Ohashi K, Nishimura M, Terasaki A G, Nakagawa H
Department of Biology, Faculty of Science, Chiba University.
J Biochem. 1994 Dec;116(6):1354-9. doi: 10.1093/oxfordjournals.jbchem.a124687.
We purified a 36-kDa protein from a low-salt, alkaline extract of chicken gizzard smooth muscle by sequential column chromatography using DEAE-Cellurofine A-800 m, hydroxylapatite, and CM-Cellurofine C-500. This protein decreased the low-shear viscosity of actin filaments and coprecipitated with them by centrifugation at 18,500 x g. Electron microscopy showed that the 36-kda peptide bundled actin filaments. Immunoblot analysis revealed that an affinity-purified antibody against the 36-kDa protein reacted exclusively with the 36-kDa protein band of smooth muscle. In indirect immunofluorescence microscopy, the affinity-purified anti-36-kDa protein antibody stained the dotty structures of isolated smooth muscle cells, while in post-embedding immunoelectron microscopy, most of the colloidal gold particles representing the 36-kDa protein were found on the dense bodies of ultrathin sections of chicken gizzard smooth muscle cells. The antibody did not stain the dense plaques of isolated smooth muscle cells. Judging from its molecular weight, the 36-kDa protein is concluded to be a new component of the dense bodies of smooth muscle.
我们通过使用DEAE - Cellurofine A - 800 m、羟基磷灰石和CM - Cellurofine C - 500进行连续柱层析，从鸡胗平滑肌的低盐碱性提取物中纯化出一种36 kDa的蛋白质。这种蛋白质降低了肌动蛋白丝的低剪切粘度，并通过18,500 x g离心与它们共沉淀。电子显微镜显示，36 kDa肽使肌动蛋白丝成束。免疫印迹分析表明，针对36 kDa蛋白质的亲和纯化抗体仅与平滑肌的36 kDa蛋白带发生反应。在间接免疫荧光显微镜下，亲和纯化的抗36 kDa蛋白抗体对分离的平滑肌细胞的点状结构进行了染色，而在包埋后免疫电子显微镜下，代表36 kDa蛋白质的大多数胶体金颗粒位于鸡胗平滑肌细胞超薄切片的致密体上。该抗体未对分离的平滑肌细胞的致密斑进行染色。从其分子量判断，36 kDa蛋白质被认为是平滑肌致密体的一种新成分。
