Screening and characterizing human NAT2 variants.

Abstract:

:Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli, can be detected by the appearance of Lac+ revertants. The mutagenesis assay is based on the reversion of an E. coli lacZ frameshift allele; the host strain for the assay is devoid of endogenous NAT activity and a plasmid vector is used for expression of human NAT2. A high-throughput version of the assay facilitates rapid screening of pools of NAT2 variants generated (for example) by random mutagenesis. Along with the methods for these assays, we present selected results of a screening effort in which mutations along the length of the NAT2 sequence have been examined. Homology modeling and simulated annealing have been used to analyze the potential effects of these mutations on structural integrity and substrate binding.

journal_name

Methods Enzymol

journal_title

Methods in enzymology

authors

Savulescu MR,Mushtaq A,Josephy PD

doi

10.1016/S0076-6879(05)00011-X

subject

Has Abstract

pub_date

2005-01-01 00:00:00

pages

192-215

eissn

0076-6879

issn

1557-7988

pii

S0076-6879(05)00011-X

journal_volume

400

pub_type

杂志文章
  • Predicting fluorescence lifetimes and spectra of biopolymers.

    abstract::Use of fluorescence in biology and biochemistry for imaging and characterizing equilibrium and dynamic processes is growing exponentially. Much progress has been made in the last few years on the microscopic understanding of the underlying principles of what controls the wavelength and quenching of fluorescence in bio...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-381270-4.00001-9

    authors: Callis PR

    更新日期:2011-01-01 00:00:00

  • Identification and quantification of protein posttranslational modifications.

    abstract::Posttranslational modifications (PTMs) of proteins perform crucial roles in regulating the biology of the cell. PTMs are enzymatic, covalent chemical modifications of proteins that typically occur after the translation of mRNAs. These modifications are relevant because they can potentially change a protein's physical ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(09)63040-8

    authors: Farley AR,Link AJ

    更新日期:2009-01-01 00:00:00

  • A guide for drug inducible transcriptional activation with HIT systems.

    abstract::Precise investigation and manipulation of gene function often require modulation in a controlled and dynamic manner. In this chapter, we describe the methods to apply HIT systems for drug inducible transcriptional activation or simultaneous activation and genome editing in human cells. Together with those for editing,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2019.02.032

    authors: Zhao C,Wei S,Wang Y

    更新日期:2019-01-01 00:00:00

  • TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

    abstract::Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1. ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2018.04.024

    authors: Young AP,Bandarian V

    更新日期:2018-01-01 00:00:00

  • Functional Study of the Vitamin K Cycle Enzymes in Live Cells.

    abstract::Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylati...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.10.015

    authors: Tie JK,Stafford DW

    更新日期:2017-01-01 00:00:00

  • Characterization of EHT 1864, a novel small molecule inhibitor of Rac family small GTPases.

    abstract::There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However,...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)00409-0

    authors: Onesto C,Shutes A,Picard V,Schweighoffer F,Der CJ

    更新日期:2008-01-01 00:00:00

  • Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    abstract::Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrome...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2016.01.007

    authors: Fan J,Baeza J,Denu JM

    更新日期:2016-01-01 00:00:00

  • Electrochemical biosensing with odorant binding proteins.

    abstract::The development of sensors that mimic the natural smell sensing mechanism and selectively recognizes the odorants remains highly challenging. Electrochemical based sensing approaches aiming at monitoring molecular recognition events between surface receptors and analytes in solution or in the gas phase, are one possib...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2020.04.071

    authors: Szunerits S,Boukherroub R,Vasilescu A

    更新日期:2020-01-01 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

  • Molecular tools for investigating ANME community structure and function.

    abstract::Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabol...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-385112-3.00004-4

    authors: Hallam SJ,Pagé AP,Constan L,Song YC,Norbeck AD,Brewer H,Pasa-Tolic L

    更新日期:2011-01-01 00:00:00

  • Chapter 9 the duffy antigen receptor for chemokines.

