Effect of beta-D-xyloside and cycloheximide on the synthesis of two types of proteochondroitin sulfate in chick embryo cartilage.

p-Nitrophenyl fi-o-xyloside, an artificial initiator of chondroitin sulfate synthesis, and cycloheximide, an inhibitor of protein synthesis, were used in studies of proteoglycan synthesis by chick embryo epiphyseal cartilage in media containing [?3lsulfate or [Wlglucose. In the absence of the drugs, the tissue showed incorporation of the labeled substrates into two different proteochondroitin sulfate populations (termed PCS-H and PCS-L). The products were readily separated from each other by zone sedimentation on a sucrose gradient in the presence of 4 M guanidine hydrochloride (sedimentation rate, PCS-H > PCS-L). Increasing the concentration of p-nitrophenyl P-D-xyloside in the culture medium resulted in a progressive reduction in the rate of PCS-H synthesis so that at 1.0 mM P-o-xyloside the incorporation of the labeled substrates was inhibited almost completely (>98%). Nevertheless, the incorporation into PCS-L proceeded with less significant reduction in its rate, and even at 1.0 mM P-o-xyloside the reaction continued at approximately one-fourth its original rate. Similarly, a preferential reduction in the rate of PCS-H synthesis was observed when the tissue was exposed to varying concentrations of cycloheximide. These results suggest that PCS-II and PCS-L may occur as separate molecular species, produced perhaps by different biosynthetic systems. The changes in the rate of synthesis were accompanied by alterations in the structure of newly synthesized proteoglycans. Thus, as the p-o-xyloside levels were raised, the tissue produced PCS-H and PCS-L with progressively decreased molecular size. Cycloheximide had the reverse effect, i.e. increasing the concentration of cycloheximide resulted in a production of PCS-H and PCS-I, with progressively increased molecular size. Gel chromatography of the chondroitin sulfates from these proteoglycan fractions indicated that the observed changes in molecular size were due to alterations in the average length of the chondroitin sulfate chains attached to core protein. In both PCS-H and PCS-L, the chondroitin sulfate chains showed a trend toward decreasing (in the presence of P-n-xyloside) or increasing (in the presence of cycloheximide) their average chain length as the inhibitor concentrations were raised. The results