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Among clostripain hydrolysate peptides of beef pancreas tryptophanyl-tRNA synthetase the peptide Ile-Ser-Phe-Pro-Ala-Ile-Asn-Gln-Phe-Ala-Ala-Pro-Ser-Gln-Phe-Ser-Ile-Arg was revealed which contains the continuous antigenic determinant for monoclonal antibody Am1. This antibody specifically cross-reacts with tryptophanyl-tRNA synthetases of procaryotes, eucaryotes and archebacteriae. The synthetic peptide with identical amino acid sequence plus N-terminal Arg residue (S-peptide), being immobilized on enzyme immunoassay (EIA) microtitration plate, also binds with Am1. Am1 affinity constant (M-1) measured by non-competitive EIA was (3.0 +/- 0.3).10(7) for S peptide and (1.4 +/- 0.3).10(9) for the native enzyme. The sequence of immunoreactive peptide adopts with high probability the secondary structure including beta-turn(s) and antiparallel beta-sheet composed of inverted repeats. At the same time, the analysis of circular dichroism spectrum (in the far UV) of the peptide dissolved in water comes closest to 16% beta-turn and only 8% beta-sheet. The binding of Am1 with peptide was not observed in aqueous solution.

A procedure for detecting protein-protein interactions is proposed. It is based on blotting of electrophoretically separated protein mixtures followed by detection of the proteins interacting with a given 125I-labelled protein used as a probe. Application of this approach to lysates of cultured mammalian cells enabled us to reveal a unique 37 kDa polypeptide interacting with 125I-labelled bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2).

A method for localization of antigenic determinants in a polypeptide chain of unknown primary structure was proposed. A protein is modified at NH2-terminal and epsilon-NH2-groups of lysine residues with maleic anhydride and then is subjected to partial enzymatic cleavage. Newly formed NH2-terminal groups are tagged with radioiodinated Bolton--Hunter's reagent. The labeled fragments of the antigen are then demaleylated. Comparison of the two longest labeled fragments, only one of which still binds monoclonal antibody, makes it possible to define the location of the antigenic determinant along the polypeptide chain. The method was tested on the bovine tryptophanyl-tRNA synthetase using earlier prepared monoclonal antibodies against this enzyme.