Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract

To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [35S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 M-guanidinium chloride (GuCl) fraction and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40 and 4% of the incorporated [35S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of 35S in chondroitin sulfate to that in heparan sulfate was 4-7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more 35S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of .apprx. 12% of the glycosaminoglycans in the saline extracts and .apprx. 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 M-urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.