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.
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.
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来自胚胎鸡胸骨软骨的 X 型胶原天然组织形式的生化特征，并鉴定其三螺旋结构域内的胰凝乳蛋白酶敏感位点。
DOI：
10.1042/bj2730333
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Jimenez,SA
中科院分区：
文献类型：
--
作者：
Reginato,AM;Jimenez,SA
我们分离和表征的完整的原生组织形式的X型胶原蛋白从推定的钙化区域的lavitic鸡胚胸骨和器官培养物中孵育的β-氨基丙腈（β APN）的存在。体内施用β-APN大大增加了X型胶原的溶解度，并允许在非变性非蛋白水解条件下提取定量的这些分子。体外生物合成研究表明，在标记过程中添加β APN导致新合成的X型胶原蛋白的可提取性增加4倍。从组织中提取或在器官培养物中生物合成的完整X型胶原的生化表征表明，X型胶原由59，000-Mr链组成，其不经历转化为较短的多肽。尽管在施用β APN后X型胶原显著增溶，但相当大的比例仍然与组织结合，并且只能通过采用蛋白水解消化然后进行二硫键还原来提取。这些发现表明，组织中的X型胶原蛋白至少通过两种不同的机制来稳定，一种涉及β APN敏感性交联，另一种通过与二硫键结合的蛋白质相互作用。用糜蛋白酶对含有1.0 M-NaCl不溶性X型胶原的组织进行有限的蛋白水解消化，导致其完全溶解。用胰凝乳蛋白酶提取的大多数X型胶原分子约为。10%短于有限的胃蛋白酶消化后获得的那些（Mr 40，000对Mr 45，000），并显示出单一的CNBr-裂解肽的选择性损失。这些研究结果表明存在的三螺旋结构域的分子内的胰凝乳蛋白酶敏感的网站。
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.