Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin).
Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin).
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DOI：
10.1083/jcb.109.3.1153
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发表时间：
1989-09
期刊：
影响因子：
--
通讯作者：
Sheetz MP
中科院分区：
文献类型：
--
作者：
Mooseker MS;Conzelman KA;Coleman TR;Heuser JE;Sheetz MP
小肠刷状缘（BB）微绒毛内的肌动蛋白束通过由BB肌球蛋白I组成的桥与膜横向相连。鸡BB肌球蛋白I，以前称为110 K-钙调蛋白，由一条表观分子量为110 kD的重链和三至四个钙调蛋白轻链分子组成。“最近的研究表明，这种复合物与肌球蛋白具有许多特性，包括机械化学活性。在这份报告中，分离和表征的膜部分，丰富的结合BB肌球蛋白I的描述。这种膜部分，被称为微绒毛膜盘，纯化的非离子洗涤剂处理的微绒毛从禽肠道BB制备的ATP提取物。超微结构分析显示，这些膜是平坦的盘状片，具有突起，其在形态上与纯化的BB肌球蛋白I相同。这些盘表现出肌动蛋白激活的Mg-ATP酶活性，并以ATP依赖的方式结合和交联肌动蛋白丝。膜盘的机械化学活性使用Nitella珠移动测定（Sheetz，M. P.的人，和J.A.。斯普迪奇1983.自然[伦敦]。303：31-35）。这些制备物被证明不受常规BB肌球蛋白的显著污染。微绒毛膜盘包被的乳胶珠以肌球蛋白样方式沿着丽藻肌动蛋白索以12-60 nm/s的速率移动（平均速率为33 nm/s）;与纯化的BB肌球蛋白I不同，在含有低Ca 2+的缓冲液中最可重复地观察到膜盘包被的珠的移动。
The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross- link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.