Ohlendieck K
Department of Pharmacology, University College Dublin, Belfield, Ireland.
Biochim Biophys Acta. 1996 Sep 4;1283(2):215-22. doi: 10.1016/0005-2736(96)00102-2.
Due to its restricted localisation to the neuromuscular junction and based on sequence homology to cytoskeletal proteins, the dystrophin-related protein utrophin is thought to be an important constituent of the membrane cytoskeleton of the postsynaptic muscle membrane and may be involved in the clustering of acetylcholine receptors at the neuromuscular junction. However, due to the low density of utrophin in microsomal muscle membranes, it is difficult to analyse the biochemical properties of the skeletal muscle isoform of utrophin. To overcome these technical difficulties, we used here immunoblot analysis of highly purified muscle surface membranes enriched even in sarcolemma markers of very low density such as ecto-5' nucleotidase and the calmodulin-sensitive Ca(2+)-ATPase. This enabled us to analyse the membrane biochemical properties of this dystrophin isoform of extremely low abundance. Since alkaline treatment released utrophin from the bilayer while it stayed associated with the insoluble pellet following detergent extraction, utrophin exhibits biochemical properties typical of a membrane cytoskeletal protein. Therefore, utrophin appears to be a specialised isoform which performs the membrane cytoskeletal function(s) of dystrophin at the postsynaptic membrane of the neuromuscular junction.
由于抗肌萎缩蛋白相关蛋白肌养蛋白局限于神经肌肉接头处，且基于其与细胞骨架蛋白的序列同源性，它被认为是突触后肌膜膜细胞骨架的重要组成部分，可能参与神经肌肉接头处乙酰胆碱受体的聚集。然而，由于肌养蛋白在微粒体肌膜中的密度较低，难以分析其骨骼肌异构体的生化特性。为克服这些技术难题，我们在此使用了对高度纯化的肌肉表面膜进行免疫印迹分析，这些膜富含低密度的肌膜标记物，如胞外5'核苷酸酶和钙调蛋白敏感的Ca(2+) -ATP酶。这使我们能够分析这种极低丰度的抗肌萎缩蛋白异构体的膜生化特性。由于碱性处理能使肌养蛋白从双层膜中释放出来，而在去污剂提取后它仍与不溶性沉淀相关联，所以肌养蛋白表现出膜细胞骨架蛋白的典型生化特性。因此，肌养蛋白似乎是一种特殊的异构体，在神经肌肉接头的突触后膜发挥抗肌萎缩蛋白的膜细胞骨架功能。
