Isolation and Properties of Proteoglycans from Bovine Aorta

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Abstract
Calf arterial tissue, preincubated under organ culture conditions for 24 h in the presence of [35S]sulfate in combination with either [3H]glucosamine or [3H]mannose, yielded 81.8% of total uronic acids, 83% of incor‐ porated [35S]sulfate and 66.8 % of incorporated [3H]glucosamine on extraction with 4 M guanidinium chloride in the presence of protease inhibitors.Two proteoglycans (A and B) differing in amino acid and glycosaminoglycan composition were purified by sequential fractionation of the guanidinium chloride extract on Sepharose 4B CL, caesium chloride density gradient centrifugation under dissociative conditions and ion‐exchange chromatography.Gel filtration of the guanidinium chloride extract resulted in the separation of two [35S]proteoglycan‐con‐ taining fractions, eluted with the void volume (fraction A) and at Kav= 0.33 (fraction B).Proteoglycan A was obtained from the Sepharose fraction A as a proteoglycan complex by a dissociative gradient. On ion‐exchange chromatography in the presence of 6 M urea the proteoglycan complex dissociated into proteoglycan A (Mr 813000) and hyaluronate (M, 81 000). Proteoglycan A contained 3‐4 chondroitin 4/6‐sulfate side chains (Mr 35000) and 10.4% protein.Proteoglycan B was purified from the Sepharose fraction B by a dissociative gradient and subsequent ion‐ exchange chromatography of the bottom fraction. Proteoglycan B was characterized as a proteoglycan monomer (M r 190000) containing 20.3 % protein with 3 ‐ 4 chondroitin sulfate/dermatan sulfate chains (chondroitin sulfate 53 %, dermatan sulfate 46 %) attached. The specific 35S radioactivity of proteoglycan B was three‐times higher than that of proteoglycan A.[3H]Mannose‐labelling of the isolated proteoglycans A and B indicated that both proteoglycans contained glycoprotein‐type oligosaccharides.Pulse‐chase experiments with [35S]sulfate as label suggested that both proteoglycans A and B are primarily produced as monomers retarded by the Sepharose gel and that proteoglycan A is successively converted to a proteoglycan complex on interaction with hyaluronate.

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