The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase.
The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase.
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发表时间：
1984-11
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通讯作者：
J. H. Collins;C. W. Borysenko
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作者：
J. H. Collins;C. W. Borysenko
鸡肠刷状边缘微绒毛中存在一种110 kda的蛋白，被认为是将形成微绒毛结构核心的肌动蛋白丝束与微绒毛质膜横向连接起来。我们用含有0.6 M KCl和5 mM ATP的溶液提取刷状边缘，然后用凝胶过滤层析、与外源性肌动蛋白络合物沉淀和羟基磷灰石层析纯化了一个110 kda的蛋白，其均匀性大于95%。110 kda蛋白-钙调蛋白复合物在没有ATP存在的情况下结合f -肌动蛋白，具有K+、edta -ATP酶（0.2 mumol/min/mg）、Ca2+-ATP酶（0.2 mumol/min/mg）活性和未被f -肌动蛋白激活的Mg2+-ATP酶活性（0.03 mumol/min/mg）。该复合物的肌动蛋白结合活性和atp酶活性与纯化的刷缘肌球蛋白相似。然而，免疫印迹分析显示110-kDa蛋白与纯化鸡刷边肌球蛋白多克隆抗体无反应性。此外，用凝乳胰蛋白酶和金黄色葡萄球菌V8蛋白酶进行有限的蛋白水解获得的110-kDa蛋白和肌球蛋白的肽图中，即使有，也几乎没有共同的肽。免疫印迹分析也表明，在制备条件下，肌球蛋白重链是稳定的。
A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.