从大鼠星形胶质细胞中快速、温和地提取、重构和鉴定微丝和神经胶质丝。

Rapid and gentle extraction, reconstitution and characterization of microfilament and glia filament from rat astrocytes.

作者信息

Ito J, Masuda M, Tada T, Tanaka R

机构信息

Department of Biochemistry, Nagoya City University Medical School, Japan.

出版信息

Neurochem Int. 1995 Jan;26(1):91-7. doi: 10.1016/0197-0186(94)00084-8.

DOI:10.1016/0197-0186(94)00084-8

PMID:7787766

Abstract

We developed gentle and rapid methods for depolymerization and extraction of both microfilament and glia filament separately from a crude cytoskeletal fraction of rat astrocytes. Electron microscopy revealed that the filament reconstituted from the microfilament extract closely resembled F-actin that was formed from G-actin of rabbit skeletal muscle. It was found by immunoblotting analysis that even the reconstituted microfilament-like filaments, which had been purified by affinity chromatography with heavy meromyosin subfragment 1 (S1)-conjugated Sepharose, contained vimentin and glia fibrillary acidic protein (GFAP) besides actin, inferring the interaction between microfilament and glia filament. The filaments (9-10 nm thick) reconstituted from the glia filament extract were composed of actin and other minor components in addition to vimentin and GFAP. Actin, GFAP, 101, 34, 32.5, 30.5, 29.5 and 28 kDa proteins found in the reconstituted glia filament-like filaments were suggested to be glia filament-associated proteins.

摘要

我们开发了温和且快速的方法，可分别从大鼠星形胶质细胞的粗细胞骨架组分中解聚并提取微丝和胶质丝。电子显微镜显示，从微丝提取物中重构的丝与由兔骨骼肌的G-肌动蛋白形成的F-肌动蛋白非常相似。通过免疫印迹分析发现，即使是通过与重酶解肌球蛋白亚片段1（S1）偶联的琼脂糖进行亲和层析纯化的重构微丝样丝，除肌动蛋白外还含有波形蛋白和胶质纤维酸性蛋白（GFAP），这暗示了微丝与胶质丝之间的相互作用。从胶质丝提取物中重构的丝（9-10纳米厚）除波形蛋白和GFAP外，还由肌动蛋白和其他次要成分组成。在重构的胶质丝样丝中发现的肌动蛋白、GFAP、101、34、32.5、30.5、29.5和28 kDa蛋白被认为是胶质丝相关蛋白。