Cellulose Thin-layer Chromatography Research Articles

Amino acid profile and potential biomass conversion of Vitellaria paradoxa fruit pulp

Vitellaria species is one of Africa's most important fruit crops with enormous human benefiting nutrients including amino acids. This study characterised Vitellaria paradoxa shea nut pulp from 5 regional zones of Ghana based on its acidity, pH and among the few for its qualitative and quantitative amino acids profile. Thin Layer Chromatography (TLC) techniques, using cellulose TLC plates were used to profile and quantify the amino acids. Fifteen essential amino acids were detected in the Vitellaria paradoxa shea nut pulp. Alanine, arginine, glutamic acid, glutamine and histidine were common in the Vitellaria paradoxa shea nut pulp. The most concentrated (41.02 g/100 g) of the 7 essential amino acids in the shea nut pulp was arginine and the least was glycine, 2.12 g/100 g. All seven 7 essential amino acids, except leucine and valine, were significantly different (P < 0.05) across the 5 shea regional zones of Ghana. The most acidic pulp, with 18.63% acidity, was from the Upper West Region. The quality and quantitative of amino acids in Vitellaria paradoxa shea nut pulp differed significantly from one region to the other. The shea nut pulp is a good source of essential amino acids and can be used fruit juice production and provide the required N for an effective fermentation to proceed. This has remarkable economic implication and significant environmental effects. Some health benefits of eating shea nut fruits consequential of its nutritional effects due to its amino acids composition have been noted to include its ability to help improve mood, boost exercise performance, prevent muscle loss and promote weight loss. Moreover, the amino acids profile of shea nut pulp may provide researchers with useful applications of the Vitellaria paradoxa shea nut pulp in the various industries.

Low-Temperature Plasma Probe Mass Spectrometry for Analytes Separated on Thin-Layer Chromatography Plates: Direct vs Laser Assisted Desorption.

Thin-layer chromatography (TLC) is a widespread technique because it allows fast, simple, and inexpensive analyte separations. In addition, direct analysis of the compounds separated on TLC plates via mass spectrometry (MS) has been shown to provide high sensitivity and selectivity while avoiding time-consuming sample extraction protocols. Here, direct desorption low-temperature plasma-mass spectrometry (LTP-MS) as well as diode laser assisted desorption (LD) LTP-MS are studied for direct spatially resolved analysis of compounds from TLC plates. Qualitative and quantitative characterization of amino acids, pharmaceuticals, and structural isomers were performed. The nature of the TLC plate stationary phase was found to have a significant influence, together with the analyte's characteristics, on the desorption efficiency. Tandem MS is shown to greatly improve the limits of detection (LODs). Direct desorption LTP-MS, without external thermal assisted desorption, demonstrates its best performance with cellulose TLC plates (LODs, 0.01 ng/mm2 to 2.55 ng/mm2) and restricted performance with normal-phase (NP) TLC plates (several analytes without observable signal). LD LTP-MS, with systematic optimization of irradiance and focal point diameter, is shown to overcome the direct-desorption limitations and reach significantly improved LODs with NP TLC plates (up to ×1000 better). In addition, a wide-ranging characterization of amino acid analytical figures of merit with LD LTP-MS shows that LODs from 84 pg/mm2 down to 0.3 pg/mm2 are achieved on NP TLC plates.

High Performance Liquid Chromatographic Determination of Anthocyanin Pigments in Some Varieties of Black Rice

Anthocyanin pigments in ten pigmented rice cultivars were isolated with cellulose thin-layer chromatography and preparative high performance liquid chromatography (HPLC), and further identified with conventional chromatographic techniques. Individual anthocyanins were then quantified by HPLC using a reversed ODS-5 column and detected at 280 nm. Ten rice varieties contained two major anthocyanins, cyanidin 3-glucoside and peonidin 3-glucoside. Total anthocyanin contents varied greatly in the range of 0-493 mg/100 g grain among varieties. Cyanidin 3-glucoside, an agent having important oxygen radical absorbing capacity (ORAC), was most abundant in Suwon #415 variety (470 mg/100 g grain), while Jawangdo (10 mg/100 g grain) and Ilpumbyeo (not detected) had the smallest amount of anthocyanin among the samples examined. As for the distribution ratio of individual anthocyanin, Suwon #415 was characterized by a relatively high percentage of cyanidin 3-glucoside (95.3%); whereas, Suwon #425 and Heugjinmi were characterized by a relatively larger amount of peonidin 3-glucoside.

