Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

Abstract

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti- ras monoclonal antibodies 142-24E05 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [α- 32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [α- 32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [α- 32P] GTP-binding protein in the plasma membrane fraction. When anti- ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.