三齿草藤(Vicia bungei Ohwi)凝集素的细胞表面受体研究

应用亲和层析法从三齿草藤(Vicia bungei Ohwi)种子中纯化的三齿草藤凝集素(VBL),可以凝集兔和豚鼠的红细胞,也可凝集人、牛和羊的精细胞,说明这些细胞表面存在有VBL的受体。用FITC和~(125)I进行标记,可得到FITC-VBL和~(125)I-VBL,其生物学活性不受影响。氯胺T法的标记率可达55％;应用FITC-VBL研究牛精细胞和兔红细胞膜上VBL受体的分布,发现二者由胞膜上受体分布据不一致。VBL与牛精细胞结合条件的正交试验表明细胞浓度的影响最大。用不同量的未标记的VBL对~(125)I-VBL与兔红细胞和人精细胞的结合实验,以Scatchard法作图,兔红细胞得一类似于双曲线的凹形曲线,提示该细胞膜上受体的性质有所不同,而人精细胞却有很大差异。若以兔红细胞膜上存在有高低亲和力两种受体进行计算,可求得结合常数和每个细胞上的受体数。应用几种单糖和外源凝集素影响~(125)I-VBL与兔红细胞的结合,当单糖(D-Man,D-Glc)浓度为0.01M时,相对结合率开始急剧下降,单糖浓度若增至0.1M时,其相对结合率仅为40％,而PHA-P和SML浓度为1mg/ml时,相对结合率开始下降,当浓度达10mg/ml时,相对结合率下降至30％左右。

Study of the Cell Surface Receptor for the Vida bungei Ohwi Lectin

From the seeds of Vida bungei Ohwi, a lectin was purified by affinity chro matography. The purified lectin is homogeneous on disc electrophoresis.It is a glycoprotein as proved by Schiff's reagent.It agglutinates erythrocytes from rabbit and guinea-pig and also agglutinates sperms from cattle, goat and man.Binding of FITC-VBL to cattle's sperms shows that most of VBL receptors on cattle's sperm exist in acrosome. The bindings of 125I-VBL to rabbit's ery-throcytes and human sperms are saturable processes .But their Scatchard plots are irregular. According to the binding assay, a binding curve and Scatchard plots were plotted.For rabbit's erythrocytes, Scatchard plots sre similar to hyperboiic curve which suggests that either there are two kinds of receptors present on thr surface or there is negative cooperative effect among VBL receptors.The number of VBL receptor sites per cell for rabbit's erythrocytes was calculated from Scatchard plots as follow: R1 = 8.47×107 Mol.per cell, R2 = 1.56×109 Mol.per cell.But irre gular Scatchard plots indicate that VBL receptors on the surface of human's sperms are very complicated.