The impacts of paper properties on matrix effects during paper spray mass spectrometry analysis of prescription drugs, fentanyl and synthetic cannabinoids

Designer drugs, drugs synthesized in a lab that mimic other drugs of abuse, have become a major cause of death in the United States. Limitations in either speed, sensitivity or selectivity of current analytical techniques hinders rapid detection of designer drugs. Paper spray mass spectrometry (PS-MS) is a rapid ambient pressure technique capable of detecting analytes in complex matrices. However, due to a lack of sample cleanup and chromatography, matrix effects can have a significant impact on the detection limits. Previous work has shown that the paper spray substrate has an impact on matrix effects, but the current literature lacks a systematic approach to studying different properties of paper with regards to matrix effects. In this work, the effect of pore size, flow rate, and thickness on ionization efficiency and recovery was assessed. Cellulose thin layer chromatography (TLC) plates were made along with a universal spray cartridge to provide a porous spray substrate similar to paper but with easily controllable properties. It was found that substrates with the highest filtration properties (thicker, slower flow rate, or smaller pore sizes) exhibited lower analyte recovery but improved ionization efficiency. This trend was verified with an offline extraction conducted with a 3D printed centrifuge extractor. Paper/solvent combinations were tested with urine samples to determine if selecting a paper and solvent with better ionization efficiency could improve detection limits. While for some drugs minimizing ionization suppression improved detection limits, other drug targets, like those that were charged at physiological pH, were largely unaffected. For analytes that showed improvement, both the paper and the solvent had an impact, although most of the improvement was due to the solvent.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry for the determination of selenomethionine and selenocysteine in algae and yeast

In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 μg L−1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.

NADPH- and NADH-dependent metabolism of and DNA adduct formation by benzo[a]pyrene catalyzed with rat hepatic microsomes and cytochrome P450 1A1.

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. Here we investigated the efficiencies of rat hepatic microsomes and rat recombinant CYP1A1 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase, epoxide hydrolase and/or cytochrome b5 in Supersomes™ to metabolize this carcinogen. We also studied the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate BaP metabolism in these systems. Up to eight BaP metabolites and two DNA adducts were generated by the systems, both in the presence of NADPH and NADH. Among BaP metabolites, BaP-9,10-dihydrodiol, BaP-4,5-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, and a metabolite of unknown structure were formed by hepatic microsomes and rat CYP1A1. One of two DNA adducts formed by examined enzymatic systems (rat hepatic microsomes and rat CYP1A1) was characterized to be 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N2-BPDE), while another adduct has similar chromatographic properties on polyethylaneimine–cellulose thin layer chromatography to a guanine adduct derived from reaction with 9-hydroxy-BaP-4,5-oxide. In the presence of either of the reductase cofactors tested, NADPH or NADH, cytochrome b5 stimulated CYP1A1-mediated formation of both BaP-DNA adducts. The results demonstrate that NADH can act as a sole electron donor for both the first and the second reduction of CYP1A1 during its reaction cycle catalyzing oxidation of BaP, and suggest that the NADH:cytochrome b5 reductase as the NADH-dependent reductase might substitute POR in this enzymatic system.Graphical abstract

Thin-layer chromatographic image analysis for the determination of sulfide ions using pyrylium cations

A highly specific and sensitive chromatographic method for the determination of sulfide ions was developed. The procedure is based on the sensitive and specific reaction of sulfide ions with 4-[p-(N,N-dimethylamino)phenyl]-2,6-diphenylpyrylium cations. The product of the reaction is a thiopyrylium derivative which can be quantitatively determined. Separations were performed on cellulose thin-layer chromatography (TLC) plates with a mixture of phosphoric buffer (pH 3.0), propan-2-ol, and acetonitrile (4:4:0.5, v/v) as the mobile phase. The thiopyrylium derivative was visible in the form of blue spots against a colorless background, which were converted into a chromatogram using TLSee software. Linearity was obtained in the range of 40–500 pmol per spot. The limits of detection and quantification were 10 pmol per spot and 33 pmol per spot, respectively. The proposed method was successfully used for the detection and determination of sulfide ions in water samples from the Busko Zdroj health resort.

Closed Anaerobic Biotransformation Products of Organoarsenic Compounds inFucus distichus

The closed anaerobic decomposition extracts ofFucus distichusincubated with seawater and sediment, and without sediment as control, were subjected to extractions and isolation on Sephadex LH 20 and Cellulose Thin Layer Chromatography. The decomposition extracts and isolates were analyzed by using both the Hydride Generation Gas Chromatography Atomic Absorption Spectrometry (HG-GC-AAS) and High Performance Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry (HPLC-ICPMS) to identify the arsenic species in the equilibrium mixtures of the seaweed and filtrates separately. In the methanol seaweed extract, equilibrium mixture of arsenosugars (AS) AS1 and AS2 and their biotransformation products of dimethylarsinoylethanol (DMAE) and dimethylarsinic acid (DMAA) were identified. In the methanol filtrate extract of the mixture, only DMAE and DMAA were identified. However, in the control methanol filtrate extract five organoarsenic species, AS1 and AS2, one unidentified hidden organoarsenic species, DMAE and DMAA were identified in the equilibrium mixture. This result confirmed that the hidden organoarsenic species inFucus distichus, AS1 and AS2, and an unidentified organoarsenic compounds are biotransformed to only DMAE and DMAA under an anaerobic condition. This also suggests that DMAE and DMAA are strong intermediate candidates for the generation of arsenobetaine, from arsenoribosides in the marine food webs.

