Kondor-Koch C, Bravo R, Fuller S D, Cutler D, Garoff H
Cell. 1985 Nov;43(1):297-306. doi: 10.1016/0092-8674(85)90035-2.
Secretion of a foreign protein--chicken oviduct lysozyme--and of endogenous proteins was studied in the polarized epithelial Madin-Darby Canine Kidney (MDCK) cell line. Cell clones that secrete enzymatically active chicken lysozyme were generated by transforming the cells with lysozyme cDNA inserted in a SV40-pBR322 recombinant vector and a dominant selectable marker gene. The kinetics and polarity of lysozyme secretion from one transformed cell clone were studied using cell monolayers grown on nitrocellulose filters. Lysozyme was secreted into the apical and the basolateral medium, demonstrating the existence of direct transport pathways to each cell surface. Control experiments excluded the effects of monolayer leakiness, reabsorption, transepithelial transport, and depolarization. In contrast, the secretion of a set of endogenous proteins of MW 30-40 kd was found to be strictly apical showing that polarized secretion also occurs in this cell line. The latter group of proteins appear to be generated from larger precursor molecules by intracellular cleavage.
在极化上皮性犬肾(MDCK)细胞系中研究了外源蛋白——鸡输卵管溶菌酶以及内源性蛋白的分泌情况。通过用插入SV40-pBR322重组载体和显性选择标记基因的溶菌酶cDNA转化细胞,生成了分泌具有酶活性的鸡溶菌酶的细胞克隆。使用在硝酸纤维素滤膜上生长的细胞单层,研究了一个转化细胞克隆中溶菌酶分泌的动力学和极性。溶菌酶分泌到顶端和基底外侧培养基中,表明存在通向每个细胞表面的直接运输途径。对照实验排除了单层渗漏、重吸收、跨上皮运输和去极化的影响。相比之下,发现一组分子量为30 - 40kd的内源性蛋白的分泌严格位于顶端,表明该细胞系中也发生极化分泌。后一组蛋白似乎是由较大的前体分子通过细胞内切割产生的。