Zelenka P, Reszelbach R, Piatigorsky J
Biochim Biophys Acta. 1979 Oct 5;556(3):447-56. doi: 10.1016/0005-2736(79)90132-9.
Urea-washed membranes from embryonic chick lenses (15 days old) and from the cortical and nuclear regions of adult chicken lenses (1 year) have been prepared by repeated centrifugation through discontinuous density gradients. The protein components of the isolated membranes have been examined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and urea. Proteins with molecular weights of 75 000, 56 000, 54 000, 48 000, 34 000, 32 000, 25 000, and 22 000 were present in all the membrane preparations, although their proportions changed during development. One additional protein, molecular weight 70 000, was seen only in the embryonic lens membranes. The greatest developmental change was the increase in 25 000 molecular weight protein from 12% in the embryonic lens to about 45% in the adult lens. Since it has been suggested that this protein is associated with gap junctions, its increase during development may reflect a corresponding increase in the number of gap junctions in the lens. The 50 000 molecular weight protein of embryonic lens membranes and membranes of adult nuclear lens fibers consisted at least partly of delta-crystallin, since delta-crystallin peptides could be identified in tryptic peptide maps of the isolated protein after in vitro radioiodination. Peptide maps of the 50 000 molecular weight protein of cortical lens fiber membranes contained no identifiable delta-crystallin peptides, although it is possible that modified delta-crystallin peptides may be present. The level of cytoplasmic contamination of the membrane fraction was estimated by preparing lens membranes in the presence of added delta-[35S]crystallin. The results indicated that cytoplasmic contamination contributes significantly to the presence of delta-crystallin in lens membrane preparations.
已通过在不连续密度梯度中反复离心，制备了来自15日龄胚胎鸡晶状体以及1岁成年鸡晶状体皮质和核区域的尿素洗涤膜。已通过在含有十二烷基硫酸钠和尿素的聚丙烯酰胺凝胶中进行电泳，对分离出的膜的蛋白质成分进行了检测。分子量为75000、56000、54000、48000、34000、32000、25000和22000的蛋白质存在于所有膜制剂中，尽管它们的比例在发育过程中发生了变化。另外一种分子量为70000的蛋白质仅在胚胎晶状体膜中可见。最大的发育变化是分子量为25000的蛋白质从胚胎晶状体中的12%增加到成年晶状体中的约45%。由于有人提出这种蛋白质与间隙连接有关，其在发育过程中的增加可能反映了晶状体中间隙连接数量的相应增加。胚胎晶状体膜和成年核晶状体纤维膜中分子量为50000的蛋白质至少部分由δ-晶状体蛋白组成，因为在体外放射性碘化后，从分离出的蛋白质的胰蛋白酶肽图谱中可以鉴定出δ-晶状体蛋白肽段。皮质晶状体纤维膜中分子量为50000的蛋白质的肽图谱中没有可识别的δ-晶状体蛋白肽段，尽管可能存在修饰的δ-晶状体蛋白肽段。通过在添加δ-[35S]晶状体蛋白的情况下制备晶状体膜，估计了膜部分的细胞质污染水平。结果表明，细胞质污染对晶状体膜制剂中δ-晶状体蛋白的存在有显著贡献。
