Fujimoto H, Mabuchi I
Department of Biology, Graduate School of Arts and Sciences, The University of Tokyo.
J Biochem. 1997 Sep;122(3):518-24. doi: 10.1093/oxfordjournals.jbchem.a021783.
We have developed a method for the isolation of cleavage furrows from dividing sea urchin eggs, which is applicable to various sea urchin species. The new method differs from that used for isolating cleavage furrows from sand dollar Clypeaster japonicus eggs [Yonemura, S., Mabuchi, I., and Tsukita, S. (1991) J. Cell Sci. 100, 73-84] in the type and concentration of detergent included in the isolation medium, the temperature during the treatment of dividing eggs with the isolation medium, and the centrifugation conditions. The contractile ring was included in the isolated cleavage furrows, as seen on rhodamine-phalloidin staining of actin filaments. When the furrows were isolated with the isolation medium containing both NaF and beta-glycerophosphate, which are potent protein phosphatase inhibitors, the isolated furrows were found to be accompanied by the mitotic apparatus. When the isolation was carried out in the absence of both NaF and beta-glycerophosphate, cleavage furrows without the mitotic apparatus were obtained. The development of a method of isolation of cleavage furrows from regular sea urchin eggs enabled us to compare protein constituents among furrows from different sea urchin and sand dollar species. We found that 32, 36, and 51 kDa proteins were concentrated in common in the cleavage furrows isolated from eggs of the sand dollars, C. japonicus and Scaphechinus mirabilis, and the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus nudus, on two-dimensional gel electrophoreses.
我们已经开发出一种从分裂的海胆卵中分离分裂沟的方法，该方法适用于各种海胆物种。这种新方法与用于从日本饼海胆（Clypeaster japonicus）卵中分离分裂沟的方法[米村，S.，马渊，I.，和筑田，S.（1991年）《细胞科学杂志》100卷，73 - 84页]在分离培养基中所含去污剂的类型和浓度、用分离培养基处理分裂卵时的温度以及离心条件方面有所不同。如在肌动蛋白丝的罗丹明 - 鬼笔环肽染色中所见，收缩环包含在分离出的分裂沟中。当用同时含有氟化钠（NaF）和β - 甘油磷酸钠（β - glycerophosphate）这两种强效蛋白磷酸酶抑制剂的分离培养基分离沟时，发现分离出的沟伴随着有丝分裂器。当在没有NaF和β - 甘油磷酸钠的情况下进行分离时，可获得没有有丝分裂器的分裂沟。从普通海胆卵中分离分裂沟方法的开发使我们能够比较不同海胆和饼海胆物种的沟之间的蛋白质成分。我们发现在二维凝胶电泳中，32 kDa、36 kDa和51 kDa的蛋白质在从日本饼海胆、奇异刻肋海胆（Scaphechinus mirabilis）的卵以及海胆、光棘球海胆（Hemicentrotus pulcherrimus）和紫球海胆（Strongylocentrotus nudus）分离出的分裂沟中共同富集。
