg of pepsin. The suspension was stirred at 4°C overnight and the undigested residue was removed by centrifugation (27 000 × g, 30 min). To inactivate pepsin, the pH of the supernatant solution was titrated to 8.0 with 5 M NaOH. After standing at 4°C overnight, collagen type II, XI and IX (HMW and LMW) were separated by differential salt precipitation in 0.5 M acetic acid at 0.9 M, 1.2 M and 2 M NaC1, respectively, as described in Reese and Mayne [21]. The final precipitate was dissolved in 0.5 M acetic acid, dialysed against 0.05 M acetic acid and lyophilized.

Metabolic labeling of chondrosarcoma in the presence of lathyrogen Pieces of tumour were rinsed three times in 10 volumes of Hanks' balanced salt solution and forced through a stainless steel sieve (mesh, 0.75 mm2). The tumour fragments were washed successively with 10 volumes each of Hanks' solution (three times) and culture medium (see below). The washed tumour fragments were incubated at 37°C in an equal volume of Eagle's MEM (sulfate-free) containing 10% (v/v) foetal calf serum, 10 mM Hepes buffer, sodium ascorbate (10 /xg/ml), sodium penicillin G (100 units/ml), streptomycin sulfate (100 /xg/ml) and 3-aminopropionitrile (100 /xg/ml) under 5% CO2/95% air atmosphere. After 30 min of preincubation, radioisotope was added to give the following final concentration: [35S]sulfate, 100 /zCi/ml; or [3H]proline, 20 /xCi/ml. Incubation was then continued for 10 h (35S) or 24 h (3H). From each culture, at the termination of labeling, medium and tumour fragments were separately collected. To the medium were added three volumes of 95% (v/v) ethanol/1.3% (w/v) potassium acetate, with stirring. The mixture was allowed to stand at 0°C for 30 min and the resultant precipitate was collected by centrifugation (12000 ×g, 0°C, 30 min) and combined with the tumour fragments for extraction and fractionation o f radiolabeled collagens (see below).

Extraction and fractionation of collagens Each combined tissue-medium sample (labeled with either 35S or 3H) prepared as above was suspended,

62 together with an equal amount of fresh (unlabeled) tumour as a carrier, in volumes of 0.05 M Tris-HC1 (pH 7.5)/1 M NaC1/protease inhibitors and left stirring for 16 h at 0°C. The insoluble residue was separated from the solubilized material by centrifugation at 12 000 x g for 30 min, and the solubilized collagens precipitated with 45%-saturated (NH4)2SO 4. The precipitate was redissolved in 0.05 M Tris-HC1 (pH 7.5)/0.5 M NaCI and subjected to salting-out procedures, according to the protocol of Duance et al. [17] for purification of type IX collagen. For preparation of a substantial amount of unlabeled type IX collagen, fresh tumours were directly homogenized and extracted as above and the resultant extract was subjected to consecutive purification procedures including salting-out, Bio-Gel A5m chromatography and DEAE-cellulose chromatography as described in "Results".

Preparation of monoclonal antibody (AK-236) specific for LMW BALB/c mice were immunized with an intraperitoneal injection of chondrosarcoma type IX collagen (the DEAE-cellulose-unbound fraction in Fig. 4), 200 /~g in 0.2 ml phosphate-buffered saline, emulsified in complete Freund's adjuvant. Injection of immunogen in incomplete Freund's adjuvant was repeated twice at 2-week intervals. Cells from the spleen were conventionally fused with NS-1 myeloma, and the hybridomas showing positive reaction on LMW were cloned by limiting dilution. One of them, designated AK-236, reacted with LMW but not with HMW, al(II), al(XI), a2(XI) and a3(XI). Using a Zymed mouse monoclonal isotyping kit, the isotype of AK-236 was determined to be IgM.

SDS-polyacrylamide gel electrophoresis and immunoblotting Polyacrylamide gel electrophoresis in the presence of 0.1% (w/v) SDS was carried out in 5% (w/v) running gel [22] under non-reducing and reducing conditions. Before electrophoresis, samples were desalted by precipitation with three volumes of 95% (w/v) ethanol/1.3% (w/v) potassium acetate at 0°C. Chondroitinase treatment before electrophoresis was performed, as previously described [23]. After electrophoresis, the proteins and proteoglycans in gels were detected by either Coomassie Blue staining or fluorography. For immunoblotting, gels were subjected to electrotransfer to nitrocellulose membranes and immunostaining following the procedure of Towbin et al. [24].

