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Monoclonal antibodies referred to as Aml, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared.Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp-tKNA synthetase activity; epitopes recognized by them are mapped close to one another and reside a t the dispensable part of the Trp-tRNA synthetase molecule. Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis.A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichiu coli. The purified enzyme interacted with Aml, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaroytic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E. coli.From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensalbe part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNATrp charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archdebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.Tryptophanyl-tRNA synthetase from beef pancreas belongs to well characterized mammalian aminoacyl-tRN A synthetases (see reviews [I -31). Trp-tRNA synthetase is composed of two identical subunits, each with a molecular mass of 60 kDa [I] and functions by the 'flip-flop' mechanism[2]. The enzyme contains a Z n Z + ion required for maintaining its structure and activity [4]. Trp-tRNA synthetase is suitable for studying the mechanism of enzyme catalysis because it can form covalent derivatives with its ligands, i. e. tryptophan [S], pyrophosphate [6, 71 and AMP [8]. The properties of TrptRNA synthetase as an antigen were also analyzed [9]; at least nine antigenic determinants were revealed for a population of polyclonal antibodies.Recent evidence indicates that monoclonal antibodies (mAbs) allow one to combine effectively molecular and cellular aspects in the investigation of the same proteins. The present work is concerned with the derivation of three C'orrcspondmw to L. L. Kisselev, Institut Molekulyarnoj Biologii, Akademiya Nauk S.S.S.R. Vavilova ulitsa 32, Moskva, USSR 1 17984Abbreviation. mAb, monoclonal antibody. Enzxmes. Tryptophanyl-1RNA synthetase (EC 6.1.1.2); trypsin (EC 3.4.21.4); clastase (EC 3.4.21.11). hybridoma cell lines producing mAbs against Trp-tRNA synthetase (Aml, Am2 and Am3), which exert a different effect on the enyzme activity and interact with different elements in the Trp-tRNA synthetase structu...

The content of Trp-tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a ' 2sI-labelled monoclonal antibody A m l , specifically interacting with any eukaryotic Trp-tRNA synthetase. Its content in the organs studied, with the exccption of bovine and sheep pancreas, was found to be 0.002-0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations werc found for sheep. The Trp-tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Rurninantia and of the other mammalians.As shown by indirect iminunofluorescence technique, bovine Trp-tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp-tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals.Aminoacyl-tRNA synthetases play a key role in protein synthcsis by catalysing the highly specific formation of aminoacyl-tRNAs [l, 21. Apart from the aminoacylation reaction some synthetases catalyse the synthesis of a dinucleotide, adenosine( 5')-tetraphospho(5')adenosine [3], which plays an unknown role in cellular metabolism. Different organs and tissues are known to vary in their level of aminoacyl-tRNA synthetase activities [4], which may be due to the intensity of protein synthesis and the amino acid composition of proteins being synthesized [5], as well as to the hypothetical regulatory functions of these enzymes. The extremely high level of TrptRNA synthetase activity was revealed in bovine pancreatic tissue long ago [6].The application of immunochemical techniques allows one to directly measure the protein content, which does not always coincide with the enzyme activity level. The content of TrptRNA synthetase was assayed in different bovine organs using polyclonal antibodies against the bovine enzyme [7, 81. A very high Trp-tRNA synthetase content in the pancreas was revealed: 2-3% of the amount of soluble proteins. This content is two orders of magnitude higher than the enzyme level in several other bovine organs such as liver, kidney and heart [8], which suggests that, besides tRNA aminoacylation, TrpCorrcspondtnce to L. L. Kisselev, Institut Molekularnoj Biologii, Akadeniiya Nauk S.S.S.R., Vavilova ulitsa 32, Moskva, USSR 1 17984Abbreviations. RIPA buffer, 20 mM Tris/HCI. pH 7.5, 1 mM EDTA, IYu Nonidet P40, 1 % sodium deoxycholate, 0.1% SDS, 0.25 M NaCI.Enzyme. Tryptophanyl-tRNA synthetase (EC 6.1.1.2).tRNA synthetase of the bovine pancreas fulfills some additional, yet unknown, function(s). The goal of the present work was to establish wheth...