Primary Deuterium Kinetic Isotope Effects From Product Yields: Rationale, Implementation, and Interpretation.

Abstract:

:A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1°DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1°DKIE may be obtained by 1H NMR analyses as the ratio of the yields of H- and D-labeled products from a reaction in 50:50 (v/v) HOH/DOD. The procedures for these 1H NMR analyses are reviewed. This product deuterium isotope effect (PDIE) is defined as 1/ϕEL for fractionation of hydrons between solvent and the transition state for the reaction examined. When the solvent is not the direct hydron donor, it is necessary to correct the PDIE for the fractionation factor ϕEL for partitioning of the hydron between the solvent and the direct donor EL. This method was used to determine the 1°DKIE on decarboxylation reactions catalyzed by wild-type orotidine 5'-monophosphate decarboxylase (OMPDC) and by mutants of OMPDC, and then in the determination of the 1°DKIE on the decarboxylation reaction catalyzed by 5-carboxyvanillate decarboxylase. The experimental procedures used in studies on OMPDC and the rationale for these procedures are described.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Amyes TL,Richard JP

doi

10.1016/bs.mie.2017.06.043

subject

Has Abstract

pub_date

2017-01-01 00:00:00

pages

163-177

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(17)30226-4

journal_volume

596

pub_type

杂志文章
  • Stem cells in the lung.

    abstract::The lung is composed of two major anatomically distinct regions-the conducting airways and gas-exchanging airspaces. From a cell biology standpoint, the conducting airways can be further divided into two major compartments, the tracheobronchial and bronchiolar airways, while the alveolar regions of the lung make up th...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)19012-6

    authors: Liu X,Driskell RR,Engelhardt JF

    更新日期:2006-01-01 00:00:00

  • Determining cellular role of G alpha 12.

    abstract::Using the expression strategies described here, we have demonstrated a model system whereby the sequential signaling events involved in cell proliferation and subsequent transformation regulated by G alpha 12 can be investigated. The model system presented here can also be used to study the temporal interrelationships...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(02)44722-2

    authors: Dermott JM,Dhanasekaran N

    更新日期:2002-01-01 00:00:00

  • A quantitative method for measuring phototoxicity of a live cell imaging microscope.

    abstract::Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-391856-7.00039-1

    authors: Tinevez JY,Dragavon J,Baba-Aissa L,Roux P,Perret E,Canivet A,Galy V,Shorte S

    更新日期:2012-01-01 00:00:00

  • Protein derivitization-expressed protein ligation.

    abstract::Expressed protein ligation (EPL) combines two methods to ligate a synthetic peptide to a recombinant protein. Native chemical ligation (NCL) is a process in which two synthesized peptides are ligated by reaction of a C-terminal thioester on one peptide with an N-terminal cysteine residue of another protein. The chemis...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-420070-8.00009-X

    authors: Mitchell SF,Lorsch JR

    更新日期:2014-01-01 00:00:00

  • Characterization of oligophrenin-1, a RhoGAP lost in patients affected with mental retardation: lentiviral injection in organotypic brain slice cultures.

    abstract::Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein, was one of the first Rho-linked MR genes identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)00419-3

    authors: Kasri NN,Govek EE,Van Aelst L

    更新日期:2008-01-01 00:00:00

  • Methods for studying oxidative regulation of protein kinase C.

    abstract::The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-405881-1.00005-7

    authors: Gopalakrishna R,McNeill TH,Elhiani AA,Gundimeda U

    更新日期:2013-01-01 00:00:00

  • Use of recombinant iron-superoxide dismutase as a marker of nitrative stress.

    abstract::Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric o...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)37031-6

    authors: Larrainzar E,Urarte E,Auzmendi I,Ariz I,Arrese-Igor C,González EM,Moran JF

    更新日期:2008-01-01 00:00:00

  • Detection and quantification of nitric oxide (NO) synthase-independent generation of NO.