Pepsin digestion For pepsin digestion of the DEAE-cellulose-unbound fraction (see Fig. 4), samples (1 mg/ml) were

equilibrated into 0.5 M acetic acid and digested with pepsin (0.1 mg/ml) for 30 h at 20°C. The digests were lyophilized for further analyses. For collagenase digestion of the DEAE-celluloseunbound fraction, samples (1 mg/ml) were equilibrated into 0.05 M Tris-HCl (pH 7.5)/2 mM CaC12 and digested with bacterial collagenase (0.01 mg/ml) for 3 h at 37°C. The digests were lyophilized and subjected to further analyses.

Estimation of the recovery of type IX collagen in c,arious fractions Samples (1 mg/ml) were equilibrated into 0.5 M acetic acid and digested with pepsin (0.1 mg/ml) for 30 h at 20°C. The digests were lyophilized and then subjected to SDS-gel electrophoresis with the known amount of standard HMW, as described above. After Coomassie Blue staining, the bands corresponding to HMW were subjected to the determination of the amount by their relative staining intensities.

Tryptic peptide mapping The radioiodination of protein samples in polyacrylamide gels followed by mapping of tryptic peptides on thin layer plates was carried out by the method of Elder et al. [25] as modified by Oike et al. [26]. Briefly, a gel slice containing a Coomassie Blue-stained protein was labelled with 125I in the presence of chloramine-T and was treated with 25 tzg of trypsin in 500/zl of 0.05 M NHaHCO 3 (pH 8.4), for 18 h at 37°C. A portion of the tryptic digests was subjected to the two dimensional mapping on a silica gel-coated thin layer plate (electrophoresis in the first dimension, and ascending chromatography in the second dimension). The peptides were visualized by radioautography with an X-ray film.

Analysis of glycosaminoglycans Glycosaminoglycans were released from proteoglycan samples by treatment with 0.2 M NaOH at room temperature for 24 h. After neutralization with acetic acid, the mixture was digested in 0.02 M Tris-HCl (pH 8.0), with Actinase E (one fiftieth the total amount of proteins) at 50°C for 12 h. Glycosaminoglycans were collected by ethanol precipitation. For determination of disaccharide unit compositions of glycosaminoglycans, chondroitinase digestion was performed, as previously described [27], and separation of the products was carried out by high performance liquid chromatography on a LiCrosorb-NH 2 column (0.26 X 25 cm), eluted with a linear gradient of NaH2PO 4 [28]. For determination of glycosaminoglycan chain sizes, samples were dissolved in 0.2 M NaC1 and subjected to high performance liquid chromatography on a column system consisting of serially connected TSK-G4000

63 PWxL, G3000 PWxL and G2500 PWxL columns (7.5 mm X 30 cm each) which had been calibrated with chondroitin sulfate standards of the known light scattering molecular weights. The column was eluted with 0.2 M NaC1 at a flow rate 0.6 ml/min. The elution of glycosaminoglycans was monitored by measuring hexuronate content.

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Results

Demonstration of a proteoglycan form of type IX collagen in rat chondrosarcoma When fragments prepared from the tumour, grown subcutaneously in rats, were metabolically labeled with either [3SS]sulfate or [3H]proline in the presence of 3-aminopropionitrile and then extracted at pH 7.5 in 1 M NaC1 containing protease inhibitors at a temperature below 4°C, approximately 85% (3Ss) or 44% (3H) of the total radioactivity incorporated into macromolecules were brought into solution. From the 1 M NaC1 extract, a protein fraction that fitted the criteria required for type IX collagen was obtained according to the salting-out procedure of Duance et al. [17]; i.e, the type IX collagen was recovered predominantly in the fraction precipitated at 4.0 M NaCI in neutral salt and 1.2 M NaC1 in 0.5 M acetic acid (designated fraction P2). The P2 fraction, thus obtained, was dialysed against 2 M urea/0.02 M NaC1/0.05 M Tris-HC1 (pH 8.0). The non-diffusible material was subjected to anion-exchange chromatography on a DEAE-cellulose column eluted with a linear salt gradient of 0.02-0.5 M NaC1 (pH 8.0). Typical elution profiles of 3H- and 3ss-labeled P2 samples are shown in Fig. 1. The proline label yielded two major fractions, an unbound fraction and a bound fraction having a peak at about 0.1 M NaC1, with a largely retarded shoulder at a higher ionic strength. The sulfate label, on the other hand, was not detected in the unbound fraction but eluted over a wide range of ionic strengths, with a main peak at about 0.3 M NaCI. The eluted fractions were combined, as indicated in Fig. 1, and aliquots (3H, 20000 cpm for lanes 1 to 10 and 10000 cpm for lanes 11 to 14; 3Ss, 3000 cpm) from the combined fractions were analysed by SDS-polyacrylamide gel electrophoresis and fluorography (Fig. 2). As Fig. 2A shows, the proline label showed a complex pattern of polypeptides, some of which had apparent molecular masses of the known collagen polypeptides; fl(II) (200 kDa), al(XI), a2(XI), a3(XI) and al(II) (100 kDa). Besides these, both DEAEcellulose-unbound and bound fractions yielded polydisperse components migrating in the 250-290 kDa region expected for type IX collagen although they were rather minor components. The component in the bound fractions tended to show an increase of molecu-

