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Isolation and partial characterization of proteoglycans from rat incisors.

ABSTRACT: Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.

SUBMITTER: Rahemtulla F

PROVIDER: S-EPMC1153418 | biostudies-other | 1984 Mar

REPOSITORIES: biostudies-other

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Project description:1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 x 10(-13) and 2.2 x 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.

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Isolation and partial characterization of proteoglycans from rat incisors.

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