Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis
Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis
复制标题
胚胎肌腱成纤维细胞的进一步表征以及使用免疫铁蛋白技术研究胶原蛋白生物合成
作者：
B. Olsen;R. Berg;Y. Kishida;D. Prockop
形态学研究是对鸡胚肌腱成纤维细胞进行的，这些细胞已被用于最近的胶原生物合成研究。细胞内质网相对丰富，含有发育良好的高尔基复合体，由小囊泡、叠膜和大液泡组成。然后设计了制备细胞片段的技术，这些细胞片段被铁蛋白抗体偶联物穿透，但保留了细胞的基本形态特征。最后，新的程序被用来进一步了解胶原蛋白是如何合成的。据其他地方报道，铁蛋白标记抗体的初步研究表明，在新分离的成纤维细胞的内质网中发现了脯氨酰羟化酶，内质网池和大高尔基空泡中都发现了前胶原。在这里描述的实验中，对细胞进行操作，使氨基酸继续融入多肽链，但分子的组装没有完成，因为脯氨酸和赖氨酸残基的羟基化被阻止了。结果表明，这些操作对脯氨酸羟化酶的分布没有影响。对与原胶原（原胶原的未羟基化形式）反应的铁蛋白偶联抗体细胞的检查表明，在脯氨酸和赖氨酸羟化酶被抑制期间，原胶原被保留在内质网的池中。用免疫学技术测定脯氨酸羟化酶表明，尽管该酶存在于内质网中，但它不与前胶原蛋白一起分泌。这些观察结果进一步证明了脯氨酸羟化酶在控制胶原生物合成中的特殊作用。
Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin- labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.