Cytochrome P450 Protocols / Edition 3

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Cytochromes P450 (CYPs) comprise a large superfamily of proteins that are of central importance in the detoxification or activation of a tremendous number of natural and synthetic hydrophobic xenobiotics, including many therapeutic drugs, chemical carcinogens and environmental pollutants. CYPs are important in mediating interactions between an organism and its chemical environment and in the regulation of physiological processes. Cytochrome P450 Protocols, Third Edition focuses on high-throughput methods for the simultaneous analysis of multiple CYPs, substrates or ligands. Although the emphasis is on CYPs of mammalian origin, it reflects an increasing interest in CYPs of bacterial species. Also included are chapters on cytochrome P450 reductase (the redox partner of CYPs) and the flavin-containing monooxygenases (FMOs), and metabolomic and lipidomic approaches for identification of endogenous substrates of CYPs (‘de-orphanizing’ CYP substrates). Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible, Cytochrome P450 Protocols, Third Edition provides a wide range of techniques accessible to researchers in fields as diverse as biochemistry, molecular biology, pharmacology, toxicology, environmental biology and genetics.

The book contains black-and-white illustrations.

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Editorial Reviews

Doody's Review Service
Reviewer: Jed N. Lampe, Ph.D.(University of Kansas Medical Center)
Description: With this third edition, the authors and contributors continue to provide what has become a tour de force of bench top manuals for the cytochrome P450 field. Building on the success of the first two editions, this book expands and diversifies a set of well-established, validated protocols for the production and analysis of cytochrome P450 enzymes (CYPs) and their diverse metabolic products.
Purpose: The authors focus on high-throughput techniques for the examination of multiple CYPs in both in vitro and in vivo models, which is a notable expansion and extension of the fundamental techniques established in the earlier two editions.
Audience: Although predominantly useful for an industrial audience due to the high-throughput nature of most of the techniques, this book should also prove of interest to graduate students and postdoctoral researchers who are new to the field of P450 science, or who would like to expand their research directions.
Features: The highlight of this edition, as well as the previous editions, is the step-by-step protocols with extensive troubleshooting notes at the end of each chapter. These are augmented with selected and appropriate color illustrations that enhance readers' understanding of each technique.
Assessment: This stands alone as the premier book for this field, which is sorely lacking in publications of this type. Researchers in the P450 field will find this to be a useful bench top guide that will provide them with the tools to explore and understand CYPs, both in vitro and in vivo.
A collection of techniques for investigating the large superfamily of proteins that are of central importance in detoxifying or activating many foreign hydrophobic compounds, among them therapeutic drugs, chemical carcinogens, and environmental pollutants. The focus is on P450 species from mammals, but the methods are also suitable for those from other sources. The techniques described are used for purifying, spectral analysis, expression in heterologous cells, determining functional capabilities, detecting and quantifying, gene expression and its regulation, and cell culture. Addressed to biologists and chemists with no previous knowledge of the techniques. Annotation c. by Book News, Inc., Portland, Or.
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Product Details

  • ISBN-13: 9781627033206
  • Publisher: Springer-Verlag New York, LLC
  • Publication date: 3/31/2013
  • Series: Methods in Molecular Biology Series, #987
  • Edition description: 3rd ed. 2013
  • Edition number: 3
  • Pages: 319
  • Product dimensions: 7.10 (w) x 10.00 (h) x 0.90 (d)

Table of Contents

1. Bioluminescent Assays for Cytochrome P450 Enzymes

Douglas S. Auld, Henrike Veith, and James J. Cali

2. In Vitro Drug-Drug Interaction Assay

Michael Zientek and Kuresh Youdim

3. High-Throughput Mass Spectrometric Cytochrome P450 Inhibition Screening

Kheng B. Lim, Can C. Ozbal, and Daniel B. Kassel

4. The Synthesis, Characterization and Application of 13C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites

Christopher McCullough, Phani Kumar Pullela, Sang-Choul Im, Lucy Waskell, Daniel Sem

5. High-Throughput Fluorescence Assay for Cytochrome P450 Mechanism-Based Inactivators

Cesar Kenaan, Haoming Zhang, and Paul F. Hollenberg

6. Identification of Endogenous Substrates of Orphan Cytochrome P450 Enzymes Through the Use of Untargeted Metabolomics Approaches

Qian Cheng and F. Peter. Guengerich

7. Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium Tuberculosis

Hugues Ouellet, Eric D. Chow, Shenheng Guan, Jeffery S. Cox, Alma L. Burlingame, and Paul R. Ortiz de Montellano

8. An Escherichia coli Expression-Based Approach for Porphyrin Substitution in Heme Proteins

Michael B. Winter, Joshua J. Woodward, and Michael A. Marletta

9. Expression in Escherichia coli of a Cytochrome P450 Enzyme with a Cobalt Protoporphyrin IX Prosthetic Group

Wesley E. Straub, Clinton R. Nishida, and Paul R. Ortiz de Montellano

10. Nanodiscs in the Studies of Membrane-Bound Cytochrome P450 Enzymes

A. Luthra, M. Gregory, Y.V. Grinkova, I.G. Denisov , and S.G. Sligar

11. Rapid LC-MS Drug Metabolite Profiling Using Bioreactor Particles

Linlin Zhao, Besnik Bajrami, and James F. Rusling

12. Fluorescence-Based Screening of cytochrome P450 activities in Intact Cells

M. Teresa Donato and M. José Gómez-Lechón

13. Screening for Cytochrome P450 Reactivity with a Reporter Enzyme

Kersten S. Rabe and Christof M. Niemeyer

14. High-Throughput Fluorescence Assay of Cytochrome P450 3A4

Qian Cheng and F. Peter. Guengerich

15. Targeted Protein Capture for Analysis of Electrophile-Protein Adducts

Rebecca E. Connor, Simona G. Codreanu, Lawrence J. Marnett, and Daniel C. Liebler

16. DNA Shuffling of Cytochrome P450 Enzymes

James B.Y.H. Behrendorff, Wayne A. Johnston, and Elizabeth M.J. Gillam

17. Measurement of P450 Difference Spectra Using Intact Cells

Wayne A. Johnston and Elizabeth M.J. Gillam

18. DNA Shuffling of Cytochromes P450 for Indigoid Pigment Production

Nedeljka N. Rosic

19. P450 oxidoreductase – Genotyping, Expression, Purification of Recombinant Protein and Activity Assessments of Wild-Type and Mutant Protein

Vishal Agrawal and Walter L. Miller

20. LICRED: A Versatile Drop-In Vector for Rapid Generation of Redox-Self-Sufficient Cytochromes P450

Federico Sabbadin, Gideon G. Grogan and Neil C. Bruce

21. Update on Allele Nomenclature for Human Cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database

Sarah C. Sim and Magnus Ingelman-Sundberg

22. Simultaneous in vivo Phenotyping of CYP Enzymes

Sussan Ghassabian and Michael Murray

23. Detection of Regulatory Polymorphisms: High-Throughput Capillary DNase-I Footprinting

Matthew Hancock and Elizabeth A. Shephard

24. Isolation of Mouse Hepatocytes

Mina Edwards, Lyndsey Houseman, Ian R. Phillips, and Elizabeth A. Shephard

25. Highly Efficient SiRNA and Gene Transfer into Hepatocyte-like HepaRG Cells and Primary Human Hepatocytes: New Means for Drug Metabolism and Toxicity Studies

Véronique Laurent, Denise Glaise, Tobias Nübel, David Gilot, Anne Corlu, and Pascal Loyer

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