Abstract
A gelatin-binding protein was isolated from porcine blood plasma by affinity chromatography in sequence on columns of Sepharose 4B coupled with gelatin, arginine, and heparin and by gel filtration through Sephadex G-200. The protein was bound strongly to the arginine column and thus could be separated from fibronectin. It reacted with concanavalin A and contained about 5% carbohydrate consisting of neutral sugars (2.6% by weight), hexosamine (1.5%), and sialic acid (0.55%). The glycoprotein also showed an interaction with hyaluronic acid, but not with unsubstituted Sepharose 4B. It consisted of 8 to 10 disulfide-linked subunits of about 47,000 daltons and contained more glycine and less proline than fibronectin. Thus we tentatively named this glycoprotein glycine-rich gelatin-binding protein (GGP). GGP did not react with antifibronectin antiserum. These results indicate that GGP is a glycoprotein which is distinct from fibronectin. However, there remains a possibility that GGP may be derived in vivo from fibronectin.
Highlights
On columns of Sepharose 4B coupled with gelatin, ar- phenylmethanesulfonyl fluoride, and 20 mM e-aminocaproicacid)
A and containedabout 5% carbohydrateconsisting of neutral sugars (2.6%by weight), hexosamine(1.5%), and sialic separatedand subjected to affinity chromatography on a gelatinSepharose column, and the 4 M urea fraction was obtained as described previously (IO).Fibronectin was isolated from this fraction by subsequent gel fitration through Sepharose 4B in 4 M urea as described previously [10]
There remains paossibility that gelatinbinding protein (GGP) may be column was dialyzedexhaustively against 0.05 M Tris-HC1buffer, pH 7.5,20 mM eaminocaproic acid, 1 mM EDTA (Buffer A), and 0.1 M NaCI, and the dialysate was applied to anarginine-Sepharose column
Summary
Scientific Research from the Ministry of Education, Science, and Culture of Japan. Thecosts of publication of this article were defrayed in part by the payment of page charges. The breakthroughfraction was found to contain fibronectin, and gel fitration afforded a pure preparation(Fig. 1, lanes 3 and 4) The 2 M NaCl-4 M urea fraction eluted from the column was further purified with heparin-Sepharose (Fig. 2b), followed by gel filtration (Fig. 2c). SDS-3%polyacrylamide gel electrophoresis showed that the GGP preparation was homogeneous, and the estimated molecular weight wasabout 450,000 (Fig. 1, lane 8),which is similar to thatof fibronectin [1,2,3,4,5,6,7,8,9,10,16]. The retarded elution even with buffer containing 2 M NaCl and 4 M urea suggested a strong interaction, not observed with other affinity gels used,and with the fibronectin-hyaluronate interaction [11]. The interaction of heparin with GGP appeared to be greater than with fibronectin, since GGP was not eluted from the column with 2 M NaCl in Buffer A, which had been shown to elute 74% of bound fibronectin [11].these binding properties to theglycosaminoglycans are similar to, but considerably different from, those of fibronectin
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