Postgraduate School of Biological Sciences, University of Bradford, BD7 1DP, Bradford, Yorkshire, UK.
Planta. 1977 Jan;134(3):267-72. doi: 10.1007/BF00384192.
Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [(35)S]methionine and the kinetics of (35)S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [(35)S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [(35)S]antigens and a concomitant increase in glyoxysomal content.
通过蔗糖密度梯度离心从蓖麻(Ricinus communis L.)胚乳中分离出乙醛酸体,将其分为基质蛋白和膜成分。用兔抗血清针对基质蛋白和十二烷基硫酸钠(SDS)溶解的膜蛋白进行了免疫。SDS-聚丙烯酰胺凝胶电泳(PAGE)分析证实,这种抗血清沉淀了各自的乙醛酸体混合抗原制剂中存在的所有主要多肽成分。此外,当用适当的乙醛酸体抗血清挑战从微粒体囊泡或溶解的微粒体膜中回收的可溶性成分时,再次发现存在血清学决定簇。完整的胚乳组织用[(35)S]甲硫氨酸孵育,并在与乙醛酸体基质部分或从微粒体中释放的可溶性部分孵育时,用抗乙醛酸体基质血清进行免疫沉淀,追踪(35)S 掺入回收的蛋白质的动力学。[(35)S]抗原迅速出现在微粒体部分,而在出现在乙醛酸体之前存在滞后期。通过添加过量未标记的甲硫氨酸中断这种动力学实验,导致微粒体中[(35)S]抗原的含量迅速下降,同时乙醛酸体中的含量增加。