    abstract::The Duffy blood group antigen is a serpentine protein with seven transmembrane domains that is not coupled to G-proteins or other known intracellular effectors. In addition to erythrocytes, it is also expressed in endothelial cells and neurons. In recent years the Duffy antigen has received much attention because of i...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(09)05409-3

    authors: Rot A,Horuk R

    更新日期:2009-01-01 00:00:00

  • The geochemical record of the ancient nitrogen cycle, nitrogen isotopes, and metal cofactors.

    abstract::The nitrogen (N) cycle is the only global biogeochemical cycle that is driven by biological functions involving the interaction of many microorganisms. The N cycle has evolved over geological time and its interaction with the oxygen cycle has had profound effects on the evolution and timing of Earth's atmosphere oxyge...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-381294-0.00022-5

    authors: Godfrey LV,Glass JB

    更新日期:2011-01-01 00:00:00

  • Regulation of cancer metabolism by oncogenes and tumor suppressors.

    abstract::Cell proliferation requires the coordination of multiple signaling pathways as well as the provision of metabolic substrates. Nutrients are required to generate such building blocks and their form of utilization differs to significant extents between malignant tissues and their nontransformed counterparts. Thus, oncog...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/B978-0-12-416618-9.00003-0

    authors: Iurlaro R,León-Annicchiarico CL,Muñoz-Pinedo C

    更新日期:2014-01-01 00:00:00

  • Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability.

    abstract::Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2017.03.018

    authors: Sannino V,Pezzimenti F,Bertora S,Costanzo V

    更新日期:2017-01-01 00:00:00

  • TM4 microarray software suite.

    abstract::Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(06)11009-5

    authors: Saeed AI,Bhagabati NK,Braisted JC,Liang W,Sharov V,Howe EA,Li J,Thiagarajan M,White JA,Quackenbush J

    更新日期:2006-01-01 00:00:00

  • Isolation and characterization of apolipoproteins A-I, A-II, and A-IV.

    abstract::A number of different analytical techniques are now available for the isolation of apoA-I, apoA-II, and apoA-IV. The choice of a particular technique is dependent on the instrumentation available, and the quantity of isolated apolipoprotein required. The isolation and characterization of the separate isoforms and the ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(86)28070-2

    authors: Brewer HB Jr,Ronan R,Meng M,Bishop C

    更新日期:1986-01-01 00:00:00

  • Characterization of the omega class of glutathione transferases.

    abstract::The Omega class of cytosolic glutathione transferases was initially recognized by bioinformatic analysis of human sequence databases, and orthologous sequences were subsequently discovered in mouse, rat, pig, Caenorhabditis elegans, Schistosoma mansoni, and Drosophila melanogaster. In humans and mice, two GSTO genes h...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(05)01005-0

    authors: Whitbread AK,Masoumi A,Tetlow N,Schmuck E,Coggan M,Board PG

    更新日期:2005-01-01 00:00:00

  • In vitro and in vivo assays for the activity of Drosha complex.

    abstract::MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(07)27005-3

    authors: Lee Y,Kim VN

    更新日期:2007-01-01 00:00:00

  • Use of Ras-transformed human ovarian surface epithelial cells as a model for studying ovarian cancer.

    abstract::The Ras gene family has been implicated in the development of many human epithelial cancers. Mutations in K-ras or its downstream mediator BRAF have been detected in about two thirds of low-grade serous carcinomas and borderline serous tumors; mutations in K-ras are also often present in benign and invasive mucinous o...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(05)07052-7

    authors: Rosen DG,Yang G,Bast RC Jr,Liu J

    更新日期:2006-01-01 00:00:00

  • Catalytic pathway of manganese superoxide dismutase by direct observation of superoxide.

    abstract::Measurement of catalysis by MnSOD using direct observation of the UV absorbance of superoxide allows determination of steady-state catalytic constants. Stabilizing superoxide in aprotic solvents such as dimethyl sulfoxide permits the use of stopped-flow spectrophotometry, although significant information is lost in th...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/s0076-6879(02)49321-4

    authors: Silverman DN,Nick HS

    更新日期:2002-01-01 00:00:00

  • Deuteration in Biological Neutron Reflectometry.

