Crouch E C, Moxley M A, Longmore W J
Department of Pathology, Jewish Hospital, St. Louis, MO 63110.
Exp Lung Res. 1988;14(5):705-24. doi: 10.3109/01902148809087838.
We have examined the matrix deposition and proteolytic processing of newly synthesized interstitial and basement membrane collagens in the isolated perfused adult rat lung. Isolated, perfused, and ventilated lungs were labeled for up to 4 h with radiolabeled proline. Collagens were partially purified from homogenates by salt fractionation and ion exchange chromatography and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The major collagenous species were identified as types I, III, and IV collagen by peptide mapping and indirect immunoprecipitation assays. Whereas extraction with neutral salts recovered radiolabeled types I and III collagen, extraction of the neutral salt residue with 2 M guanidine-HCl preferentially recovered types III and IV collagen. Reextraction of the guanidine-HCl residue in the presence of dithiothreitol selectively recovered type IV procollagen (PC) and covalently cross-linked aggregates of type IV chains. In pulse-chase experiments we observed extensive conversion of type I PC to collagen during a 4-h chase. Although type III PC was efficiently converted to p-collagen, only small amounts of fully processed chains were identified. Type IV PC did not undergo detectable proteolytic processing. The isolated perfused rat lung should prove useful for further studies of lung collagen metabolism.
我们研究了成年大鼠离体灌注肺中新合成的间质胶原和基底膜胶原的基质沉积及蛋白水解过程。将离体、灌注和通气的肺用放射性标记的脯氨酸标记长达4小时。通过盐分级分离和离子交换色谱从匀浆中部分纯化胶原，并用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）进行检测。通过肽图谱分析和间接免疫沉淀试验将主要的胶原种类鉴定为I型、III型和IV型胶原。用中性盐提取可回收放射性标记的I型和III型胶原，而用2M盐酸胍提取中性盐残渣则优先回收III型和IV型胶原。在二硫苏糖醇存在下对盐酸胍残渣进行再提取可选择性回收IV型前胶原（PC）和IV型链的共价交联聚集体。在脉冲追踪实验中，我们观察到在4小时的追踪过程中I型PC大量转化为胶原。虽然III型PC有效地转化为原胶原，但仅鉴定出少量完全加工的链。IV型PC未发生可检测到的蛋白水解过程。离体灌注的大鼠肺应可证明对进一步研究肺胶原代谢有用。
