Beef Pancreas tryptophanyl-tRNA Synthetase Research Articles

Gamma Interferon Potently Induces Tryptophanyl-tRNA Synthetase Expression in Human Keratinocytes

Incubation of human keratinocytes with gamma interferon (gamma-IFN) induces the synthesis of a 53-kDa protein of unknown nature and function. We report the identification of this protein through amino acid microsequencing. The NH2-terminal amino acid sequence of the 53-kDa antigen demonstrated that this protein was tryptophanyl-tRNA synthetase (Frolova et al, Gene 109:291-296, 1991, Genbank accession number 61715). This result was validated by the sequencing of tryptic peptides. Identification of the 53-kDa gamma-IFN-induced protein was confirmed by immunoblotting with an antiserum directed against beef pancreas tryptophanyl-tRNA synthetase. Northern blot analysis using a synthetic oligonucleotidic 32P-labeled probe evidenced a 3.1-kb transcript in gamma-IFN-treated cells indicating that the gene was regulated at the pre-translational level. These data show that gamma-IFN potently induces in keratinocytes the expression of an enzyme directly involved in protein biosynthesis. Elevated levels of tryptophanyl-tRNA synthetase in treated cultured keratinocytes might be involved in the cell-growth-inhibitory activity of gamma interferon.

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction.

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.

Negative cooperativity in adenylate formation catalysed by beef pancreas tryptophanyl-tRNA synthetase: Influence of tRNA Trp

FEBS LettersVolume 137, Issue 1 p. 95-99 Full-length articleFree Access Negative cooperativity in adenylate formation catalysed by beef pancreas tryptophanyl-tRNA synthetase Influence of tRNATrp Sergey Kh. Degtyarev, Sergey Kh. Degtyarev Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this authorSegrey F. Beresten, Segrey F. Beresten Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this authorAlexey Yu. Denisov, Alexey Yu. Denisov Institute of Organic Chemistry, Siberian Branch of the USSR Academy of Sciences, Novosibirsk 630 090, USSRSearch for more papers by this authorOl'ga I. Lavrik, Ol'ga I. Lavrik Institute of Organic Chemistry, Siberian Branch of the USSR Academy of Sciences, Novosibirsk 630 090, USSRSearch for more papers by this authorLev L. Kisselev, Lev L. Kisselev Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this author Sergey Kh. Degtyarev, Sergey Kh. Degtyarev Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this authorSegrey F. Beresten, Segrey F. Beresten Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this authorAlexey Yu. Denisov, Alexey Yu. Denisov Institute of Organic Chemistry, Siberian Branch of the USSR Academy of Sciences, Novosibirsk 630 090, USSRSearch for more papers by this authorOl'ga I. Lavrik, Ol'ga I. Lavrik Institute of Organic Chemistry, Siberian Branch of the USSR Academy of Sciences, Novosibirsk 630 090, USSRSearch for more papers by this authorLev L. Kisselev, Lev L. Kisselev Institute of Molecular Biology, USSR Academy of Sciences, Moscow USSRSearch for more papers by this author First published: January 11, 1982 https://doi.org/10.1016/0014-5793(82)80322-0Citations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume137, Issue1January 11, 1982Pages 95-99 ReferencesRelatedInformation

18] Tryptophanyl-tRNA synthetase from beef pancreas

This chapter describes the isolation of tryptophanyl-tRNA synthetase [L-tryptophan: tRNA Trp ligase (AMP-forming), EC 6.1.1.2] from beef pancreas. The purification procedure comprises of five steps and yields about 130 mg of the enzyme per kilogram of tissue. The yield of the enzyme is considerably higher than that reported by others. Various properties and inhibitors of the given enzyme are described as well. From the mentioned data on beef pancreas tryptophanyl-tRNA synthetase it is concluded that this enzyme is characterized enzymologically rather intensively. Development of primary and three-dimensional structure investigations in the nearest future is expected because of the convenient and rapid procedure for enzyme purification. The availability of the specific inhibitors of different chemical nature makes it possible to evaluate various functional groups involved in the formation of active center. The mechanism of enzymic action needs to be investigated by means of fast kinetic methods.

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.

Evidence for essential histidine residues in tryptophanyl-tRNA synthetase.

