Lindsay L L, Hedrick J L
Department of Biochemistry and Biophysics, University of California, Davis 95616.
J Exp Zool. 1988 Mar;245(3):286-93. doi: 10.1002/jez.1402450309.
Interacting egg envelope and sperm surface components were identified for Xenopus laevis using blotting methods. Sperm were extracted with sodium dodecyl sulfate (SDS), the extracted proteins separated by gel electrophoresis and blotted, and the blots treated with 125I-labeled heat solubilized envelopes. The converse experiment was also performed where envelope components were separated by gel electrophoresis, blotted, and the blots treated with 125I-labeled sperm components. Blotted sperm components with apparent molecular weights of 14K, 19K, 25K, and 35K selectively bound the solubilized envelopes. All of the envelope binding components were found to be localized on the sperm surface by radioiodinating intact sperm using Iodo-Gen. The blotted egg envelope component with an apparent molecular weight of 37K selectively bound to solubilized sperm components, and this binding was due to the protein moiety of the glycoprotein. 125I-labeled heat solubilized envelopes from unfertilized and fertilized eggs showed the same pattern of binding to blotted sperm components. Selected sulfated carbohydrates (fucoidan, dextran sulfate, and heparin, but not chondroitin sulfate) inhibited fertilization and binding of 125I-labeled heat solubilized envelopes to blotted sperm extract. Thus, the binding of heat solubilized envelopes to electrophoretically separated and blotted sperm proteins may reflect cellular interactions.
利用印迹法鉴定了非洲爪蟾相互作用的卵膜和精子表面成分。用十二烷基硫酸钠（SDS）提取精子，提取的蛋白质经凝胶电泳分离并印迹，印迹用125I标记的热溶解卵膜处理。还进行了相反的实验，即将卵膜成分经凝胶电泳分离、印迹，印迹用125I标记的精子成分处理。表观分子量为14K、19K、25K和35K的印迹精子成分选择性地结合溶解的卵膜。通过使用碘甘醚对完整精子进行放射性碘化发现，所有卵膜结合成分都定位于精子表面。表观分子量为37K的印迹卵膜成分选择性地结合溶解的精子成分，这种结合归因于糖蛋白的蛋白质部分。来自未受精卵和受精卵的125I标记的热溶解卵膜显示出与印迹精子成分相同的结合模式。选定的硫酸化碳水化合物（岩藻依聚糖、硫酸葡聚糖和肝素，但不包括硫酸软骨素）抑制受精以及125I标记的热溶解卵膜与印迹精子提取物的结合。因此，热溶解卵膜与经电泳分离和印迹的精子蛋白质的结合可能反映了细胞间的相互作用。
