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Branched-Chain Amino Acids, Part B
1st Edition - August 25, 2000
Editors: John R. Sokatch, John N. Abelson, Melvin I. Simon, Robert Adron Harris
Hardback ISBN:9780121822255
9 7 8 - 0 - 1 2 - 1 8 2 2 2 5 - 5
eBook ISBN:9780080496795
9 7 8 - 0 - 0 8 - 0 4 9 6 7 9 - 5
Volume 324 of Methods in Enzymology supplements Volume 166. It includes genetic information (cloning, gene expression) and information on human genetic diseases not available when… Read more
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Volume 324 of Methods in Enzymology supplements Volume 166. It includes genetic information (cloning, gene expression) and information on human genetic diseases not available when Volume 166 was published.General Description of the Series:The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.
Preparation of substrates and assay of enzymes
Cloning, expression, and purification of enzymes
Detection and consequences of genetic defects
Regulation and expression of enzymes
Biochemists, microbiologists, analytical chemists, geneticists, biomedical researchers, and nutritionists.
Contributors to volume 324
Preface
Volumes in Series
Section I: Preparation of Substrates, Assays of Intermediates and Enzymes, and Use of Enzyme Inhibitors
[1]: Synthesis and Gas Chromatography/Mass Spectrometry Analysis of Stereoisomers of 2-Hydroxy-3-methylpentanoic Acid
Introduction
Synthesis
[2]: Analysis of Intracellular Metabolites as Tool for Studying Branched-Chain Amino Acid Biosynthesis and Its Inhibition in Bacteria
Introduction
Determination of Intracellular Concentrations of 2-Keto Acids
Determination of Intracellular Concentrations of Acetohydroxy Acids
Discussion
Acknowledgment
[3]: Determination of Branched-Chain L-Amino-Acid Aminotransferase Activity
Introduction
Assay Using Branched-Chain L-Amino Acid Substrate
Materials and Methods
Assays Using Branched-Chain 2-Oxo Acid Substrates
Materials and Methods
Sample Preparation
Tissue Activities and Kinetic Properties
[4]: Analysis of (S)- and (R)-3-Methyl-2-oxopentanoate Enantiomorphs in Body Fluids
Introduction
Principle of Method
Materials and Methods
Performance of Method
Concentrations in Human Body Fluids
[5]: Spectrophotometric Assay for Measuring Branched-Chain Amino Acids
Introduction
Perspectives
Acknowledgments
[6]: Determination of Branched-Chain α-Keto Acid Dehydrogenase Activity State and Branched-Chain α-Keto Acid Dehydrogenase Kinase Activity and Protein in Mammalian Tissues
[18]: Expression of E1 Component of Human Branched-Chain α-Keto Acid Dehydrogenase Complex in Escherichia coli by Cotransformation with Chaperonins GroEL GroES
Introduction
Induction of Mammalian Maltose-Binding Protein–E1 Fusion by Cotransformation with Chaperonins GroEL/GroES
Efficient Expression and Purification of Hexahistidine-Tagged Human E1
Isolation and Separation of an αβ Heterodimeric Assembly Intermediate
Expression of Mutant Human E1 in Aberrant Assembly State
[19]: Production of Recombinant Mammalian Holo-E2 and E3 and Reconstitution of Functional Branched-Chain α-Keto Acid Dehydrogenase Complex with Recombinant E1
Production of Functional E2 and E3 Componants
Radiochemical Assays for E1 or E2 Based on Overall Reaction of a Reconstituted Recombinant Branched-Chain α-Keto Acid Dehydrogenase Complex
[20]: Production of Recombinant E1 Component of Branched-Chain α-Keto Acid Dehydrogenase Complex
Introduction
Construction of Prokaryotic Expression Vector for Branched-Chain α-Keto Acid Dehydrogenase E1
Site-Directed Mutagenesis of Branched-Chain α-Keto Acid Dehydrogenase E1α
Expression and Purification of Recombinant Branched-Chain α-Keto Acid Dehydrogenase E1
Assay of Branched-Chain α-Keto Acid Dehydrogenase Activity
Reconstitution of Branched-Chain α-Keto Acid Dehydrogenase E1/E2 Complex
Assay of Branched-Chain α-Keto Acid Dehydrogenase E2 Binding by Mutant Branched-Chain α-Keto Acid Dehydrogenase E1 Enzymes
Characteristics of Wild-Type and Mutant Branched-Chain α-Keto Acid Dehydrogenase E1s
Purification of Native Methylmalonate-semialdehyde Dehydrogenase from Rat Liver
Activity Assay
Cloning Strategy Used to Obtain Methylmalonate-semialdehyde Dehydrogenase cDNA
Expression of Wild-Type Methylmalonate-semialdehyde Dehydrogenase in Escherichia coli
Preparation and Expression of Methylmalonate-semialdehyde Dehydrogenase Mutants
Characteristics of Native Methylmalonate-semialdehyde Dehydrogenase cDNA Encoding Rat Methylmalonate-semialdehyde Dehydrogenase, and Wild-Type Recombinant Methylmalonate-semialdehyde Dehydrogenase
