Plant Biotechnology Institute, National Research Council of Canada, S7N 0W9, Saskatoon, Sask., Canada.
Planta. 1985 Mar;163(3):317-27. doi: 10.1007/BF00395141.
Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant "clathrin", had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.
从悬浮培养的大豆（ Glycine max （L.） Merr.）细胞原生质体制备得到富含被膜小泡的级分。通过在垂直转子中用线性蔗糖梯度对原生质体匀浆进行等密度离心，初步进行了富集。从该梯度中收集被膜小泡级分，并以速率区带离心通过第二个线性蔗糖梯度，以去除较大的污染膜小泡。用十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳测定，被膜小泡级分中最主要的多肽是植物“网格蛋白”，相对分子质量约为 190kDa。其他富集的多肽包括 105、100、96、64、50、38 和 32kDa 处的条带。该方法与利用蔗糖分步梯度从大豆原生质体制备被膜小泡的方法进行了比较。还测试了等密度-速率区带离心程序用于制备牛脑被膜小泡的效果。
