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 (betaAPN). 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 betaAPN 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 59000-M(r) chains that do not undergo conversion into shorter polypeptides. Despite the marked solubilization of type X collagen upon administration of betaAPN, 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 betaAPN-sensitive cross-links and the second through interactions with disulphidebonded 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 (M(r) 40 000 versus M(r) 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.