Olsen B R, Berg R A, Kishida Y, Prockop D J
J Cell Biol. 1975 Feb;64(2):340-55. doi: 10.1083/jcb.64.2.340.
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.
对鸡胚肌腱成纤维细胞进行了形态学研究，这些细胞已用于近期多项关于胶原蛋白生物合成的研究。这些细胞内质网相对丰富，含有由小泡、堆叠膜和大液泡组成的发达高尔基体。然后设计了制备细胞碎片的技术，这些碎片可被铁蛋白 - 抗体结合物穿透，但保留了细胞的基本形态特征。最后，采用这些新方法进一步了解胶原蛋白的合成方式。如其他地方所报道，用铁蛋白标记抗体进行的初步研究表明，在新鲜分离的成纤维细胞内质网中发现了脯氨酰羟化酶，并且在内质网池和大的高尔基体液泡中都发现了前胶原。在此处描述的实验中，对细胞进行处理，使氨基酸继续掺入多肽链，但由于脯氨酰和赖氨酰残基的羟化被阻止，分子组装未完成。结果表明，这些操作不会改变脯氨酰羟化酶的分布。用与原胶原（前胶原的未羟化形式）反应的抗体偶联的铁蛋白检查细胞，结果表明在脯氨酰和赖氨酰羟化酶受抑制期间，原胶原保留在内质网池中。用免疫技术测定脯氨酰羟化酶表明，虽然该酶存在于内质网内，但它不会与前胶原一起分泌。这些观察结果为脯氨酰羟化酶在胶原蛋白生物合成控制中的特殊作用提供了进一步的证据。
