Nuclear genome sequence of the plastid-lacking cryptomonad Goniomonas avonlea provides insights into the evolution of secondary plastids.

BACKGROUND: The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea-the first for any goniomonad-to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily. RESULTS: We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~ 92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida. CONCLUSION: We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic "rewiring" that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.

Role of immune microenvironment in gastrointestinal stromal tumours.

AIMS: The immune microenvironment is a prognostic factor for various malignancies. The significance of key players of this immune microenvironment, including tumour-infiltrating lymphocytes (TILs) and expression of programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase (WARS) in gastrointestinal stromal tumours (GISTs) is largely unknown. METHODS AND RESULTS: Tissue microarrays were constructed from pathology files, 1996-2016. Immunohistochemistry for PD-L1, IDO and WARS was correlated with tumour size, mitoses and outcomes. TILs expressing CD3, CD4, CD8, FoxP3 and GBP5 were counted. A total of 129 GISTs were analysed. Mean patient age was 62.5 years; 52.0% were male. Tumour location included 89 stomach (69.0%), 33 small bowel (25.6%) and seven other (5.4%). Mean tumour size was 5.6 cm; mean mitoses were 7.2 per 50 high-power field. Nineteen patients (15.0%) developed disease progression, to abdominal wall (n = 8), liver (n = 6) and elsewhere (n = 5). Median progression-free survival was 56.6 months; five patients died of disease. PD-L1 was positive in 88 of 127 tumour samples (69.0%), 114 of 127 tumours were IDO-positive (89.8%) and 60 of 127 were positive for WARS (47.2%). PD-L1 was associated with increased size (P = 0.01), necrosis (P = 0.018) and mitoses (P = 0.006). Disease progression was not associated with PD-L1 (P = 0.44), IDO (P = 0.14) or WARS (P = 0.36) expression. PD-L1-positive GISTs with CD8+ or CD3+ TILs were significantly smaller than tumours with CD8+ or CD3+ TILs. CONCLUSIONS: PD-L1 expression was associated with increased size and mitoses. High CD8+ or CD3+ TIL counts were associated with decreased PD-L1/IDO+ GIST size. PD-L1 and IDO could be significant in GIST tumour biology, which invites consideration of immunotherapy as a potential treatment option.

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins.

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.

Human tryptophanyl-tRNA synthetase can efficiently complement the Saccharoymces cerevisiae homologue, Wrs1P.

Aminoacyl-tRNA synthetases corresponding to each of the 20 amino acids are essential proteins for nearly all cells. The tryptophan-specific enzyme in the cytoplasm of Saccharomyces cerevisiae (ScWRS) is a 49-kDa protein encoded by the WRS1 gene and required for survival. The human enzyme (HsWRS) is a 54-kDa protein with 46% sequence identity to ScWRS. HsWRS has a kinase domain in the N-terminal region and a serine phosphorylation site near the C-terminus not present in the yeast enzyme. To determine if the gene encoding the human protein could functionally complement the WRS1 gene, HsWRS cDNA was cloned in the expression vectors pVT100U and pYES then transformed into a diploid yeast where one copy of WRS1 had been replaced with a G418(R) gene. Tetrads derived from these transformants were dissected and spores germinated on media selecting for the presence of the plasmids. Haploid colonies were then tested for resistance to G418. G418(R) cells were unable to grow in the presence of 5-fluoroorotic acid, indicating their dependence on the plasmids for viability. Polymerase chain reaction analysis of these cells confirmed the presence of the HsWRS gene and the absence of WRS1. Growth rates of cells expressing HsWRS are essentially identical to the parent. This demonstrates that the human enzyme can function in yeast and effectively replace the ScWRS protein in spite of the presence of two unique functions and a >50% overall difference in amino acid sequence.

Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases.

Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials. Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain. Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes. We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive. The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant. Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo. Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme. Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2. Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.

Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.

Preparation of enantiomerically pure L-7-azatryptophan by an enzymatic method and its application to the development of a fluorimetric activity assay for tryptophanyl-tRNA synthetase.