A simple and rapid method for histamine analysis in fish and fishery products by TLC determination

A simple and rapid thin-layer chromatography (TLC)-densitometry method for histamine analysis in fish and fishery products was established. Histamine was extracted from seafood with 80% ethanol. The sample extracts and standard solutions of histamine were applied to cellulose TLC plate and developed by ascending chromatography with ammonia–ethanol 1:3 (v/v) as mobile phase. Histamine was visualized with pauly’s reagent, the intensity of the colored spots was digitized and calculated by image processing method software, Lane & Spot Analyzer (ATTO, Tokyo, Japan). Successful separation of histamine from other (imidazole compounds) pauly’s reagent-positive components in 80% ethanol fish extracts was achieved. The linearity of the histamine estimation using this method was good within the range from 30 ng to 1000 ng of histamine (r2 = 0.9997). Histamine can be satisfactorily detected and completely separated from histidine, 4-methyl-imidazole and other pauly’s reagent-positive compounds. This method does not need expensive instrument, any tedious pretreatment to eliminate potential interference other imidazole compounds such as histidine or carnosine. It is also less reagents compared with HPLC method. Moreover, it is a simple, rapid, sensitive, reproducible. Therefore this method is suitable for monitor of histamine in multiple fish and fishery products simultaneously that contain as little as 20 ppm histamine (2.0 mg/100 g).

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3'-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.

Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser(846) and Ser(868), which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser(846) or Ser(868) regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser(846) phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.

Evaluation of thin-layer chromatography systems for analysis of amino acids in complex mixtures

Summary Different thin-layer chromatography (TLC) systems were evaluated for analysis of 21 biologically important essential and nonessential amino acids in complex mixtures such as biological tissues and fluids. Amino acids were visualized on the layers by derivatization with ninhydrin reagent, and RF values were determined by slit-scanning densitometry. The five systems found to be most useful for analysis of amino acids were cellulose and silica gel high-performance TLC (HPTLC) plates developed with either 2-butanol-pyridine-glacial acetic acid-deionized water, 39:34:10:26, or 2-butanol-pyridine-25% ammonia-deionized water, 39:34:10:26, and ion exchange TLC plates developed with citrate buffer, pH 3.3. Using these five systems with ninhydrin detection, identification of all amino acids except for leucine and isoleucine in complex mixtures is possible, and quantification can be achieved if the amino acid to be quantified is well separated from adjacent components of the mixture. Example chromatograms are illustrated for separation and identification of amino acids in a snail tissue sample on a cellulose HPTLC plate.

Phosphorylation of Viral RNA-dependent RNA Polymerase and Its Role in Replication of a Plus-strand RNA Virus

Central to the process of plus-strand RNA virus genome amplification is the viral RNA-dependent RNA polymerase (RdRp). Understanding its regulation is of great importance given its essential function in viral replication and the common architecture and catalytic mechanism of polymerases. Here we show that Turnip yellow mosaic virus (TYMV) RdRp is phosphorylated, when expressed both individually and in the context of viral infection. Using a comprehensive biochemical approach, including metabolic labeling and mass spectrometry analyses, phosphorylation sites were mapped within an N-terminal PEST sequence and within the highly conserved palm subdomain of RNA polymerases. Systematic mutational analysis of the corresponding residues in a reverse genetic system demonstrated their importance for TYMV infectivity. Upon mutation of the phosphorylation sites, distinct steps of the viral cycle appeared affected, but in contrast to other plus-strand RNA viruses, the interaction between viral replication proteins was unaltered. Our results also highlighted the role of another TYMV-encoded replication protein as an antagonistic protein that may prevent the inhibitory effect of RdRp phosphorylation on viral infectivity. Based on these data, we propose that phosphorylation-dependent regulatory mechanisms are essential for viral RdRp function and virus replication.

