Life Sciences Commons^™

Evidence For Direct Roles Of Two Additional Factors, Secp43 And Soluble Liver Antigen, In The Selenoprotein Synthesis Machinery, Xue-Ming Xu, Heiko Mix, Bradley A. Carlson, Paula J. Grabowski, Vadim N. Gladyshev, Marla J. Berry, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA ^[Ser]Sec, but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2’-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA ^[Ser]Sec, and consequently reduced glutathione peroxidase …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis*, Nicole R. Buan, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobAand FldA interact, a computer model of aCobA∙FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA …

Different Catalytic Mechanisms In Mammalian Selenocysteine- And Cysteine-Containing Methionine-R-Sulfoxide Reductases, Hwa-Young Kim, Vadim Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are …

[Accepted Article Manuscript Version (Postprint)] Identification And Functional Characterization Of Arabidopsis Peroxin4 And The Interacting Protein Peroxin22, Bethany Zolman, Melanie Monroe-Augustus, Illeana Silva, Bonnie Bartel

Biology Department Faculty Works

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitinconjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression …

Process For The Preparation Of Citalopram Intermediate, Aslam A. Malik, Hasan Palandoken, Joy A. Stringer, Dershing Huang, Antonio Romero, Olivier Dapremont

Chemistry and Biochemistry

The present invention provides, inter alia, a novel process for the preparation of Citalopram, a known antidepressant.

Loss Of Editing Activity During The Evolution Of Mitochondrial Phenylalanyl-Trna Synthetase, Hervé Roy, Jiqiang Ling, Juan D. Alfonzo, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA^Phe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA^Phe, an activity enhanced in …

A Novel Cysteine-Rich Domain Of Sep15 Mediates The Interaction With Udp-Glucose:Glycoprotein Glucosyltransferase, Vyacheslav M. Labunskyy, Andrew D. Ferguson, Dmitri E. Fomenko, Yogarany Chelliah, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 copurifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. …

Performance Of Binary-Encounter-Bethe (Beb) Theory For Electron-Impact Ionization Cross Sections Of Molecules Containing Heavy Elements (Z > 10), Gregory E. Scott, Karl K. Irikura

Chemistry and Biochemistry

The binary-encounter-Bethe (BEB) theory developed by Kim and coworkers has been successful for computing electron-impact ionization cross sections of many molecules. However, some recent publications have stated that BEB theory performs poorly for molecules that contain heavier elements such as chlorine and sulfur. We have found that the BEB calculations in those publications were performed incorrectly. When performed correctly, BEB predictions are as good for heavy-element molecules as for light-element molecules. We recommended recently that an alternative, less-confusing procedure be used for molecules that contain heavier elements. The alternative procedure, based upon effective core potentials (ECPs), does not require explicit …

Crystal Structures Of Oxidized And Reduced Mitochondrial Thioredoxin Reductase Provide Molecular Details Of The Reaction Mechanism, Ekaterina I. Biterova, Anton A. Turanov, Vadim N. Gladyshev, Joseph J. Barycki

Vadim Gladyshev Publications

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine- containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a …

A Facile Synthesis Of (Tert-Alkoxy)Amines, Hasan Palandoken, Chris M. Bocian, Michelle R. Mccombs, Michael H. Nantz

Chemistry and Biochemistry

Tertiary alcohols react with stoichiometric BF₃·Et₂O and N-hydroxyphthalimide to yield N-alkoxyphthalimides. Subsequent hydrazinolyses afford the title compounds.

Fractals Analysis Of Cardiac Arrhythmias, Mohammed Saeed

Department of Biological & Biomedical Sciences

Heart rhythms are generated by complex self-regulating systems governed by the laws of chaos. Consequently, heart rhythms have fractal organization, characterized by self-similar dynamics with long-range order operating over multiple time scales. This allows for the self-organization and adaptability of heart rhythms under stress. Breakdown of this fractal organization into excessive order or uncorrelated randomness leads to a less-adaptable system, characteristic of aging and disease. With the tools of nonlinear dynamics, this fractal breakdown can be quantified with potential applications to diagnostic and prognostic clinical assessment. In this paper, I review the methodologies for fractal analysis of cardiac rhythms and …

Charge-Switch Nucleotides, John G. K. Williams, Gregory R. Bashford, Jiyan Chen, Dan Draney, Nara Narayanan, Bambi L. Reynolds, Pamela Sheaff

Biomedical Imaging and Biosignal Analysis Laboratory

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation Without interference from unincorporated NPs and Without illuminating the polymerase-DNA complex.