Beef pancreas tryptophanyl‐tRNA synthetase (Mr 60000 × 2) reacts with diethyl pyrocarbonate at pH 6.0. A differential spectrum of modified versus native enzyme as well as the restoration of the initial spectrum after NH2OH treatment of the modified enzyme prove the formation of carbethoxy histidine residues. In 8 M urea, 22 histidine residues are carbethoxylated by diethyl pyrocarbonate in accord with the total histidine content of the enzyme. In the native enzyme only 4–5 histidine residues react at equal rate whereas the others are resistant to modification. The circular dichroism spectra of the native and modified protein are similar. The modified enzyme does not dissociate into subunits.After modification of accessible histidine residues the enzymatic activity sharply decreases both in the reaction of ATP‐[32P]pyrophosphate exchange and that of tRNA aminoacylation. The modified enzyme is almost completely reactivated after treatment with NH2OH. The rate of the enzyme inactivation in the aminoacylation reaction approaches quite closely that of carbethoxylation of the histidine residues; a complete loss of the ability of tryptophanyl‐tRNA synthetase to catalyze tryptophanyl‐tRNA synthesis is brought about by the modification of 2–3 histidine residues per dimer. The modified enzyme losses almost completely its ability to bind tryptophanyl‐tRNA or uncharged tRNA. A conclusion is made that the accessible histidine residues are involved directly or indirectly in the tRNA binding. The ATP‐[32P]pyrophosphate exchange is less affected with modification of the enzyme by diethyl pyrocarbonate than the tRNA acylation reaction. All substrates protect the tryptophanyl‐tRNA synthetase against inactivation with diethyl pyrocarbonate; the most effective protectors are tryptophan and tryptophan plus ATP‐Mg2+ for the exchange reaction. Modified peptide(s) was isolated by diagonal electrophoresis.

Beef pancreas tryptophanyl-tRNA synthetase. Order of substrate binding in ATP-( 32P)-pyrophosphate exchange reaction

Summary The order of substrate binding to tryptophanyl-tRNA synthetase purified from beef pancreas was studied. Kinetics of 32 P PP i -ATP isotope exchange reaction catalyzed by the enzyme were measured in a concentration range of each substrate and also in the presence of various concentrations of a substrate analog, tryptamine. The set of data obtained was treated by means of so called «sequential planning of experiment which allows one to extract fully the information from experimental kinetic data. Three possible orders of substrate binding were considered, namely: (1) ATP first, tryptophan second — (2) tryptophan first, ATP second — (3) random order. Among these sequences the probability of the first one (ATP first, tryptophan second) was found to be close to 100 p. cent whereas two thers were close to zero probability. The high degree of discrimination between various mechanisms was achieved only in the case where all the kinetic data obtained, i.e. both in the presence and absence of the inhibitor, were taken into account in the treatment of the set of experimental points. The discriminatory ability of the method developed is insufficient if the experimental points obtained in the presence of inhibitor are neglected. The procedure developed for the treatment of kinetic data is versatile and can be used for the determination of the sequence of substrate binding for any purified aminoacyl-tRNA synthetase. Any other hypothetical mechanism for the isotope exchange reaction catalyzed by the same enzyme could be treated in a similar manner. The established order of substrate binding should not be extrapolated a priori to other synthetases and to the same enzyme in the presence of tRNA.

Purification of mammalian tryptophan tRNA Fluorescence properties of the free and the enzymatically acylated tryptophan tRNA

Highly purified rabbit liver tryptophan tRNA (tRNA Trp) has been obtained by a slight modification of the Maxwell's procedure using beef pancreas tryptophanyl-tRNA synthetase as acylating enzyme. The accepting activity of this tRNA has been found to be ⩾ 1300 pmoles per A 260 nm unit ★ ★ A 260 nm unit refers to absorbance per ml as measured in a 1-cm light path at 260 nm. Fluorescence spectra performed on the purified tRNA Trp revealed a specific response at 400 nm; the wavelength of maximum excitation was determined at 300 nm. At the characteristic emission fluorescence wavelength of Trp, it has been shown that the chromophore Trp is almost completelyquenched when bound to the tRNA Trp.

Beef pancreas tryptophanyl-tRNA synthetase. Molecular weight, composition and spectral properties.

The molecular weight of beef pancreatic tryptophanyl‐tRNA synthetase has been determined by Sephadex filtration, by equilibrium sedimentation according to Yphantis and to Van Holde and Baldwin, and by light scattering. These different techniques indicate an average molecular weight of 108000. The amino acid composition has been determined after total acid hydrolysis of the performic acid oxidized and of the carboxymethylated reduced protein. Spectrophotometric measurements, according to Edelhoch, have been used for tryptophan determination. The cysteine and cystine content have been measured before and after reduction by sodium borohydride in urea. From this study it is concluded that tryptophanyl‐tRNA synthetase contains no disulfide bridge. Its molecular extinction coefficient at 280 nm is 9 × 104. The optical rotatory dispersion of the enzyme indicates that it contains about a third of its polypeptide chain in helical conformation.