Purification of dyneins from sperm flagella.
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.
Inaba
Kazuo
K
Shimoda Marine Research Center, University of Tsukuba, Shizuoka, Japan.
Mizuno
Katsutoshi
K
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D002938:Ciona intestinalis / Q000378:metabolism; D004398:Dyneins / Q000302:isolation & purification*; D005399:Fishes / Q000378:metabolism; D008297:Male; D012617:Sea Urchins / Q000378:metabolism; D013082:Sperm Tail / Q000737:chemistry / Q000378:metabolism*
D004398:Dyneins