Manjunath C K, Goings G E, Page E
Am J Physiol. 1984 Jun;246(6 Pt 2):H865-75. doi: 10.1152/ajpheart.1984.246.6.H865.
Gap junctions were purified from rat hearts in the presence of absence of proteolysis inhibitors and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy of thin sections. In absence of proteolysis inhibitors or in presence of ethylenediaminetetraacetic acid or leupeptin, gap junctions contained a single major protein band at relative molecular weight (Mr) 29,500 and minor bands at Mr 44,000-47,000, 17,750, and 16,500 and showed smooth cytoplasmic surfaces in electron micrographs. SDS-PAGE of junctions prepared with phenylmethylsulfonylfluoride (PMSF) showed markedly decreased intensity of the Mr 29,500 band and increased intensity of bands at Mr 44,000, 45,500, and 47,000; electron microscopy of these gap junctions showed presence of a fuzzy layer on their cytoplasmic surfaces. Urea (8 M) could not remove this fuzzy layer. In electron micrographs of rat ventricular myocytes, cytoplasmic surfaces of gap junctions were fuzzy. We conclude that rat heart gap junction protein consists of an intramembrane component (Mr 29,500) that extends into the "gap" and a cytoplasmic surface component (Mr 14,500-17,500) that corresponds to the fuzzy layer and is hydrolyzable by a serine protease.
在有或没有蛋白酶解抑制剂的情况下，从大鼠心脏中纯化间隙连接，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和超薄切片电子显微镜进行检查。在没有蛋白酶解抑制剂的情况下，或在存在乙二胺四乙酸或亮抑酶肽的情况下，间隙连接含有一条相对分子量（Mr）为29,500的主要蛋白带以及Mr为44,000 - 47,000、17,750和16,500的次要蛋白带，并且在电子显微镜照片中显示出光滑的细胞质表面。用苯甲基磺酰氟（PMSF）制备的连接的SDS-PAGE显示Mr为29,500的条带强度明显降低，而Mr为44,000、45,500和47,000的条带强度增加；这些间隙连接的电子显微镜检查显示其细胞质表面存在模糊层。尿素（8M）无法去除该模糊层。在大鼠心室肌细胞的电子显微镜照片中，间隙连接的细胞质表面是模糊的。我们得出结论，大鼠心脏间隙连接蛋白由延伸到“间隙”中的膜内成分（Mr 29,500）和对应于模糊层且可被丝氨酸蛋白酶水解的细胞质表面成分（Mr 14,500 - 17,500）组成。
