Inaba Kazuo, Mizuno Katsutoshi
Shimoda Marine Research Center, University of Tsukuba, Shizuoka, Japan.
Methods Cell Biol. 2009;92:49-63. doi: 10.1016/S0091-679X(08)92004-5. Epub 2009 Nov 21.
Metazoan spermatozoa, especially those from marine invertebrates and fish, are excellent sources for isolating axonemal dyneins because of their cellular homogeneity and the large amounts that can be collected. Sperm flagella can be easily isolated by homogenization and subsequent centrifugation. Axonemes are obtained by demembranation of flagella with the nonionic detergent Triton X-100. The outer arm dyneins have been most widely studied because they are specifically extracted by a high-salt solution and can be isolated as a relatively pure fraction of ~20S two-headed dynein by sucrose density gradient centrifugation. Only a few reports have described the isolation of inner arm dyneins from sperm and the protocol has room for improvement. Sperm show clear changes in motility at fertilization, which are exerted through the regulation of axonemal dyneins by protein phosphorylation and Ca(2+) binding. Therefore dyneins from sperm flagella are an excellent biochemically tractable source for studying the regulation of axonemal dyneins. Here we describe protocols used for purification of flagellar dyneins from sperm of tunicates, sea urchins, and fish. The techniques described here could be applied to other species with appropriate modifications.
后生动物的精子，尤其是来自海洋无脊椎动物和鱼类的精子，因其细胞同质性和可收集的大量样本，是分离轴丝动力蛋白的极佳来源。精子鞭毛可通过匀浆和随后的离心轻松分离。轴丝是通过用非离子去污剂Triton X - 100去除鞭毛的膜而获得的。外臂动力蛋白得到了最广泛的研究，因为它们可被高盐溶液特异性提取，并且通过蔗糖密度梯度离心可作为相对纯的约20S双头动力蛋白组分分离出来。仅有少数报告描述了从精子中分离内臂动力蛋白，且该方案还有改进空间。精子在受精时运动性会发生明显变化，并通过蛋白磷酸化和Ca(2+)结合对轴丝动力蛋白的调节来实现。因此，来自精子鞭毛的动力蛋白是研究轴丝动力蛋白调节的一种极佳的易于生化处理的来源。在这里，我们描述了用于从被囊动物、海胆和鱼类精子中纯化鞭毛动力蛋白的方案。这里描述的技术经过适当修改后可应用于其他物种。