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Fig. 1. DEAE-cellulose chromatography of a type IX collagen-enriched fraction (P2) obtained from metabolically radiolabeled chondrosarcoma. Samples (P2 fractions obtained from rat chondrosarcoma that had been metabolically labeled with either [3H]proline or [aSS]sulfate) were dialysed against 2 M urea/0.02 M NaCI/0.05 M Tris-HCl (pH 8.0), and applied onto a DEAE-cellulose column (2x 10 cm) equilibrated in the 2 M urea buffer. After the column was washed with the above buffer (200 ml), a linear gradient elution of 0.02-0.5 M NaC1 (500 ml) was applied. Elution profiles of 3H-label (e) and 3ss-label (o) are shown in one figure. In each case, fractions were pooled for further analysis, as indicated by the boxes (a-k) above curves.

lar mass as a function of increasing salt concentrations on anion-exchange chromatogram (lanes 5, 7, 9, 11 and 13). On digestion with chondroitinase ABC, all of them converted to a 250 kDa component (lanes 6, 8, 10, 12 and 14), a protein corresponding in molecular mass to the DEAE-cellulose-unbound fraction, although the only subtle conversion was observed in the fractions eluted with low salt concentrations. No other [3H]proline-labeled components showed such a susceptibility to chondroitinase ABC. The results, coupled with observations with mercaptoethanol-treated samples (see Fig. 6 for the results of reduction of unlabelled samples), suggest that the 250 kDa component in the DEAE-cellulose unbound fraction is a type IX collagen, devoid of the chondroitin sulfate chain, and that the components of higher molecular masses in the bound fraction are type IX collagen-proteoglycans with different lengths of chondroitin sulfate chains. Consistent with this assignment, the major sulfate label in the fractions e-g was shown to consist of the molecules migrating in the region for type IX collagen-proteoglycan (260-290 kDa)(Fig. 2, lanes 19-23). When the 3ss-labeled samples were digested with chondroitinase ABC before electrophoresis, all the sulfate label of the 265-290 kDa components was released, causing the bands to disappear on SDS-gel (Fig. 2, lanes 20-24). Type IX collagen-proteoglycans with still higher molecular masses were detected on the 35S fluorogram of strongly anionic fractions from the DEAE-cellulose

64 column (Fig. 2, lanes 25 and 27), suggesting polydispersity of the newly synthesized type IX collagen-proteoglycan molecules with respect to chain length a n d / o r number of the chondroitin sulfate attached. However, there is an alternative. The labeled components with higher molecular masses, especially at the origin, might be contaminated with other proteoglycan(s).

Isolation and characterization of native forms For further characterization of rat chondrosarcoma type IX collagen, we studied the purification procedure for isolating a substantial amount of type IX collagen from flesh (unlabelled) chondrosarcoma. Earlier studies with tumours grown in normal and lathyritic rats indicated that the amount of a 1 M NaCl-soluble form of type II collagen was far larger in the lathyritic tumours than in the non-lathyritic tumours [16]. Since our preliminary attempts to isolate type IX collagen from 1 M NaC1 extracts of the lathyritic tumours were hampered by contamination with an exceedingly large amount of type II collagen (see Fig. 2), advantage was taken in the present study (Scheme I) of the fact that the ratio of type IX to type II collagen dissolved in 1 M NaC1 was much higher in the non-lathyritic than in the lathyritic tumours (see below for the yield of type IX collagen from the non-lathyritic tumours). The 1 M NaC1 extract from non-lathyritic tumours was subjected to a modification of the NaCl-fractiona-

