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A series of small peptides corresponding to the amino termini of the fibrin α- and β-chains has been synthesized. The peptides glycyl-L-prolyl-L-arginyl-L-proline and glycyl-L-prolyl-L-arginylsarcosine are potent inhibitors of fibrin polymerization. Moreover, these peptides have a natural stability stemming from their inherent resistance to proteolysis because of the involvement of amino acids in each of their peptide bonds. The peptide glycyl-L-prolyl-L-arginyl-L-proline binds to fibrinogen and to fragment D, in both cases with an association constant of approximately 5 × 10 4 ; it does not bind to fragment E. The number of binding sites is two for fibrinogen and one for fragment D. The tripeptide glycyl-L-prolyl-L-arginine binds less tightly and is less than half as effective in preventing polymerization. The peptide glycyl-L-histidyl-L-arginyl-L-proline, which corresponds exactly to the amino terminus of the fibrin β-chain, does not inhibit the aggregation of fibrin monomers under the conditions used. It does bind weakly to fibrinogen, however, suggesting the involvement of sites other than those binding the α-chain analogues. Various other peptides were found not to inhibit polymerization; these included glycine-L-proline, L-prolyl-L-arginine and glycyl-L-prolyl-L-seryl-L-proline. The last-named corresponds to the serine/arginine amino acid replacement previously reported for a defective human fibrinogen.

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Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-l cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per ,ug of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-rat), with a peak activity of 2,400 foci per ,ug of DNA when 305 amino acids were deleted. Deletions of >383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extent of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.

Antibodies specific for the amino-and carboxy-terminal portions of simian virus 40 large tumor (I) antigen were obtained by immunization of rabbits with synthetic peptides corresponding to these regions. The amino-terminal synthetic peptide has the sequence Ac-Met-Asp-Lys-Val-Leu-AsnArg-(Tyr). The tyrosine residue was introduced in order to couple the peptide to bovine serum albumin with bis-diazotized benzidine. The carboxy-terminal peptide has the sequence LysPro-Pro-Thr-Pro-Pro-Pro-Glu-ProGlu-Thr. It was coupled to bovine serum albumin with glutaraldehyde. The antisera against both peptides reacted with large T antigen. The specificity of the immune reaction was demonstrated by inhibition experiments using excess synthetic peptides. Furthermore, fragments of T antigen encoded by the nondefective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4, which contain the carboxy terminus and lack the amino terminus of large T antigen, were precipitated only with antiserum to the carboxy-terminal peptide. Small T antigen was not precipitated with either serum, suggesting that the amino terminus of small T antigen has a conformation different from that of large T antigen or that it is sterically hindered by a host protein.The procedures used here are of general importance for identification and characterization of gene products.The early region of the simian virus 40 (SV40) genome encodes two structurally related proteins, large tumor (T) antigen of apparent molecular weight 95,000 (1-5) and small T antigen of apparent molecular weight 17,000 (4). Large T antigen is encoded by two discontinuous sections from 0.65 to 0.59 and from 0.54 to 0.17 map units on the genome; small T antigen maps between 0.65 and 0.54 units (6, 7). Thus, both proteins share a common amino-terminal sequence (8-10), but they have unique carboxy-terminal regions. Both antigens have been identified and characterized with antisera from animals bearing SV4O-induced tumors. These antisera are not specific in that they contain antibodies against both antigens. Antisera specific for either large or small T antigen have not been described. They would be useful, however, for analyzing the cellular localization of the antigens and for studies of structure-function relationships. One approach for obtaining such specific antibodies might be Kohler and Milstein's technique for the production of monoclonal antibodies by hybridomas (11). An alternative approach is to synthesize peptides corresponding to specific regions of large or small T antigen and to raise antibodies against these peptides. In principle, the latter approach became possible as a result of the recent determination of the entire DNA sequence of the SV40 genome (12, 13), from which the amino acid sequence of the antigens can be inferred. In this paper we describe the preparation of antibodies specific for the ends of large T antigen, with synthetic peptides as antigens.MATERIALS AND METHODS TC7 cells were used for infection with large-plaque SV40 and HeLa S3 cells for infe...

Phosphorylation of the carboxyl terminus of pp60C'T, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60111 inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized-peptides corresponding to the carboxyl-terminal 13 residues of pp60'w phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp6OC`src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide innobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp6OvSrc, the transforming protein of Rous sarcoma virus. Only traces of pp60v7 and pp6Oc 527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60'1 bound much less efficiently to the phosphorylated peptide than did pp60`8)c(FS27). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60cSrc(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp6OC4re peptide markedly inhibited phosphorylation of pp604rC(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60C`rc binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.

The synthesis of both transferrin receptor (TfR) and ferritin is regulated post-transcriptionally by iron. This is mediated by iron responsive elements (IREs) in the 5'- and 3'-untranslated regions, respectively, of TfR and ferritin mRNAs. Although these IREs have different sequences, they both form a characteristic stem-loop. We used competition assays and partial peptide mapping of UV-crosslinked ferritin and TfR IRE-protein complexes to show that the cytosolic protein binding to the ferritin 5'-IRE, the iron-responsive element binding protein (IRE-BP), also binds to TfR 3'-IREs. To identify the structural requirements necessary for RNA-protein binding, ferritin IRE RNAs were synthesized which contained altered secondary structures and base substitutions. Affinities of these RNAs for IRE-BP were assayed in RNA-protein binding gels. Substitutions disrupting base-pairing of the stem prevented IRE-BP binding. Substitutions which restored base-pairing also restored IRE-BP binding. We conclude that the IRE-BP binds to both ferritin and TfR IREs and recognizes a particular IRE conformation.

The affinity of the amino terminal tetrapeptide of the beta chain of fibrin, Gly-His-Arg-Pro, for fibrinogen dramatically increases in the presence of 2 millimolar calcium ion. In contrast, there is no significant increase in the affinity of peptides beginning with the amino terminal sequence of the fibrin alpha chain, Gly-Pro-Arg, in the presence of calcium ions, although the number of binding sites increases. In the latter case, the increased number of sites is due to the alpha chain analogs binding to the site ordinarily occupied by the beta chain analogs. These results indicate that structures at the amino terminus of the fibrin beta chain play a more important role in fibrin polymerization when calcium ions are present.