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Physical and immunochemical characterization of proteoglycans synthesized during chondrogenesis in the chick embryo
Abstract:
Chick limb buds from 4- to 7-day-old embryos and sterna from 14-day-old embryos were labeled with [35S]sulfate, and the sulfated proteoglycans (PGS) were extracted either with associative (0.5 M guanidine . HCl) or dissociative (4.0 M guanidine . HCl) solvents. The existence of several populations of PGS is revealed in each age group when the limb extracts are analyzed on sucrose density gradients under dissociative conditions. The major component of 7-day limbs has a faster sedimentation rate than does that of 4-day limb buds. These major components of 4- and 7-day extracts chromatograph in the void volume of a controlled-pore glass (CPG) 1400 column. They differ from each other immunologically. The CPG 1400 void volume material from 4-day limb mesenchyme (PGS(LM)-I) shows no cross-reactivity with antiserum against PGS from juvenile cartilage (A1-D1-1400 V0). In contrast, the CPG 1400 void volume material from 7-day limbs, which contain cartilage [PGS(LC)-I], gave a cross-reaction of 70%. When PGS(LM)-I is chromatographed on CPG 2500, 45% of the labeled material chromatographs in the void volume. When PGS(LM)-I is sedimented in a cesium chloride gradient under dissociative conditions, it can be shown that the PGS in the bottom fraction (D1) has a monomer-aggregate relationship. In the absence of hyaluronic acid, this fraction is included on CPG 2500, whereas in the presence of hyaluronic acid it is excluded. Limb mesenchyme, therefore, synthesizes a PGS molecule which can interact with hyaluronic acid to form an aggregate. The endogenous material of a 4-day limb mesenchyme which causes the aggregation of PGS cannot be separated from PGS by chromatography on CPG 240 or 1400 under dissociative conditions. In contrast, the aggregating material from sterna can be separated from PGS under these conditions. These observations are interpreted to mean that the aggregating material of limb mesenchyme is larger than is that of cartilage.