Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin
Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin
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微绒毛 110K-钙调蛋白：核苷酸对分离细胞骨架的影响以及纯化复合物与 F-肌动蛋白的相互作用
DOI：
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发表时间：
1985
影响因子：
7.8
通讯作者：
A. Bretscher
中科院分区：
文献类型：
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作者：
K. Verner;A. Bretscher
从肠上皮细胞分离的微绒毛含有与钙调蛋白（110 K-CM）复合的细胞骨架Mr 110，000多肽，据信其将微丝核心束横向连接到质膜。以前的工作表明，生理水平的ATP可以部分溶解分离的微绒毛细胞骨架或分离的微绒毛的110 K-CM复合物。然而，一旦被提取，110 K-CM复合物已被发现难以在水性缓冲液中保持稳定可溶。这是由于微绒毛细胞骨架制剂中存在内源性ATP酶（37 ℃时约100 nmol Pi/min/mg），其消耗ATP，随后沉淀110 K-CM。向此类沉淀物中添加ATP可使110 K-CM重新溶解。在来自细胞骨架或膜结合刷状缘的110 K-CM的溶解中包含ATP再生系统增加了溶解的110 K-CM的量。发现110 K-CM从微绒毛细胞骨架中溶解需要二价阳离子（Mg 2+、Mn 2+或Co 2+，但不需要Zn 2+）和核苷三磷酸（ATP、GTP、CTP或ITP）。ADP不增溶110 K-CM，但可部分抑制ATP依赖的增溶作用。溶解的110 K在用[γ-32 P]ATP提取微绒毛核心的过程中被磷酸化，但这与110 K-CM的溶解无关，因为内源性激酶对ATP是特异性的，而溶解不是。使用在ATP再生系统存在下的刷状缘细胞骨架的ATP提取、溶解的提取物的凝胶过滤、ATP耗尽步骤以特异性地用F-肌动蛋白沉淀110 K-CM、以及再溶解，随后通过磷酸纤维素层析来纯化110 K-CM。纯化的复合物在ATP存在和不存在下均稳定地溶于水性缓冲液中。它结合几乎定量的F-肌动蛋白在ATP的情况下，并显示从F-肌动蛋白的核苷酸溶解特性类似于从微绒毛核心的110 K-CM的溶解。在低ATP水平下，ADP的存在增加了与F-肌动蛋白的结合.这些结果表明，纯化的复合物已经以天然形式分离。这些数据证实并扩展了Howe和Mooseker的研究（1983，J. Cell Biol.，97：974-985），并进一步强调110 K-CM是稳定的水溶性复合物而不是完整的膜蛋白。
Microvilli isolated from intestinal epithelial cells contain a cytoskeletal Mr 110,000 polypeptide complexed with calmodulin (110K-CM) that is believed to link the microfilament core bundle laterally to the plasma membrane. Previous work has shown that physiological levels of ATP can partially solubilize the 110K-CM complex from isolated microvillus cytoskeletons or isolated microvilli. However, once extracted, the 110K-CM complex has been found to be difficult to maintain stably soluble in aqueous buffers. This is due to the presence of an endogenous ATPase (approximately 100 nmol Pi/min per mg at 37 degrees C) in microvillus cytoskeletal preparations that depletes the ATP with subsequent precipitation of 110K-CM. Addition of ATP to such precipitates resolubilizes 110K-CM. Inclusion of an ATP regenerating system in the solubilization of 110K-CM from cytoskeletons, or membrane- bound brush borders, increases the amount of 110K-CM solubilized. Solubilization of 110K-CM from microvillus cytoskeletons was found to require a divalent cation (Mg2+, Mn2+, or Co2+, but not Zn2+) and a nucleoside triphosphate (ATP, GTP, CTP, or ITP). ADP did not solubilize 110K-CM, but could partially inhibit ATP-dependent solubilization. Solubilized 110K was phosphorylated during extraction of microvillus cores with [gamma-32P]ATP, but this was unrelated to the solubilization of 110K-CM as the endogenous kinase was specific for ATP, whereas the solubilization was not. The 110K-CM was purified using ATP extraction of brush border cytoskeletons in the presence of an ATP regenerating system, gel filtration of the solubilized extract, an ATP depletion step to specifically precipitate 110K-CM with F-actin, and resolubilization followed by phosphocellulose chromatography. The purified complex was stably soluble in aqueous buffers both in the presence and absence of ATP. It bound almost quantitatively to F-actin in the absence of ATP, and showed nucleotide solubilization characteristics from F-actin similar to that found for solubilization of 110K-CM from microvillus cores. At low ATP levels, the binding to F- actin was increased in the presence of ADP. These results suggest that the purified complex has been isolated in a native form. The data confirm and extend the studies of Howe and Mooseker (1983, J. Cell Biol., 97:974-985) using a partially purified preparation of 110K-CM and further emphasize that 110K-CM is a stably water soluble complex and not an integral membrane protein.