Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody.
Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody.
复制标题
原位细胞内禽类 X 型胶原并使用构象依赖性单克隆抗体测定其热稳定性。
DOI：
10.1016/0014-4827(86)90504-5
复制
发表时间：
1986
影响因子：
3.7
通讯作者：
Schmid,TM
中科院分区：
文献类型：
--
作者：
Linsenmayer,TF;Gibney,E;Schmid,TM
在鸡胚脊椎软骨的肥大区域内原位检测细胞内和基质相关的X型胶原的螺旋结构域的热稳定性。为此，我们采用了在逐渐升高的温度下反应的未固定组织切片的间接免疫荧光组织化学（Linsenmayer等人，J cell biol 99（1984）1405）[18]与构象依赖性单克隆抗体（XCR-AC 9）（Schmid & Linsenmayer，J cell biol 100（1985）598）[19]。最近启动X '型合成的肥大软骨细胞并没有立即分泌它，而是将其保留在细胞内的细胞质细胞器中。这使得细胞内的X射线型清晰可见。在细胞内的X-型胶原库中，抗体识别的表位在高达55° C时是稳定的，但在60° C时被破坏。这比当分子在中性溶液中时表位的热稳定性高5-10° C（如通过竞争ELISA测定的）。基质相关的X型胶原至少在65-67.5° C下稳定。我们的结论是，在原位的胶原蛋白螺旋在其正常的细胞内环境的稳定性是相当大的比可能从测量的分子在溶液中预测。
The thermal stability of the helical domain of intracellular and matrix-associated type X̄ collagen was examined in situ within the hypertrophic region of embryonic chick vertebral cartilages. For this we employed indirect immunofluorescence histochemistry of unfixed tissue sections reacted at progressively higher temperatures (Linsenmayer et al., J cell biol 99 (1984) 1405)[18] with a conformation-dependent monoclonal antibody (X ̄-AC 9)(Schmid & Linsenmayer, J cell biol 100 (1985) 598)[19]. The hypertrophic chondrocytes which had most recently initiated synthesis of type X̄ did not immediately secrete it, but instead retained it intracellularly within cytoplasmic organelles. This allowed for clear visualization of the intracellular type X̄. Within the pool of intracellular type X̄ collagen, the epitope recognized by the antibody was stable up to 55° C, but was destroyed at 60° C. This is 5–10° C higher than the thermal stability of the epitope when the molecule is in neutral solution (as determined by competition ELISA). The matrix-associated type X̄ collagen is stable at least to 65–67.5° C. We conclude that in situ the stability of the collagen helix in its normal intracellular environment is considerably greater than might be predicted from measurements made on molecules in solution.