Vater C A, Jackson R C
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Dev Biol. 1989 Sep;135(1):111-23. doi: 10.1016/0012-1606(89)90162-0.
A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.
通过对来自紫海胆（Strongylocentrotus purpuratus）卵的纯化皮质分泌囊泡进行蔗糖密度梯度分级分离，制备了一种膜级分。纯化的皮质囊泡膜级分的磷脂与蛋白质之比为1.76，并且如通过电子显微镜观察到的那样，呈现出生物膜典型的形态。通过SDS-聚丙烯酰胺凝胶电泳分析纯化膜的蛋白质组成，结果表明其与卵、细胞表面复合物、皮质囊泡、受精产物和卵黄小板的蛋白质组成不同。外周膜蛋白的碱性提取（pH 11.0）使磷脂与蛋白质之比增加到2.55，并去除了几种多肽。对分离的皮质囊泡膜级分的免疫印迹分析显示，两种主要的皮质囊泡内容物蛋白的污染水平较低。还分离了富含卵质膜和卵黄小板膜的级分，并通过SDS-聚丙烯酰胺凝胶电泳与皮质囊泡膜进行比较。发现这三种膜级分的蛋白质组成几乎没有重叠，这表明皮质囊泡膜制剂相对没有来自这些可能的非皮质囊泡膜源的污染。通过免疫印迹发现质膜和皮质囊泡膜样品均含有肌动蛋白。
