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 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 (p21K(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 p21K(4A) expressed in Escherichia coli (esh-p21K(4A)) and mammalian cell (mam-p21K(4A)). One monoclonal antibody, designated SARA-K1, reacted selectively with esh-p21K(4A). The epitope for SARA-K1 was defined on tryptic peptide (177-184), containing Cys180, of the K(4A)-peptide. Pulse-chase experiments of mam-p21K(4A) synthesis at 24 degrees 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-p21K(4A). Furthermore, in Triton X-114 partitioning experiments using mammalian cells pre-treated with Mevalotin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor, SARA-K1 precipitated [35S]methionine-labeled, [3H]mevalonic acid-unlabeled mam-p21K(4A) in the aqueous phase, but did not precipitate [3H]mevalonic acid-labeled mam-p21K(4A) in either aqueous or detergent phase. The data presented clearly show that the SARA-K1 specifically recognizes the primary translational product pro-p21K(4A).
Ishihara
H
H
Laboratory for Cell Biology, Hoechst Japan Ltd., Kawagoe.
Kimura
M
M
Kuzumaki
N
N
Ono
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011498:Protein Precursors / Q000737:chemistry / Q000276:immunology*; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D016283:Proto-Oncogene Proteins p21(ras)