    abstract::Neutron reflectometry (NR) is uniquely positioned in structural biology, because of its ability to characterize biomolecular interfacial architectures like lipid membranes and membrane-associated proteins nondestructively and in their native environment. Mimicking biological processes, samples can be manipulated and t...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.05.019

    authors: Heinrich F

    更新日期:2016-01-01 00:00:00

  • Measurement of lipolysis products secreted by 3T3-L1 adipocytes using microfluidics.

    abstract::Glass microfluidic devices have been fabricated to monitor the secretion of glycerol or fatty acids from cultured murine 3T3-L1 adipocytes. In the current studies, adipocytes are perfused in a reversibly sealed cell chamber, and secreted products are analyzed by enzyme assay on either a single- or dual-chip device. Th...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/B978-0-12-800280-3.00011-6

    authors: Dugan CE,Kennedy RT

    更新日期:2014-01-01 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

  • Anti-inflammatory and cytoprotective properties of hydrogen sulfide.

    abstract::Hydrogen sulfide is an endogenous gaseous mediator that plays important roles in many physiological processes in microbes, plants, and animals. This chapter focuses on the important roles of hydrogen sulfide in protecting tissues against injury, promoting the repair of damage, and downregulating the inflammatory respo...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2014.11.034

    authors: Gemici B,Wallace JL

    更新日期:2015-01-01 00:00:00

  • Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.

    abstract::Biochemical analysis of the components of the mRNA decay machinery is crucial to understand the mechanisms of mRNA decay. The Lsm1p-7p-Pat1p complex is a key activator of decapping in the 5' to 3'-mRNA decay pathway that is highly conserved in all eukaryotes. The first step in this pathway is poly(A) shortening that i...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(08)02603-7

    authors: Tharun S

    更新日期:2008-01-01 00:00:00

  • Selective amino acid segmental labeling of multi-domain proteins.

    abstract::The steady technical advances of nuclear magnetic resonance (NMR) over the past decades enabled a significant increase in the molecular size of protein particles that can be subjected to a structural and functional characterization in solution. The larger molecular weight of such proteins is accompanied with an increa...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/bs.mie.2015.05.028

    authors: Michel E,Allain FH

    更新日期:2015-01-01 00:00:00

  • High-speed atomic force microscopy techniques for observing dynamic biomolecular processes.

    abstract::Atomic force microscopy (AFM) enables direct visualization of single-protein molecules in liquids at submolecular resolution. High-speed AFM further makes it possible to visualize dynamic biomolecular processes at subsecond resolution. However, dynamic imaging of biomolecular processes imposes various requirements on ...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/S0076-6879(10)75020-5

    authors: Yamamoto D,Uchihashi T,Kodera N,Yamashita H,Nishikori S,Ogura T,Shibata M,Ando T

    更新日期:2010-01-01 00:00:00

  • Structural and biophysical methods to analyze clock function and mechanism.

    abstract::Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttrans...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/bs.mie.2014.10.004

    authors: Egli M

    更新日期:2015-01-01 00:00:00

  • Isolation and identification of neuroendocrine peptides from milk.

    abstract::Hormone, hormone-like substances, and neuroendocrine peptides are natural constituents of milk, and they may have an important physiological and pharmacological role in neonate development. The concentration of these peptides in milk greatly exceeds those in serum and implies an active concentration mechanism in the m...

    journal_title:Methods in enzymology

    pub_type: 杂志文章

    doi:10.1016/0076-6879(89)68038-x

    authors: Hazum E

    更新日期:1989-01-01 00:00:00

  • Identification by MS/MS of disulfides produced by a functional redox transition.

    abstract::Among posttranslational modifications of proteins entailed with signal transduction, the redox transition is today brought to the focus as a major biochemical event accounting for the signaling functions of reactive oxygen species. Thermodynamic and kinetic criteria highlight hydroperoxides and protein disulfides as s...

    journal_title:Methods in enzymology

    pub_type: 杂志文章,评审

    doi:10.1016/S0076-6879(10)73011-1

    authors: Mauri P,Toppo S,De Palma A,Benazzi L,Maiorino M,Ursini F

    更新日期:2010-01-01 00:00:00