Fractions Chondroitinase

12 a -,

34 56 b c - 4. - ,

78 d -,

tion procedure of Duance et al. [17], as outlined in Scheme 1. In the modified procedure, the P2 fraction of the original procedure was further partitioned into two fractions, P2-1 and P2-2, precipitated at 0.9 M NaCI and 1.2 M NaCI, respectively, in 0.5 M acetic acid. Cartilage characteristic proteoglycan (CSPG) never precipitated under these conditions (unpublished observations). Examination of P2-1 and P2-2 by SDS-gel electrophoresis showed that most of the al(II) protein in P2 was recovered in P2-1, whereas 81% of the type IX collagen was in P2-2 (results not shown). Fraction P2-2 still contained several other" protein components with lower molecular masses. To remove these contaminating proteins, P2-2 was chromatographed on Bio-Gel A5m and the void volume fraction was collected, as shown in Fig. 3. The protein, thus obtained, may actually represent type IX collagen (see below) and accounts for about 36% of the total type IX collagen in the tumours. The total type IX collagen accounted for about 0.45% of the tumor dry weight and about 1.7% of the total proteoglycans in the tumor. This calculation was based on the total amounts of peptic fragments, HMW and LMW, yielded from the void volume fraction (Fig. 3) and from other fractions including the 1 M NaC1 (pH 7.4)-insoluble residue (Scheme 1; determined photometrically using type II collagen as a standard; see below for details) as described in the Materials and Methods section. The void volume fraction

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Fig. 2. S D S / 5 % (w/v)-polyacrylamide gel electrophoresis of fractions from the DEAE-cellulose columns (see Fig. 1). The samples are: 1, 3, 5, 7, 9, 11 and 13, fractions a, b, c, d, e, f and g, respectively, from the DEAE-cellulose column for [3H]proline-labeled P2 preparation; 2, 4, 6, 8, 10, 12 and 14, as in 1, 3, 5, 7, 9, 11 and 13, respectively, but treated with chondroitinase ABC before electrophoresis; 15, 17, 19, 21, 23, 25, and 27, fractions c, d, e, f, g, h and i, respectively, from the DEAE-cellulose column for [35S]sulfate-labeled P2 preparation; 16, 18, 20, 22, 24, 26 and 28, as in 15, 17, 19, 21, 23, 25 and 27, respectively, but treated with chondroitinase ABC before electrophoresis. Fluorographs are shown. Fractions h and i from the DEAE-cellulose column of [3H]proline-labeled P2 preparation did not contain enough radioactivity for subsequent fluorography. Molecular masses estimated from the positions of standard /3(11) (200 kDa), a l ( l I ) (100 kDa) and chick embryo cartilage a3(IX) (68 kDa) samples are indicated by the arrowheads. The arrows denote the positions expected for the indicated molecules.

65 Rat chondrosarcoma Extracted with I M NaCI/ 50 mM Tris-HCI, pH 7.4, 4°C, 16 h

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Scheme I. Flow diagram of salt-fractionation procedure for the purification of rat chondrosarcoma type IX collagen (a modification of the method of Duance et al., [18]).

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from the Bio-Gel A5m column was analysed by anionexchange chromatography on DEAE-cellulose (Fig. 4). The protein was separated into two major fractions, an unbound fraction accounting for about 84% of the total A230 and a bound fraction (16%) eluted in a salt concentration range from 0.02 M to 0.5 M NaC1. Fractions were pooled, as shown in Fig. 4. The unbound protein (fraction A) migrated as a diffuse band, with a molecular mass about 250 kDa (Fig. 5, lane 1). Treatment with chondroitinase ABC prior to electrophoresis had no effect on its mobility (lane 2). Treatment with bacterial collagenase,'on the other hand, caused complete disappearance of the protein band (not shown). It is noteworthy that the unbound component (250 kDa) is significantly smaller in molecular mass than the corresponding component of chick embryo cartilage type IX (270 kDa). The DEAE-cellulose-bound fraction (fractions B and C, Fig. 4) also appeared as a diffuse band, but their molecular masses were in somewhat higher range (250 kDa-270 kDa). Digestion with chondroitinase ABC reduced the apparent molecular masses to about 250 kDa (lanes 4 and 6), suggesting that the somewhat higher molecular mass band represents a proteoglycan with a 250 kDa core protein, to which chondroitin sulfate chain(s) are covalently linked. Fractions B and C accounted for about 11% and 5% of the total A23 o and about 26% and 74% of the total hexuronte. Therefore, polydispersity of the DEAE-cellulose-bound fraction may be mainly due to the presence of covalently bound glycosaminoglycans with different chain lengths (see below).

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Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma.

Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was fu...
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