Post-Translational Regulation Of Adipose Differentiation-Related Protein By The Ubiquitin/Proteasome Pathway, John Tansey

Chemistry Faculty Scholarship

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate
fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic
lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the
presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked
by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments
confirmed that ADRP degradation is inhibited by MG132. Conditions that …

Expression Of G-Protein Inwardly Rectifying Potassium Channels (Girks) In Lung Cancer Cell Lines, Howard Plummer 3rd, Madhu Dhar, Maria Cekanova Ms, Rndr, Phd, Hildegard Schuller

Faculty Publications and Other Works -- Biochemistry, Cellular and Molecular Biology

BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small …

The Clearance Of Protein-Bound Solutes By Hemofiltration And Hemodiafiltration, Timothy W. Meyer, Jason L. Walther, Maria Enrica Pagtalunan, Andres W. Martinez, Ali Torkamani, Patrick D. Fong, Natalie S. Recht, Channing R. Robertson, Thomas H. Hostetter

Chemistry and Biochemistry

Background. Hemofiltration in the form of continuous venovenous hemofiltration (CVVH) is increasingly used to treat acute renal failure. Compared to hemodialysis, hemofiltration provides high clearances for large solutes but its effect on protein-bound solutes has been largely ignored.

Methods. Standard clinical systems were used to remove test solutes from a reservoir containing artificial plasma. Clearances of the protein-bound solutes phenol red (C_PR) and indican (C_IN) were compared to clearances of urea (C_UREA) during hemofiltration and hemodiafiltration. A mathematical model was developed to predict clearances from values for plasma flow Q_p, …

Regulatable Gene Expression Systems For Gene Therapy Applications: Progress And Future Challenges, Shyam Goverdhana, Mariana Puntel, Weidong Xiong, Jeffrey Zirger, Carlos Barcia, James Curtin, Eric Soffer, Sonali Mondkar, Gwendalyn King, Jinwei Hu, Marianela Candolfi, Diane Greengold, Pedro Lowenstein, Maria Castro

Articles

Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life …

A Role For Mitogen-Activated Protein Kinase (Erk1/2) Activation And Non-Selective Pore Formation In P2x7 Receptor-Mediated Thymocyte Death, Rodolphe Auger, Iris Motta, Karim Benihoud, David M. Ojcius, Jean M. Kanellopoulos

All Dugoni School of Dentistry Faculty Articles

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. …

Lack Of Association Of Methylenetetrahydrofolate Reductase 677c>T Mutation With Coronary Artery Disease In A Pakistani Population, M Perwaiz Iqbal, Tasneem Fatima, Siddiqa Parveen, Farzana A. Yousuf, Majid Shafiq, Naseema Mehboobali, Abrar H. Khan, Iqbal Azam, Philippe M. Frossard

Department of Biological & Biomedical Sciences

Pakistanis belong to the South Asian population which has the highest known rate of coronary artery disease. Folic acid deficiency also appears to be highly prevalent in this population. Methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism decreases the activity of this enzyme and can be associated with mild to moderate hyperhomocysteinemia in homozygotes, particularly when there is folic acid deficiency, as well as with coronary artery disease. To assess the value of genotyping the MTHFR 677C>T dimorphism, we carried out a case-control study of dimorphism 677C>T for putative association with myocardial infarction (MI) among Pakistani nationals. We investigated a …

Evolution Of Selenium Utilization Traits, Hector Romero, Yan Zhang, Vadim Gladyshev, Gustavo Salinas

Vadim Gladyshev Publications

Background: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is …

A Modified Poisson–Boltzmann Analysis Of The Capacitance Behavior Of The Electric Double Layer At Low Temperatures, Douglas Henderson, L. B. Bhuiyan, C. W. Outhwaite

Faculty Publications

The modified Poisson–Boltzmann theory is used to analyze the anomalous behavior of the electric double layer capacitance for small surface charge at low temperatures and densities. Good agreement is found with simulation and recent density-functional theory results. Negative adsorption is also found in line with theory and simulation. An unsatisfactory feature is the relatively poor structure in this region due to the inherent approximations in the theory. This feature is unimportant in relation to the capacitance results but has implications when calculating adsorption properties.

Hard-Sphere Radial Distribution Function Again, Andrij Trokhymchuk, Douglas Henderson, Ivo Nezbeda, Jan Jirsak

Faculty Publications

A theoretically based closed-form analytical equation for the radial distribution function, g(r), of a fluid of hard spheres is presented and used to obtain an accurate analytic representation. The method makes use of an analytic expression for the short- and long-range behaviors of g(r), both obtained from the Percus-Yevick equation, in combination with the thermodynamic consistency constraint. Physical arguments then leave only three parameters in the equation of g(r) that are to be solved numerically, whereas all remaining ones are taken from the analytical solution of the Percus-Yevick equation.

