Characterization of newly synthesized proteoglycans from rabbit menisci in organ culture

Abstract

Rabbit menisci were incubated with Na2 35SO4 in short-term organ culture to label newly synthesized proteoglycans. The radioactive products present in both tissue and culture medium were characterized separately with respect to distribution after ultracentrifugation in CsCl isopycnic density gradients, hydrodynamic size, interaction with hyaluronic acid, and glycosaminoglycan composition (types, size and content). Analysis of proteoglycan size by gel-filtration chromatography of the most-dense CsCl fractions (A1) on Sephacryl S-500 (associative conditions) resolved three species. A peak with Kav. approx. 0.7 was present in each chromatogram, and constituted the principal component in tissue extracts. Two other peaks with Kav. values of approx. 0.2 and 0.45 were also found. When the A1 fraction from tissue was subjected to CsCl-density-gradient ultracentrifugation under dissociative conditions, 71% of the recovered radioactivity was present in the most dense (A1D1) fraction. Incubation with hyaluronic acid of either A1 or A1D1 fraction from associative extract did not alter the apparent size of the labelled product, indicating a lack of aggregate formation. Meniscal proteoglycans showed an unusual and marked tendency to adsorb irreversibly to agarose and agarose-containing gel-filtration-chromatography media. High-pressure liquid-chromatographic analyses indicated that the sulphated glycosaminoglycans consisted of chondroitin 6-sulphate (72%), chondroitin 4-sulphate (19%) and dermatan sulphate (5%). Endo-beta-galactosidase (keratanase) digestion of the material failed to detect the presence of keratan sulphate. Of the labelled glycosaminoglycans, 95% was eluted from Sephacryl S-400 as a single symmetrical peak with a Kav. of 0.5. The results of studies with tissue extracts and culture medium were similar.