Specific detection of the precursor of ras p21 with a mouse monoclonal anti-C-terminal peptide antibody, SARA-K1

Abstract

In an attempt to clarify the post-translational modifications of ras oncogene product p21, we have established a mouse monoclonal antibody specific for the precursor of p21. The C-terminal peptide (156–188) of K(4A)- ras oncogene product p21 (p21 K(4A)), termed K(4A)-peptide, was used as the immunogen. In Western blotting, monoclonal antibodies were examined for their differential reactivity between two types of p21 K(4A) expressed in Escherichia coli (esh-p21 K(4A)) and mammalian cell (mam-p21 K(4A)). One monoclonal antibody, designated SARA-K1, reacted selectively with esh-p21 k(4a). The epitope for SARA-K1 was defined on tryptic peptide (177–184), containing Cys 180, of the K(4A)-peptide. Pulse-chase experiments of mam-p21 K(4A) synthesis at 24 °C revealed that SARA-K1 precipitated a 21 kDa protein within a 7 min chase but not after a 10 min chase, indicating that SARA-K1 recognizes the precursor of mam-p21 k(4a). Furthermore, in Triton X-114 partitioning experiments using mammalian cells pre-treated with Mevalotin, 3-hydroxy-3-methylglutaryl cocnzyme A (HMG CoA) reductase inhibitor, SARA-K1 precipitated [ 35S]methionine-labeled, [ 3H]mevalonic acid-unlabeled mam-p21 K(4A) in the aqueous phase, but did not precipitate [ 3H]mevalonic acid-labeled mam-p21 K(4A) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically recognizes the primary translational product pro-p21 K(4A).