Singh G, Katyal S L, Gottron S A
Biochim Biophys Acta. 1985 Jun 10;829(2):156-63. doi: 10.1016/0167-4838(85)90184-0.
In an earlier publication we had reported the preparation of a rabbit antiserum specific for rat Clara cell secretory proteins. This rabbit anti-rat Clara cell serum was found to react with two proteins in rat lung lavage by crossed-immunoelectrophoresis. Immunoblotting of rat lung lavage proteins, after sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis, disclosed three bands of reactivity with anti-Clara cell serum. The relative molecular masses of these three proteins were about 200 (protein A) 55 (protein B) and about 12 kDa (protein C). Anti-Clara cell antibodies eluted from Sepharose-4B-linked protein C (as well as the antiserum raised by immunizing rabbits with protein C) reacted with proteins A and C. Anti-Clara cell antiserum unbound to proteins A and C (as well as antiserum raised by immunizing rabbits with protein B) reacted with protein B only. In non-SDS polyacrylamide gel electrophoresis, protein B migrated as a single band, slightly cathodic to albumin; protein C resolved into three bands, all anodic to albumin. Immunoblots of isoelectric focusing gels showed three bands (pI 5.2-5.7) that reacted with antibody to protein C, and four bands corresponding to protein B were seen in the pI range 4.6-5.0. As determined by immunoperoxidase staining of paraformaldehyde fixed methacrylate embedded 1 micron thick sections of rat lung, protein(s) A (and protein C) and protein B were present in the same cells and in the same granules. Protein B was resistant to trypsin digestion, whereas proteins A and C were readily degraded by trypsin. Rat Clara cell secretory proteins consist of at least two antigenic types that appear to be functionally distinct, and each antigenic type displays charge microheterogeneity.
在较早的一篇出版物中，我们报道了一种针对大鼠克拉拉细胞分泌蛋白的兔抗血清的制备。通过交叉免疫电泳发现，这种兔抗大鼠克拉拉细胞血清能与大鼠肺灌洗中的两种蛋白质发生反应。在十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳后，对大鼠肺灌洗蛋白进行免疫印迹分析，发现与抗克拉拉细胞血清有三条反应带。这三种蛋白质的相对分子质量分别约为200（蛋白质A）、55（蛋白质B）和约12 kDa（蛋白质C）。从与琼脂糖-4B连接的蛋白质C上洗脱的抗克拉拉细胞抗体（以及用蛋白质C免疫兔子产生的抗血清）与蛋白质A和C发生反应。未与蛋白质A和C结合的抗克拉拉细胞抗血清（以及用蛋白质B免疫兔子产生的抗血清）仅与蛋白质B发生反应。在非SDS聚丙烯酰胺凝胶电泳中，蛋白质B迁移为单一一条带，略向阴极迁移至白蛋白位置；蛋白质C分离为三条带，均向阳极迁移至白蛋白位置。等电聚焦凝胶的免疫印迹显示有三条带（pI 5.2 - 5.7）与蛋白质C的抗体发生反应，在pI范围4.6 - 5.0内可见对应蛋白质B的四条带。通过对用多聚甲醛固定、甲基丙烯酸酯包埋的1微米厚大鼠肺切片进行免疫过氧化物酶染色确定，蛋白质A（和蛋白质C）以及蛋白质B存在于同一细胞和同一颗粒中。蛋白质B对胰蛋白酶消化有抗性而蛋白质A和C很容易被胰蛋白酶降解。大鼠克拉拉细胞分泌蛋白至少由两种功能上似乎不同的抗原类型组成，并且每种抗原类型都表现出电荷微不均一性。
