Khayat M, Lubzens E, Tietz A, Funkenstein B
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel.
Gen Comp Endocrinol. 1994 Feb;93(2):205-13. doi: 10.1006/gcen.1994.1024.
The vitellogenic ovary of Penaeus semisulcatus contains mRNA specific for vitellin (Vt), low levels of which are found in nonvitellogenic ovaries, and is absent from testes. Immunoisolation from cell-free translation of poly(A)+ RNA using antiserum against purified Vt produced a 35-kDa polypeptide. No differences were found between Vt precipitated from the translation products of the rabbit reticulocyte system and that from the wheat germ extract. The specificity of the immunoprecipitation reaction was demonstrated by the absence of precipitation with nonimmunized rabbit serum and with antibodies prepared against purified hemocyanin or lipoprotein I (LPI), which are crustacean hemolymph proteins. In addition, competition with the radioactively labeled translation product occurred only in the presence of purified Vt, but not BSA or LPI. Vt synthesized in the translation system had a significantly lower molecular weight than that of the purified Vt or that synthesized by ovaries incubated in vitro. The possibility that this difference may be because of the non-glycosylated nature of a cell-free translation product was tested. The removal of oligosaccharides from purified Vt by enzymatic digestion with N-glycosidase F resulted in the appearance of a smaller polypeptide of 36 kDa, which reacted immunologically with Vt antiserum in a Western blot. The size of this fragment is very close to the molecular weight of the translation product.
半滑舌鳎的卵黄生成期卵巢含有卵黄蛋白（Vt）特异性mRNA，非卵黄生成期卵巢中也有少量该mRNA，而精巢中则没有。使用抗纯化Vt的抗血清从多聚腺苷酸（poly(A)+）RNA的无细胞翻译产物中进行免疫分离，得到了一条35 kDa的多肽。从兔网织红细胞系统翻译产物中沉淀的Vt与从小麦胚芽提取物中沉淀的Vt之间没有差异。通过用未免疫的兔血清以及针对纯化血蓝蛋白或脂蛋白I（LPI，甲壳动物血淋巴蛋白）制备的抗体进行沉淀实验，证明了免疫沉淀反应的特异性。此外，只有在存在纯化Vt的情况下，才会与放射性标记的翻译产物发生竞争，而牛血清白蛋白（BSA）或LPI则不会。在翻译系统中合成的Vt分子量明显低于纯化的Vt或体外培养卵巢合成的Vt。测试了这种差异可能是由于无细胞翻译产物的非糖基化性质的可能性。用N-糖苷酶F酶解去除纯化Vt中的寡糖后，出现了一条36 kDa的较小多肽，该多肽在蛋白质免疫印迹中与Vt抗血清发生免疫反应。该片段的大小与翻译产物的分子量非常接近。