Import and Processing of Preproteins by Mitochondria
Conclusions
Acknowledgments
[32]: Cloning, Expression, and Purification of Mammalian 4-Hydroxyphenylpyruvate Dioxygenase/α-Ketoisocaproate Dioxygenase
Introduction
Materials and Methods
Purification of 4-Hydroxyphenylpyruvate Dioxygenase
Comments on Cloning, Isozymes, Expression, and Purification of 4-Hydroxyphenylpyruvate Dioxygenase
Conclusions
[33]: Mammalian Branched-Chain Aminotransferases
Enzyme Assay
Cloning of Mammalian Mitochondrial and Cytosolic Branched-Chain Aminotransferases
Insertion of Human Branched-Chain Aminotransferase Isoenzyme cDNAs into Expression Plasmid
Transformation Protocol
Expression of Mitochondrial and Cytosolic Branched-Chain Aminotransferases
Purification of Mitochondrial Branched-Chain Aminotransferase
[34]: Branched-Chain-Amino-Acid Transaminases of Yeast Saccharomyces cerevisiae
Introduction
Assays
[35]: Purification, Properties, and Sequencing of Aminoisobutyrate Aminotransferases from Rat Liver
Introduction
Assay Method
Purification of D- and L-AIBATs
Properties
Interconversion between D- and L-Enantiomers of β-Aminoisobutyrate
Cloning and Sequencing of Rat D-AIBAT and L-AIBAT
[36]: Branched-Chain Keto Acid Dehydrogenase of Yeast
Introduction
Materials and Reagents
Assay Method
Growth and Handling of Yeast
Harvesting and Breaking Yeast
Purification of Yeast Branched-Chain α-Keto Acid Dehydrogenase Complex
Properties
Acknowledgments
[37]: β-Alanine Synthase an Enzyme Involved in Catabolism of Uracil and Thymine
Enzyme Assays
Isotope Assay
Purification of β-Alanine Synthase
Properties of Enzyme
Section III: Detection and Consequences of Genetic Defects in Genes Encoding Enzymes of Branched-Chain Amino Acid Metabolism
[38]: Diagnosis and Mutational Analysis of Maple Syrup Urine Disease Using Cell Cultures
Enzyme Analysis of Maple Syrup Urine Disease with Intact Cultured Lymphoblasts or Fibroblasts
Identification and Mutational Analysis of Affected Branched-Chain α-Keto Acid Dehydrogenase Subunits in Maple Syrup Urine Disease Cells
[39]: Detection of Gene Defects in Branched-Chain Amino Acid Metabolism by Tandem Mass Spectrometry of Carnitine Esters Produced by Cultured Fibroblasts
Introduction
Procedure
Application
Discussion
[40]: Molecular and Enzymatic Methods for Detection of Genetic Defects in Distal Pathways of Branched-Chain Amino Acid Metabolism
Assay of Short-Chain 3-Ketoacyl-CoA Thiolase Ketothiolase (β-Ketothiolase) Activity
Mutation Analysis of Short-Chain 3-Ketoacyl-CoA Thiolase
Assay of 3-Methylcrotonyl-CoA Carboxylase and Propionyl-CoA Carboxylase
Mutation Analysis of PCCB Gene
Assay of 3-Methylglutaconyl-CoA Hydratase
Assay of 3-Hydroxy-3-Methylglutaryl-CoA Lyase
Methods for Analyses of Defects in L-Valine Catabolic Pathway
Acknowledgments
[41]: Genetic Defects in E3 Component of α-Keto Acid Dehydrogenase Complexes
Introduction
Identification of Genetic Defects in E3-Deficient Patients
Findings in E3-Deficient Subjects and Their Families
Acknowledgments
[42]: Targeting E3 Component of α-Keto Acid Dehydrogenase Complexes
Introduction
Cloning of Mouse Dld Gene
Construction of Dld Gene Targeting Vector
Targeted Disruption of Dld Gene in Embryonic Stem Cells
Analysis of Targeted Embryonic Stem Cells
Establishment of Chimeric Mice
Identification of Genotype of Disrupted Dld Gene
Analysis of Dld+/– Animals
Identification of the Dld–/– Embryos
Analysis of Dld–/– Embryos
Prospect
Acknowledgments
Section IV: Regulation and Expression of Enzymes of Branched-Chain Amino Acid Metabolism
[43]: Regulation of Expression of Branched-Chain α-Keto Acid Dehydrogenase Subunits in Permanent Cell Lines
General Methodology
Results
Summary
Acknowledgments
[44]: Expression of Murine Branched-Chain α-Keto Acid Dehydrogenase Kinase
General Methods
Remarks
Acknowledgments
[45]: Regulation of Branched-Chain α-Keto Acid Dehydrogenase Kinase Gene Expression by Glucocorticoids in Hepatoma Cells and Rat Liver
Introduction
H4IIE Cell Culture
Animal Model
Promoter Studies of BCKD Kinase Gene
Primary Hepatocytes
Remarks
Author Index
Subject Index
No. of pages: 550
Language: English
Published: August 25, 2000
Imprint: Academic Press
Hardback ISBN: 9780121822255
eBook ISBN: 9780080496795
JS
John R. Sokatch
Affiliations and expertise
University of Texas, Austin, U.S.A.
JA
John N. Abelson
Affiliations and expertise
California Institute of Technology, Division of Biology, Pasadena, U.S.A.