Flurkey W H
Department of Chemistry, Indiana State University, Terre Haute, Indiana 47809.
Plant Physiol. 1985 Oct;79(2):564-7. doi: 10.1104/pp.79.2.564.
Poly A(+) mRNA was isolated from Vicia faba leaves and translated in vitro using a rabbit reticulocyte translation system. From analysis of the total translation products, the major proteins synthesized in vitro were 32 kilodaltons and 20 kilodaltons. When antibodies to Vicia faba polyphenoloxidase were added, a specific immunoprecipitable protein was observed. This protein's molecular weight was shown to be similar to that of the isolated enzyme (45 kilodaltons). The isolated enzyme successfully competed with the in vitro synthesized product for antipolyphenoloxidase. In addition, the in vitro synthesized product was not immunoprecipitated with antitomato peroxidase and comigrated with isolated and/or iodinated enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis. Using in vitro translation and specific immunoprecipitation, a primary translation product corresponding to Vicia faba polyphenoloxidase was identified as a 45 kilodaltons protein.
从蚕豆叶片中分离出多聚腺苷酸（Poly A）阳性mRNA，并使用兔网织红细胞翻译系统进行体外翻译。通过对总翻译产物的分析，体外合成的主要蛋白质分子量为32千道尔顿和20千道尔顿。当加入抗蚕豆多酚氧化酶的抗体时，观察到一种特异性的可免疫沉淀蛋白。该蛋白的分子量显示与分离出的酶（45千道尔顿）相似。分离出的酶成功地与体外合成产物竞争抗多酚氧化酶。此外，体外合成产物不能被抗番茄过氧化物酶免疫沉淀，并且在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中与分离的和/或碘化的酶迁移率相同。通过体外翻译和特异性免疫沉淀，鉴定出与蚕豆多酚氧化酶相对应的初级翻译产物是一种45千道尔顿的蛋白质。