Mammalian Selenoprotein Thioredoxin-Glutathione Reductase, Dan Su, Sergey V. Novoselov, Qi-An Sun, Mohamed E. Moustafa, You Zhou, Richard Oko, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization …

The Cell Cycle–Regulated Genes Of Schizosaccharomyces Pombe, Anna Oliva, Adan Rosebrock, Francisco Ferrezuelo, Haiying Chen, Saumyadipta Pyne, Steve Skiena, Bruce Futcher, Janet Leatherwood

Department of Molecular Genetics and Microbiology Faculty Publications

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 …

Carbon Cycling In Lake Superior, N. R. Urban, M. T. Auer, Sarah A. Green, X. Lu, D. S. Apul, K. D. Powell, L. Bub

Department of Chemistry Publications

Carbon (C) cycling in Lake Superior was studied within the Keweenaw Interdisciplinary Transport Experiment in Superior (KITES) project to assess (1) whether the lake is net heterotrophic, (2) sources, sinks and residence time for dissolved organic carbon (DOC), (3) importance of terrigenous organic C subsidies, and (4) factors limiting C flow through bacteria. During 3 years of fieldwork, measurements were made of spatial and temporal distributions of C pools and rates of photosynthesis, community respiration, and bacterial production. Measurements were made of the composition of dissolved organic matter (DOM), rates of DOM photolysis, lability of DOM toward microbial consumption, and …

An Analysis Of The Chemical Processes In The Smoke Plume From A Savanna Fire, Jorg Trentmann, Robert J. Yokelson, Peter V. Hobbs, Tanja Winterrath, Ted J. Christian, Meinrat O. Andreae, Sherri A. Mason

Chemistry and Biochemistry Faculty Publications

[1] Photochemistry in young plumes from vegetation fires significantly transforms the initial fire emissions within the first hour after the emissions are injected into the atmosphere. Here we present an investigation of field measurements obtained in a smoke plume from a prescribed savanna fire during the SAFARI 2000 field experiment using a detailed photochemical box-dilution model. The dilution used in the model simulations was constrained by measurements of chemically passive tracers (e.g., CO) near and downwind of the fire. The emissions of the dominant carbonaceous compounds, including oxygenated ones, were taken into account. The field measurements revealed significant production of …

A Revised Nomenclature For Mammalian Acyl-Coa Thioesterases/Hydrolases, Mary Hunt, Junji Yamada, Lois Maltais, Mathew Wright, Ernesto Podesta, Stefan Alexson

Articles

Acyl-CoA thioesterases, also known as acyl-CoA hydrolases, are a group of enzymes that hydrolyze CoA esters such as acyl-CoAs (saturated, unsaturated, branched chain), bile acid-CoAs, CoA esters of prostaglandins etc, to the corresponding free acid and coenzyme A. There is however significant confusion regarding the nomenclature of these genes. In agreement with the HUGO Gene Nomenclature Committee (HGNC) and the Mouse Genomic Nomenclature Committee (MGNC), a revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases has been suggested for the 12 member family. The family root symbol is ACOT, with human genes named ACOT1-12, and rat and mouse named Acot1-12. Several of the …

A Critical Role For Kalirin In Ngf Signaling Through Trka, Kausik Chakrabarti, Rong Lin, Noraisha I. Schiller, Yanping Wang, David Koubi, Ying-Xin Fan, Brian B. Rudkin, Gibbes R. Johnson, Martin R. Schiller

Life Sciences Faculty Research

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low …

Glycoprotein Gp130 Of Dictyostelium Discoideum Influences Macropinocytosis And Adhesion, Catherine P. Chia, Sujatha Gomathinayagam, Robert J. Schmaltz, Laura K. Smoyer

Department of Biochemistry: Faculty Publications

Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed Mr of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained …

Pyrrolysine And Selenocysteine Use Dissimilar Decoding Strategies, Yan Zhang, Pavel V. Baranov, John F. Atkins, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic …

Association Between Archaeal Prolyl- And Leucyl-Trna Synthetases Enhances TrnaPro Aminoacylation, Mette Praetorius-Ibba, Theresa E. Rogers, Rachel Samson, Zvi Kelman, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA^Pro charging showed that the catalytic efficiency (k_cat/K_m) of ProRS increased 5-fold …