    abstract::Nitric oxide formation in ischemia-reperfusion injury can be identified and measured directly using EPR of nitroso-heme complexes comprising NO bound to either Mb or Hb-Fe2+. This article described the successful use of this method to detect and quantify the generation of NO formed independently of nitric oxide syntha...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(02)59182-5

    authors: Lepore DA

    更新日期:2002-01-01 00:00:00

  • Mod-seq: A High-Throughput Method for Probing RNA Secondary Structure.

    abstract::It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.01.012

    authors: Lin Y,May GE,Joel McManus C

    更新日期:2015-01-01 00:00:00

  • Near-infrared photoactivatable nitric oxide donors with photoacoustic readout.

    abstract::In this chapter, we motivate the need for photoactivatable NO donor molecules and give a brief survey of the existing chemical tools in the field. We then provide detailed protocols for the synthesis and validation of a near-infrared light-activated NO donor molecule, photoNOD-1, developed in our research group. With ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.05.003

    authors: Zhou EY,Knox HJ,Reinhardt CJ,Partipilo G,Chan J

    更新日期:2020-01-01 00:00:00

  • Preparation, Biochemical Analysis, and Structure Determination of the Bromodomain, an Acetyl-Lysine Binding Domain.

    abstract::The bromodomain (BrD) represents an evolutionarily conserved protein domain whose function mostly is to recognize acetylated lysine residues in histones and nuclear proteins in regulation of gene transcription in chromatin. The highly conserved BrD structure features an unusual left-handed, antiparallel four-helix bun...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.01.018

    authors: Ren C,Zeng L,Zhou MM

    更新日期:2016-01-01 00:00:00

  • Membrane protein expression and analysis in yeast.

    abstract::This protocol describes the expression and analysis of membrane proteins produced in yeast, as illustrated with Yarrowia lipolytica and Pichia pastoris. Step by step, we explain how to generate a yeast strain expressing the membrane protein of interest, how to prepare a membrane protein sample from yeast, and how to a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2014.12.010

    authors: Claes K,Guerfal M,Callewaert N

    更新日期:2015-01-01 00:00:00

  • Engineering Short Preorganized Peptide Sequences for Metal Ion Coordination: Copper(II) a Case Study.

    abstract::Peptides are multidentate chiral ligands capable of coordinating different metal ions. Nowadays, they can be obtained with high yield and purity, thanks to the advances on peptide/protein chemistry as well as in equipment (peptide synthesizers). Based on the identity and length of their amino acid sequences, peptides ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.04.007

    authors: Lima LM,Iranzo O

    更新日期:2016-01-01 00:00:00

  • In vitro and tissue culture methods for analysis of translation initiation on the endoplasmic reticulum.

    abstract::For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)31004-5

    authors: Stephens SB,Nicchitta CV

    更新日期:2007-01-01 00:00:00

  • RAG and HMGB1 proteins: purification and biochemical analysis of recombination signal complexes.

    abstract::Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA d...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)08032-3

    authors: Bergeron S,Anderson DK,Swanson PC

    更新日期:2006-01-01 00:00:00

  • Characterization of parvalbumin and polcalcin divalent ion binding by isothermal titration calorimetry.

    abstract::The elucidation of structure-affinity relationships in EF-hand proteins requires a reliable assay of divalent ion affinity. In principle, isothermal titration calorimetry (ITC) should be capable of furnishing estimates for Ca2+- and Mg2+-binding constants in these systems. And because the method yields the binding ent...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)04210-9

    authors: Henzl MT

    更新日期:2009-01-01 00:00:00

  • GEF and glucosylation assays on liposome-bound Rac.

    abstract::Rac binds tightly to lipid membranes through a lipid modification. The influence of the lipid membrane environment on the multiple interactions of Rac has not been well documented. In this chapter, we detail a method to prepare geranyl-geranylated Rac bound to liposomes of defined composition. With this method, one ca...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)06006-X

    authors: Mesmin B,Antonny B

    更新日期:2006-01-01 00:00:00

  • Exploration of self-renewal and pluripotency in ES cells using RNAi.

