Isolation and Characterization of Glycoproteins in Proteoglycan Aggregates of Calf Rib Cartilage†

Abstract

Proteoglycan aggregates were prepared under associative conditions from calf rib cartilage extracts isolated with 4 M guanidine.cntdot.HCl in the presence of proteinase inhibitors. Five Coomassie blue positive bands including 2 link proteins could be detected after treatment of proteoglycan aggregates with 2-mercaptoethanol. After dissociating these proteoglycan aggregates with 4 M guanidine.cntdot.HCl, 3 fractions were obtained by chromatography on Sepharose 2B under the same conditions. A large complex obtained in the excluded volume was partially soluble in the presence of 0.1% sodium dodecyl sulfate, but did not enter 7.5% polyacylamide gel. It split into 7 bands; 5 of them were not found in proteoglycan aggregates after 2-mercaptoethanol treatment. Neither link proteins nor hyaluronate could be detected in this complex, but chrondroitin sulfate was present. Proteoglycan monomers and 1 included fraction were isolated in addition to this complex. From the included fraction, 8 glycoproteins including 2 link proteins were isolated by preparative polyacrylamide gel electrophoresis and their amino acid and sugar contents determined. These glycoproteins possessed the ability to bind hyaluronate. Results indicate the presence of other glycoproteins besides the 2 link proteins and a glycoprotein-proteoglycan complex in proteoglycan complex in proteoglycan aggregates of calf rib cartilage.