从鸡胚中分离和鉴定合成胶原蛋白的多核糖体。

Isolation and characterization of collagen-synthesizing polysomes from chick embryos.

作者信息

Wang L, Andrade H F, Silva S M, Simões C L, D'Abronzo F H, Brentani R

出版信息

Prep Biochem. 1975;5(1):45-57. doi: 10.1080/00327487508061561.

Abstract

Collagen-synthesizing polysomes were isolated by low-speed centrifugation of the post-mitochondrial supernatant of chick homogenates. Electron microscopy of the fraction thus isolated shows it to be exclusively composed of ribosomes. Amino acid incorporation in vitro showed that these particles were efficient in the incorporation of proline, but not tryptophan, as opposed to ribosomes obtained from the supernatant of the low-speed centrifugation. The incorporation process was highly dependent on GTP, and exibited an optimal Mg2+concentration of 5.6mM. The reaction was inhibited by RNase, elongation inhibitors as anysomycin, sparsomycin, fusidic acid and GDPCP. It was also moderately inhibited by initiation inhibitors such as aurintricarboxilic acid and pyrocatechol violet. The product of the incorporation was characterized as collagen by its sensitivity towards purified collagenase, lack of tryptophan, chromatography in CM-cellulose and molecular sieve chromatography in Sephadex G-200.

摘要

通过对鸡匀浆线粒体后上清液进行低速离心，分离出了合成胶原蛋白的多核糖体。对如此分离得到的组分进行电子显微镜观察，结果显示其仅由核糖体组成。体外氨基酸掺入实验表明，与从低速离心上清液中获得的核糖体不同，这些颗粒能有效地掺入脯氨酸，但不能掺入色氨酸。掺入过程高度依赖鸟苷三磷酸（GTP），并且表现出最佳镁离子（Mg2+）浓度为5.6毫摩尔。该反应受到核糖核酸酶（RNase）、伸长抑制剂如茴香霉素、稀疏霉素、夫西地酸和GDP-胞苷二磷酸（GDPCP）的抑制。它也受到起始抑制剂如金精三羧酸和邻苯二酚紫的适度抑制。通过其对纯化胶原酶的敏感性、缺乏色氨酸、在羧甲基纤维素（CM-纤维素）上的色谱分析以及在葡聚糖凝胶G-200上的分子筛色谱分析，将掺入产物鉴定为胶原蛋白。