    abstract::Embryonic stem cells (ESCs) have the ability to expand indefinitely in vitro and give rise to cells of all three germ layers as well as germ cells. For these reasons, ESCs hold great promise for biomedicine. In order to harness the potential of pluripotent cells, it is necessary to first understand the molecular mecha...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)77018-X

    authors: Schaniel C,Lee DF,Lemischka IR

    更新日期:2010-01-01 00:00:00

  • Yeast osmoregulation.

    abstract::Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(07)28002-4

    authors: Hohmann S,Krantz M,Nordlander B

    更新日期:2007-01-01 00:00:00

  • Determination of glycosylation sites and disulfide bond structures using LC/ESI-MS/MS analysis.

    abstract::Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been so...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)15007-7

    authors: Yen TY,Macher BA

    更新日期:2006-01-01 00:00:00

  • Modeling and simulation of the immune system as a self-regulating network.

    abstract::Numerous aspects of the immune system operate on the basis of complex regulatory networks that are amenable to mathematical and computational modeling. Several modeling frameworks have recently been applied to simulating the immune system, including systems of ordinary differential equations, delay differential equati...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)67004-X

    authors: Kim PS,Levy D,Lee PP

    更新日期:2009-01-01 00:00:00

  • Expression, purification, and assay of cytosolic (catalytic) domains of membrane-bound mammalian adenylyl cyclases.

    abstract::The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha. They have served a...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(02)45012-4

    authors: Hatley ME,Gilman AG,Sunahara RK

    更新日期:2002-01-01 00:00:00

  • Measuring mitochondrial uncoupling protein-2 level and activity in insulinoma cells.

    abstract::Mitochondrial uncoupling protein-2 (UCP2) regulates glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells-the physiological role of the beta cell UCP2 remains a subject of debate. Experimental studies informing this debate benefit from reliable measurements of UCP2 protein level and activity. In this ch...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-405881-1.00015-X

    authors: Barlow J,Hirschberg V,Brand MD,Affourtit C

    更新日期:2013-01-01 00:00:00

  • Chemical biology of glycoproteins: From chemical synthesis to biological impact.

    abstract::Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained us...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.02.030

    authors: Li Y,Tran AH,Danishefsky SJ,Tan Z

    更新日期:2019-01-01 00:00:00

  • Rabring7: a target protein for rab7 small g protein.

    abstract::Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)03059-4

    authors: Mizuno K,Sakane A,Sasaki T

    更新日期:2005-01-01 00:00:00

  • Predicting G-protein-coupled receptors families using different physiochemical properties and pseudo amino acid composition.

    abstract::G-protein-coupled receptors (GPCRs) initiate signaling pathways via trimetric guanine nucleotide-binding proteins. GPCRs are classified based on their ligand-binding properties and molecular phylogenetic analyses. Nonetheless, these later analyses are in most case dependent on multiple sequence alignments, themselves ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-407865-9.00004-2

    authors: Rehman ZU,Mirza MT,Khan A,Xhaard H

    更新日期:2013-01-01 00:00:00

  • Purification and characterization of Escherichia coli and human nucleotide excision repair enzyme systems.

    abstract::Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been stu...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(06)08012-8

    authors: Reardon JT,Sancar A

    更新日期:2006-01-01 00:00:00

  • Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum.

    abstract::In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, additi...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(92)19028-5

    authors: Rexach MF,Schekman RW

    更新日期:1992-01-01 00:00:00

  • Image intensification techniques for detection of proteins in cultured cells.

    abstract::We describe here the components and uses of two image intensifier systems. The SIT camera system is convenient, relatively inexpensive, readily adaptable to most microscopes, and reliable. It suffers from lack of resolution at high gain levels and the inability to extend its sensitivity by accumulating an image over t...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(83)98154-5

    authors: Willingham MC,Pastan IH

    更新日期:1983-01-01 00:00:00

  • Production and characterization of monoclonal idiotypes and anti-idiotypes for small ligands.

    abstract::The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availabili...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(89)78005-8

    authors: Kussie PH,Albright G,Linthicum DS

    更新日期:1989-01-01 00:00:00