Orlicky D J, Miller G J, Evans R M
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
Prostaglandins Leukot Essent Fatty Acids. 1990 Sep;41(1):51-61. doi: 10.1016/0952-3278(90)90131-4.
A bovine corpora luteal membrane glycoprotein which coelutes from multiple chromatographic procedures with bound tritiated prostaglandin F2a ([3H]PGF2 alpha) has been identified and purified to homogeneity. The properties of this molecule include: an apparent molecular mass by polyacrylamide gel electrophoresis (PAGE) of 135 kD; glycosylation which resists endoglycosidases D and H but is susceptible to cleavage by the exoglycosidase sialidase; binding of the molecule to Wheat Germ Agglutinin Sepharose but not to Concanavalin A Sepharose or Soybean Agglutinin Sepharose; migration on O'Farrell 2-D PAGE (pI 3-10) to the acidic side of the gel; binding to DEAE-Cellulose at pH 7.5 which can be displaced with NaCl at concentrations above approximately 100 mM; and, when solubilized with Triton X-100, binding to Phenyl-Sepharose or Octyl-Sepharose columns. Lastly, a rabbit polyclonal antibody against this [3H]PGF2 alpha binding protein has been made which allows both Western blotting of the 135 kD protein as well as immunohistochemical staining of ovarian tissue in a manner expected from previous binding studies. Problems associated with membrane solubilization of the receptor and receptor renaturation are discussed.
一种牛黄体膜糖蛋白已被鉴定并纯化至同质，该蛋白在多种色谱分析过程中与结合的氚标记前列腺素F2α（[3H]PGF2α）共洗脱。该分子的特性包括：聚丙烯酰胺凝胶电泳（PAGE）显示的表观分子量为135 kD；糖基化可抵抗内切糖苷酶D和H，但易被外切糖苷酶唾液酸酶切割；该分子与麦胚凝集素琼脂糖结合，但不与刀豆球蛋白A琼脂糖或大豆凝集素琼脂糖结合；在O'Farrell双向PAGE（pI 3 - 10）上向凝胶的酸性侧迁移；在pH 7.5时与DEAE - 纤维素结合，当氯化钠浓度高于约100 mM时可被取代；并且，用Triton X - 100溶解后，可与苯基琼脂糖或辛基琼脂糖柱结合。最后，制备了一种针对这种[3H]PGF2α结合蛋白的兔多克隆抗体，该抗体可用于对135 kD蛋白进行蛋白质印迹分析以及对卵巢组织进行免疫组织化学染色，染色方式与先前的结合研究预期一致。还讨论了与受体膜溶解和受体复性相关的问题。