Phosphorylation of Coronin 1B by Protein Kinase C Regulates Interaction with Arp2/3 and Cell Motility

Coronins are a conserved family of WD repeat-containing, actin-binding proteins that regulate cell motility in a variety of model organisms. Our results show that Coronin 1B is a ubiquitously expressed member of the mammalian Coronin gene family that co-localizes with the Arp2/3 complex at the leading edge of fibroblasts, and co-immunoprecipitates with this complex. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated by protein kinase C (PKC) phosphorylation. Coronin 1B is phosphorylated by PKC both in vitro and in vivo. Using tryptic peptide mapping and mutagenesis, we have identified serine 2 (Ser-2) on Coronin 1B as the major residue phosphorylated by PKC in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to phorbol 12-myristate 13-acetate (PMA) and increased speed in single cell tracking assays. Cells expressing the Coronin 1B S2D mutant have attenuated PMA-induced ruffling and slower cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed, whereas expression of the S2D mutant renders cells hypersensitive to its effects. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts, and that its ability to control motility and interactions with the Arp2/3 complex are regulated by PKC phosphorylation at Ser-2. Furthermore, Coronin 1B phosphorylation is responsible for a significant fraction of the effects of PMA on fibroblast motility.

Cooperation between Small Nuclear RNA-activating Protein Complex (SNAPC) and TATA-box-binding Protein Antagonizes Protein Kinase CK2 Inhibition of DNA Binding by SNAPC

Protein kinase CK2 regulates RNA polymerase III transcription of human U6 small nuclear RNA (snRNA) genes both negatively and positively depending upon whether the general transcription machinery or RNA polymerase III is preferentially phosphorylated. Human U1 snRNA genes share similar promoter architectures as that of U6 genes but are transcribed by RNA polymerase II. Herein, we report that CK2 inhibits U1 snRNA gene transcription by RNA polymerase II. Decreased levels of endogenous CK2 correlates with increased U1 expression, whereas CK2 associates with U1 gene promoters, indicating that it plays a direct role in U1 gene regulation. CK2 phosphorylates the general transcription factor small nuclear RNA-activating protein complex (SNAP(C)) that is required for both RNA polymerase II and III transcription, and SNAP(C) phosphorylation inhibits binding to snRNA gene promoters. However, restricted promoter access by phosphorylated SNAP(C) can be overcome by cooperative interactions with TATA-box-binding protein at a U6 promoter but not at a U1 promoter. Thus, CK2 may have the capacity to differentially regulate U1 and U6 transcription even though SNAP(C) is universally utilized for human snRNA gene transcription.

Protein Kinase C Phosphorylation of the Metabotropic Glutamate ReceptormGluR5 on Serine 839 Regulates Ca2+Oscillations

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.

Screening method for wood extractives: direct cellulose thin-layer chromatography plate

A method for screening wood extractives was developed using cellulose thin-layer chromatography plate (Cell-TLC) separation and was directly applied to bioassays. Wood meal (≪60 mesh) from nangka (Artocarpus heterophyllus Lamk) heartwood was extracted with hot methanol. The crude extract was separated using a Cell-TLC plate (50 × 50 mm). Two broad bands with R f values of 0.46 and 0.96 were found, and the bands showed completely different effects against the pest termite Coptotermes formosanus Shiraki and the decay fungus Fomitopsis palustris. The band with R f 0.46 was preferentially consumed by workers of C. formosanus, and it did not show any growth inhibition against F. palustris when the Cell-TLC plate was directly exposed to the organism. In contrast, the band with R f 0.96 appeared to repel strongly the feeding by C. formosanus and inhibited the growth of F. palustris. It was concluded that the Cell-TLC system was applicable for screening wood extracts consisting of many compounds.

Stimulation of glycolysis in anaerobic elongation of pondweed (Potamogeton distinctus) turions

Stem segments prepared from pondweed (Potamo geton distinctus A. Benn.) turions (overwintering buds) elongate in anaerobic conditions, whereas there is almost no elongation in air. The anaerobic elongation was accompanied by a decrease in dry weights of stem segments, mainly due to consumption of storage starch in the amyloplasts of stem cells. On the other hand, total contents of amino acids increased in stem segments, in which contents of alanine, valine, leucine, and isoleucine increased, but contents of asparatic acid decreased. Moreover, contents of lactate in stem tissues increased at an early stage of anaerobic incubation. In tracer experiments with 14C-glucose, 14C incorporation into stem tissues in anoxia was only half of that in normoxia. However, conversion of 14C to ethanol occurred exclusively in anoxia. 14C-labelled metabolites were analysed by two-dimensional cellulose thin-layer chromatography. 14C incorporation into sucrose and alanine was significantly increased in anoxia. The activity of alanine aminotransferase was enhanced by anoxia, suggesting that pyruvate is a precursor of alanine synthesis. The results suggest that pondweed turions produce energy necessary for anaerobic elongation by activating conversion of storage starch in the amyloplasts to ethanol, alanine and lactate.

A Single Amidotransferase Forms Asparaginyl-tRNA and Glutaminyl-tRNA in Chlamydia trachomatis

Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

Recovering antibody secretion using a hapten ligand as a chemical chaperone.

Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.