The reaction of D,L-7-azatryptophan (D,L-7AW) with tryptophanyl-tRNA synthetase (TrpRS), adenosine triphosphate (ATP), and Mg2+ in the presence of inorganic pyrophosphatase results in the formation of a highly fluorescent l-7AW-adenylate complex. Detection of this complex is based on its enhanced fluorescence at 315 nm excitation and 360 nm emission after the addition of ATP. This stereoselective reaction was used to develop an activity assay for TrpRS using commercially available racemic D,L-7AW. The assay can be used to determine the activity of TrpRS from samples which contain less than 1 nmol of enzyme in 250 microL of sample. Thus the enzyme activity can be assessed without resorting to a radioactive assay of tRNATrp acylation. A secondary use of the stereoselective assay was for confirming the presence of pure L-7AW, D-7AW, or mixtures of the two enantiomers. D-7AW and L-7AW were prepared by reacting D,L-7AW with chloroacetic anhydride to form N-chloroacetyl-D,L-7AW (ClAc-7AW) followed by stereospecific proteolytic digestion of ClAc-7AW using carboxypeptidase A to produce the free L-7AW. The L-7AW could be separated from unreacted N-chloroacetyl-7AW by reverse-phase HPLC. The TrpRS-based assay was able to unambiguously discriminate between the two enantiomers of 7AW. The assay was then used to identify which enantiomer of 7AW was present in resolved fractions of the tripeptide L-lysyl-D,L-7-azatryptophyl-L-lysine. Digestion of the resolved tripeptides with protease enzymes produced the free L or D enantiomer of 7AW, which was easily identified using the TrpRS assay procedure.

Effects of cytokines on the gamma interferon-induced tryptophanyl-tRNA synthetase expression by human keratinocytes.

Incubation of human keratinocytes with gamma interferon (gamma-IFN) has been shown to potently induce the synthesis of a 53 kDa protein which was recently identified as tryptophanyl-tRNA synthetase (TRS). However, in spite of the high sensitivity of cultured keratinocytes to TRS induction by gamma-IFN, the study of inflammatory skin lesions has allowed the detection of the protein only in a few cases, suggesting regulatory mechanisms from soluble endogenous mediators with antagonistic activity on the induction of TRS by gamma-IFN. Among these mediators, we wondered whether cytokines selected for possible anti-inflammatory activity and potentially derived from activated resident skin cells, such as IL-4, IL-10, TNF-alpha and TGF-beta, may be involved in the modulation of the keratinocyte TRS expression. To assess this possibility, we investigated the modulation of the synthesis of TRS by human cultured keratinocytes upon stimulation by various gamma-IFN/cytokine combinations. The effects were evaluated by immunoblotting assay revealed by enhanced chemiluminescence, with the aid of a specific antibody to the TRS protein. Results failed to demonstrate any effect of the tested cytokines, whether on the basal level of the TRS, or on the gamma-IFN-induced enzyme expression in keratinocytes. It is thus unlikely that such cytokines can account for the infrequency of the TRS detection in inflammatory skin processes. Further investigations of alternative working hypothesis should help elucidate the regulation of TRS in human keratinocytes.

Compartmentalization of certain components of the protein synthesis apparatus in mammalian cells.

A comparative study on the localization of free cytosolic tryptophanyl-tRNA synthetase (TrpRS) and several components of the multi-aminoacyl-tRNA synthetase (ARS) complex (glutamyl-prolyl-tRNA synthetase (GluProRS), arginyl-tRNA synthetase (ArgRS)), and two non-synthetase polypeptides p38 and p43 has been carried out on ultrathin sections of cultured rabbit kidney cells by the immunogold technique using monoclonal antibodies raised against appropriate polypeptides. It has been shown that GluProRS, ArgRS and p38 polypeptide are distributed in the cells similarly to TrpRS and are located mainly in the vicinity of ribosomes. A smaller but significant portion of these proteins has been observed in the nuclei in the diffuse chromatin regions and in the vicinity of interchromatin granules. On the contrary, the main part of p43 protein was found in the cell nuclei; this indicates that this protein may exist in the cell separately from the cytoplasmic multi-ARS complex. Our results argue in favor of compartmentalization of both free ARS (such as TrpRS) and the multi-ARS complex in the vicinity of ribosomes. At the same time, the detection of some ARS in the diffuse chromatin regions in the nucleus implies that these enzymes may exhibit some non-canonical functions in addition to their role in protein synthesis.

Overcoming non-isomorphism by phase permutation and likelihood scoring: solution of the TrpRS crystal structure.

Entropy maximization to maximum likelihood, constrained jointly by the best available experimental phases and by a sufficiently good envelope, can bring about substantial model-independent map improvement, even at medium (3.1 A) resolution [Xiang, Carter, Bricogne & Gilmore (1993). Acta Cryst. D49, 193-212]. In the crystal structure determination of the Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS), however, the following had to be dealt with simultaneously: (1) a serious lack of isomorphism in the heavy-atom derivatives, resulting in large starting-phase errors; and (2) an initially poorly known molecular envelope. Because the constraints--both phases and envelope--were insufficiently well determined at the outset, maximum-entropy solvent flattening as previously applied was unsuccessful. Rather than improving the maps, it led to a deterioration of their quality, accompanied by a dramatic decrease of the log-likelihood gain as phases were extended from about 5 A resolution to the 2.9 A limit of the diffraction data. This deadlock was broken by the identification of strong reflections, which were initially unphased and which were inaccessible by maximum-entropy extrapolation from the phased ones, and by permutation of the phases of these reflections so as to sample the space of possible electron-density and envelope modifications they represented. Permutation was carried out by successive full and incomplete factorial designs [Carter & Carter (1979). J. Biol. Chem. 254, 12219-12223] for 28 strong reflections selected in decreasing order of their 'renormalized' structure-factor amplitudes. The permuted reflections included one reflection for which the probability distribution from multiple isomorphous replacement with anomalous scattering (MIRAS) indicated an incorrect phase with a high figure of merit and which consequently had a large renormalized structure factor. A similar permutation was carried out for six different binary choices related to the calculation and description of the molecular envelope. Permutation experiments were scored using the log-likelihood gain and contrasts for each main effect were analyzed by multiple-regression least squares. Student t tests provided significant and reliable indications for a large majority of the permuted reflections and for all six hypotheses related to the molecular envelope. The resulting phase improvement made it possible to assign positions (hitherto unobtainable) for nine of the ten selenium atoms in an isomorphous difference Fourier map for selenomethionine-substituted TrpRS crystals and hence to solve the structure. Phase-permutation methods continued to be useful in producing improved maps from all the available isomorphous-replacement phase information and therefore played a critical role in solving the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoelectron microscopic location of tryptophanyl-tRNA synthetase in mammalian, prokaryotic and archaebacterial cells.

Monoclonal antibody Am1 against conservative epitope of tryptophanyl-tRNA synthetase (WRS) was labeled with colloidal gold particles and used to localize the enzyme on ultrathin sections of eubacteria (Escherichia coli), archaebacteria (Methanococcus halophilus), rat pancreas tissue and rat fibroblasts (cell line RAT1). In all cell types immunoelectron microscopy revealed predominant cytoplasmic location of gold particles, as this could be expected from known biochemical data. In particular, in mammalian cells intensive labeling was observed in cytoplasmic regions rich in polysomes and free ribosomes. At the same time, the label was virtually absent in cytoplasmic regions where microfilament bundles were present. Significant concentrations of gold particles were found in mitochondria and nuclei. In the latter case, gold particles were located over diffuse chromatin regions and were virtually absent over compact chromatin. The density of diffuse chromatin in labeling may amount to about 50% of that found in the cytoplasm. Distribution of labeled antibodies over E. coli cells looks rather similar to that found for M. halophilus: gold particles are preferably concentrated over the cytoplasm and "boundary zone", i.e., a 30 nm wide cytoplasmic zone adjacent to the nucleoid border, while the label over nucleoid is virtually absent. Two main conclusions are drawn: (i) although in the animal cell homogenates WRS is recovered mainly as a soluble cytosolic enzyme, in intact cells it is associated with defined cellular organelles and compartments; this may be an evolutionarily acquired feature probably typical for multicellular organisms; (ii) the considerable density of labeling in diffuse (not compact) chromatin regions may be indicative of WRS involvement in the active chromatin functions (transcription, processing, transfer of gene products, etc.).

Depletion of tryptophan is not involved in expression of tryptophanyl-tRNA synthetase mediated by interferon.

Gamma interferon (IFN-gamma) affects tryptophan metabolism by mediating the expression of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase. In the present study, we investigated the role of indoleamine 2,3-dioxygenase-mediated tryptophan depletion in the induction of tryptophanyl-tRNA synthetase by IFN-gamma. The addition of excess tryptophan to the culture medium did not affect the induction of tryptophanyl-tRNA synthetase by IFN-gamma, indicating that tryptophan degradation is not directly involved in the IFN-gamma-mediated expression of tryptophanyl-tRNA synthetase.

[Immune electron microscope determination of the localization of tryptophanyl-tRNA-synthetase in bacteria and higher eukaryotes]. / Immuno-élektronno-mikroskopicheskoe opredelenie lokalizatsii triptofanil-tRNK-sintetazy v kletkakh bakterii i vysshikh éukariot.

Localization of tryptophanyl-tRNA synthetase (TRS) was studied on ultrathin (UT) sections of Escherichia coli cells and of rat fibroblasts fixed with glutaraldehyde and embedded in "Lowicryl K4M" resin at -35 degrees C. The UT sections were treated with the complexes of monoclonal and/or polyclonal antibodies against TRS with colloidal gold 15 and 8 nm in size. In both types of the cells cytoplasm was the most intensely labelled. In fibroblast cytoplasm, zones with a greater amount of ribosomes were mainly labelled, the gold particles being found over both the cysternae of granular endoplasmic reticulum and the areas of localization of free ribosomes. In the zones of microfilament localization TRS was not detected. A great amount of TRS was found in mitochondria and in the fibroblast nuclei. In the latter case, the label was concentrated over the diffuse chromatin localization regions, a minimal binding being observed over compact chromatin. The number of particles observed over diffuse chromatin equals to 50-80% against the label in fibroblast cytoplasm. In contrast, the label used to be absent over the E. coli nucleoid. The presence of TRS in the fibroblast nucleus may evidence in favour of a possible regulatory role of TRS in eukaryots.

Molecular and cellular studies of tryptophanyl-tRNA synthetase using monoclonal antibodies. Evaluation of a common antigenic determinant in eukaryotic, prokaryotic and archaebacterial enzymes which maps outside the catalytic domain.

Monoclonal antibodies referred to as Am1, 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-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at 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 Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaryotic 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 dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.

Molecular and cellular studies of tryptophanyl-tRNA synthetases using monoclonal antibodies. Remarkable variations in the content of tryptophanyl-tRNA synthetase in the pancreas of different mammals.

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 125I-labelled monoclonal antibody Am1, specifically interacting with any eukaryotic Trp-tRNA synthetase. Its content in the organs studied, with the exception 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 were 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 Ruminantia and of the other mammalians. As shown by indirect immunofluorescence 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.

[Immunocytochemical localization of tryptophanyl-tRNA-synthetase in a line of bovine kidney cells and in sublines with elevated enzyme levels]. / Immunotsitokhimicheskaia lokalizatsiia triptofanil-tRNK-sintetazy v linii kletok pochki byka i v subliniiakh s povyshennym soderzhaniem fermenta.

Intracellular localization of tryptophanyl-tRNA synthetase (E. C. 6.1.1.2) has been studied immunocytochemically using monospecific antibodies in cultured bovine kidney cells (strain MDBK) and in substrains with elevated enzyme levels. Both light and electron microscopy were used and native or detergent-treated cells were examined. The product of cytochemical reaction was revealed on free polyribosomes, polyribosomes attached to membranes of granular endoplasmic reticulum, on cytofilaments and in the nucleus as well.

[Localization of antigenic determinants using monoclonal antibodies and alpha-NH2-terminal labeling of polypeptide chains]. / Lokalizatsiia antigennykh determinant s ispol'zovaniem monoklonal'nykh antitel i vvedeniia alpha-NH2-kontsevoi metki v polipeptidnye tsepi.

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.

On the recovery of Cys-containing peptides during peptide mapping by HPLC. Tryptic peptides of Trp-tRNA synthetase of E.coli.

Conditions are presented for separating the major tryptic peptides of E.coli tryptophanyl-transfer RNA synthetase by reversed-phase liquid chromatography using a water-methanol gradient in the presence of 0.1% trifluoroacetic acid. Three of the peptides contain cysteine and are recovered in good yields if alkylated, but otherwise cannot be detected. A convenient post-digestion alkylation procedure is appropriate for use with small samples of protein which can be digested under reducing conditions. These results will be of interest for studies of the labeling of sulfhydryl groups in other proteins.

Limited proteolysis of tryptophanyl-tRNA synthetase from beef pancreas.

Treatment of purified tryptophanyl-tRNA synthetase with either chymotrypsin, papain, subtilisin or elastase converts all the enzyme into a high-molecular-weight intermediate. This protease-resistant core molecule has the same dimeric structure as the native protein and possesses the ability to bind substrates (tryptophan, ATP and tRNATrp) but is catalytically inactive. The monomer molecular weight of the protease-treated enzyme is 39000 compared to 54000 for the intact molecule. Chemical studies indicate that proteases excise the amino-terminal part of the polypeptide chain. It has been demonstrated previously that removal of a 13000-dalton fragment from the amino-terminal region of the tryptophanyl-tRNA synthetase converts the native enzyme to another active form. Cleavage of 20 additional amino acids produces the inactive protease-resistant core.

Multiple forms of tryptophanyl-tRNA synthetase from beef pancreas.

Three different forms of tryptophanyl-tRNA synthetase can be isolated from pancreatic extracts. Structural, immunological and catalytic properties of these various forms have been compared. The native enzyme is a dimeric molecule of molecular weight 108000. Two other forms, of molecular weight 85000 and 82000, are composed of two polypeptide chains identical with the carboxyl terminal region of the native subunits. These molecules are supposed to derive from the original protein by removal, from the amino-terminal part of each subunit, of a fragment of 11000 to 13000 molecular weight. Such removal modifies the shape and the stability of the molecule and decreases its specific acitvity. The origin of the derived forms is attributed to proteolysis. In fact, limited proteolysis of purified tryptophanyl-tRNA synthetase, in its native form, by elastase, results in the formation of an active compound, similar to one of the tryptophanyl-tRNA synthetase derived forms. Furthermore, incubation with "elastolytic fractions" prepared from pancreatic extracts presenting a particularly high level of proteolytic activity produces the same cleavage in tryptophanyl-tRNA synthetase polypeptide chain.