Wolosin J M, Okamoto C, Forte T M, Forte J G
Biochim Biophys Acta. 1983 Dec 13;761(2):171-82. doi: 10.1016/0304-4165(83)90226-x.
A quantitative assessment of the distribution and state of microfilament-related proteins in the heterocellular fundic gastric epithelium was carried out. Actin content, as determined by the DNAase inhibition assay, ranged from 29 to 42 micrograms/mg of tissue protein, depending upon the tissue source. About 60% of the total actin existed in fresh tissue in the polymeric form (F-actin). The distribution of fluorescent-labelled phallicidin demonstrated that F-actin was concentrated predominantly in the acid-secreting oxyntic cells. The patterns of distribution corresponded to the location of the numerous elongated apical surface microvilli seen within oxyntic cell canaliculi. In the isolated apical membrane, actin represented about 10% of the total protein and was present entirely as F-actin. After mild treatment of membranes with Triton X-100, filaments could be readily visualized by negative staining. More extensive Triton X-100 extraction solubilized intrinsic membrane protein and yielded an insoluble residue highly enriched in actin and containing several additional polypeptides. Homogenization and fractionation of the gastric epithelium in low ionic strength media led to the depolymerization of a significant proportion of the tissue actin which was recovered in the homogenate supernatant. When purified by DNAase affinity chromatography, this gastric actin displayed structural and functional properties similar to muscle actin. Incubation of the homogenate supernatant in KCl-Mg2+ induced the formation of actin-rich gels. The gels contained myosin as well as several other peptides that may be actin-binding proteins.
对异细胞胃底上皮中微丝相关蛋白的分布和状态进行了定量评估。根据DNA酶抑制试验测定，肌动蛋白含量范围为每毫克组织蛋白29至42微克，具体取决于组织来源。新鲜组织中约60%的总肌动蛋白以聚合形式（F-肌动蛋白）存在。荧光标记的鬼笔环肽分布表明，F-肌动蛋白主要集中在分泌酸的壁细胞中。其分布模式与在壁细胞小管内看到的众多细长顶端表面微绒毛的位置相对应。在分离的顶端膜中，肌动蛋白占总蛋白的约10%，并且完全以F-肌动蛋白形式存在。用Triton X-100对膜进行温和处理后，通过负染色可以很容易地观察到细丝。更广泛的Triton X-100提取可溶解内在膜蛋白，并产生高度富含肌动蛋白且含有几种其他多肽的不溶性残渣。在低离子强度介质中对胃上皮进行匀浆和分级分离导致相当一部分组织肌动蛋白解聚，这些解聚的肌动蛋白在匀浆上清液中回收。当通过DNA酶亲和层析纯化时，这种胃肌动蛋白显示出与肌肉肌动蛋白相似的结构和功能特性。在KCl-Mg2+中孵育匀浆上清液会诱导富含肌动蛋白的凝胶形成。这些凝胶含有肌球蛋白以及其他几种可能是肌动蛋白结合蛋白的肽。
