Antisera specific for rap 1 proteins distinguish between processed and nonprocessed rap 1b.
Polyclonal antisera were generated against synthetic peptides corresponding to distinct regions of the rap 1 protein sequences. A "rap 1-common" antiserum, prepared against an 18-amino acid segment of the rap 1a protein near the proposed GTP-binding region, reacted with both rap 1a and rap 1b recombinant proteins expressed in Escherichia coli and with two low molecular weight GTP-binding proteins of 22 and 24 kDa in unstimulated human platelets. An antiserum raised against a carboxyl-terminal peptide of rap 1b containing the putative site of post-translational processing reacted strongly with bacterial-expressed recombinant rap 1b and with a 24-kDa GTP-binding protein in platelets, but not with recombinant rap 1a or a 22-kDa GTP-binding protein. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this rap 1b immunoreactive protein coincided with that of bacterial-expressed rap 1b and not with the faster migrating form of rap 1b that incorporates radioactivity from [3H]mevalonic acid in the insect/baculovirus system. This suggests that our rap 1b-specific antiserum recognizes only one form of rap 1b, that which has not undergone carboxyl-terminal post-translational processing.
Winegar
D A
DA
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Ohmstede
C A
CA
Chu
L
L
Reep
B
B
Lapetina
E G
EG
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D016367:Baculoviridae; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000145:classification / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011499:Protein Processing, Post-Translational; D016283:Proto-Oncogene Proteins p21(ras) / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020668:rap GTP-Binding Proteins
D010455:Peptides; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins