Helmly R Bruce, Brown Kenneth Michael
303 Lisner Hall, Department of Biological Sciences, The George Washington University, 2023 G Street N.W., 20052, Washington DC, USA.
Rouxs Arch Dev Biol. 1987 Apr;196(4):262-267. doi: 10.1007/BF00376351.
Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na/K ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.
通过对Jacobson和Branton（1977年）以及Jacobson（1980年）的方法进行改进，从紫海胆（Strongylocentrotus purpuratus）的囊胚细胞表面膜分离出了聚阳离子珠。本研究是该程序在胚胎系统中的首次应用。胚胎细胞附着在聚赖氨酸包被的聚丙烯酰胺珠上并裂解，胚胎细胞表面膜仍附着在珠上，细胞质颗粒从膜的暴露内表面冲洗掉。细胞表面膜片从珠上解吸并通过离心收集。解离的胚胎细胞的细胞表面膜分别约8%和5%在珠上和膜沉淀中回收。细胞表面膜部分中[H]伴刀豆球蛋白A结合以及细胞表面标记酶碱性磷酸酶和钠钾ATP酶的比活性分别比胚胎细胞匀浆高16倍、19倍和32倍。从电子显微镜照片和酶分析判断，膜相对不含细胞质污染物。细胞质标记酶细胞色素c氧化酶、过氧化氢酶、酸性磷酸酶、NADP和NADPH - 细胞色素c还原酶以及乙酰胆碱酯酶在膜部分中的活性明显低于匀浆水平。整个过程可在4小时内完成。由于这种细胞表面膜分离技术仅依赖于带负电荷的细胞粘附到带正电荷表面的趋势，它比大多数其他方法更不可能表现出物种和发育阶段特异性，并且应该在研究胚胎阶段特异性膜成分的发育作用中证明是有用的。
