SPECIFIC DETECTION OF THE PRECURSOR OF RAS P21 WITH A MOUSE MONOCLONAL ANTI-C-TERMINAL PEPTIDE ANTIBODY, SARA-K1

In an attempt to clarify the post-translational modifications of ras o ncogene product p21, we have established a mouse monoclonal antibody s pecific 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 wer e examined for their differential reactivity between two types of p21( K(4A)) expressed in Escherichia coli (esh-p21(K(4A))) and mammalian ce ll (mam-p21(K(4A))). One monoclonal antibody, designated SARA-K1, reac ted selectively with esh-p21(K(4A)). The epitope for SARA-K1 was defin ed on tryptic peptide (177-184), containing Cys(180), of the K(4A)-pep tide. Pulse-chase experiments of mam-p21(K(4A)) synthesis at 24 degree s 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 recognize s the precursor of mam-p21(K(4A)). Furthermore, in Triton X-114 partit ioning experiments using mammalian cells pre-treated with Mevalotin, 3 -hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, SA RA-K1 precipitated [S-35]methionine-labeled, [H-3]mevalonic acid-unlab eled mam-p21(K(4A)) in the aqueous phase, but did not precipitate [H-3 ]mevalonic acid-labeled mam-p21(K(4A)) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically r ecognizes the primary translational product pro-p21(K(4A)).