Biochemical characterization of the native tissue form of type X collagen from embryonic chick sternal cartilage and identification of a chymotrypsin-sensitive site within its triple-helical domain.

We isolated and characterized the intact native tissue form of type X collagen from the presumptive calcification region of lathyritic chick-embryo sterna and from organ cultures incubated in the presence of beta-aminopropionitrile (beta APN). The administration of beta-APN in vivo greatly increased the solubility of type X collagen and allowed the extraction of quantitative amounts of these molecules under non-denaturing non-proteolytic conditions. Biosynthetic studies in vitro showed that the addition of beta APN during labelling resulted in a 4-fold increase in the extractability of the newly synthesized type X collagen. Biochemical characterization of the intact type X collagen extracted from the tissues or biosynthesized in the organ cultures showed that type X collagen is composed of 59,000-Mr chains that do not undergo conversion into shorter polypeptides. Despite the marked solubilization of type X collagen upon administration of beta APN, a substantial proportion remained tissue-bound and could only be extracted by employing proteolytic digestion followed by disulphide bond reduction. These findings indicate that type X collagen in the tissues is stabilized by at least two different mechanisms, one involving beta APN-sensitive cross-links and the second through interactions with disulphide-bonded proteins. Limited proteolytic digestion with chymotrypsin of tissues containing 1.0 M-NaCl-insoluble type X collagen resulted in its complete solubilization. The majority of type X collagen molecules extracted with chymotrypsin were approx. 10% shorter than those obtained after limited pepsin digestion (Mr 40,000 versus Mr 45,000) and showed the selective loss of a single CNBr-cleavage peptide. These findings indicate the existence of chymotrypsin-sensitive sites within the triple-helical domain of the molecules.