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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:

9 7 8 - 0 - 1 2 - 1 8 2 2 2 5 - 5

eBook ISBN:

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.

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
  - Introduction
  - Principle
  - Branched-Chain α-Keto Acid Dehydrogenase Complex Activity
  - Branched-Chain α-Keto Acid Dehydrogenase Kinase Activity
  - Activities of Branched-Chain α-Keto Acid Dehydrogenase Complex and Kinase in Various Mammalian Tissues
  - Remarks
  - Acknowledgments
- [7]: Simultaneous Quantification of Plasma Levels of α-Ketoisocaproate and Leucine by Gas Chromatography–Mass Spectrometry
  - Introduction
  - Procedures
  - Conclusions
- [8]: Synthesis of Methacrylyl-CoA and (R)- and (S)-3-Hydroxyisobutyryl-CoA
  - Introduction
  - Synthesis and Purification of Methacrylyl-CoA
  - Enzymatic Synthesis of (S)-3-Hydroxyisobutyryl-CoA
  - Chemical Synthesis of (R)- and (S)-3-Hydroxyisobutyryl-CoA
  - Analysis of Methacrylyl-CoA and 3-Hydroxyisobutyryl-CoA by Electrospray Ionization Mass Spectrometry
  - 1H Nuclear Magnetic Resonance Analysis of 3-Hydroxyisobutyryl-CoA
  - Acknowledgments
- [9]: Pathways of Leucine and Valine Catabolism in Yeast
  - Introduction
  - Culture Conditions and Handling of Yeast
  - Rationale of 13C-Labeling Experiments and General Requirements
  - Sample Preparation for Analysis by 13C Nuclear Magnetic Resonance
  - Identification of Signals in Nuclear Magnetic Resonance Spectrum
  - Analysis of Isoamyl and Isobutyl Alcohols by Gas Chromatography–Mass Spectrometry
  - Assay of α-Ketoisocaproate Reductase Activity in Yeast
  - Elucidation of Catabolic Pathways
  - Concluding Remarks
Section II: Cloning, Expression, and Purification of Enzymes of Branched-Chain Amino Acid Metabolism
- [10]: Isolation of Subunits of Acetohydroxy Acid Synthase Isozyme III and Reconstitution of Holoenzyme
  - Introduction
  - Isolation of Subunits of Acetohydroxy Acid Synthase Isozyme III
  - Reconstitution of Acetohydroxy Acid Synthase III from Its Subunits
  - Discussion
  - Acknowledgment
- [11]: Branched-Chain Amino-Acid Aminotransferase of Escherichia coli
  - Overview
  - Assay Method
  - Cloning of ilvE Gene
  - Purification Procedures
  - Properties
- [12]: Purification of Sodium-Coupled Branched-Chain Amino Acid Carrier of Pseudomonas aeruginosa
  - Bacteria and Plasmids
  - Overproduction of LIV-II Carrier
  - Preparation of Membranes
  - Preparation of Immunoaffinity Column
  - Purification of LIV-II Carrier
  - Reconstitution of LIV-II Carrier
  - Transport Assays
  - Concluding Remarks
- [13]: Reconstitution of Pseudomonas aeruginosa High-Affinity Branched-Chain Amino Acid Transport System
  - Introduction
  - Methods for Preparation of BraC
  - Methods for Preparation of Proteoliposomes
  - Transport Assay
  - Conclusion
- [14]: Purification of Pseudomonas putida Branched-Chain Keto Acid Dehydrogenase E1 Component
  - Introduction
  - Materials and Methods
  - Comments on Purification Procedure
- [15]: Pseudomonas mevalonii 3-Hydroxy-3-methylglutaryl-CoA Lyase
  - Introduction
  - Assay Methods
  - Expression of Active HMG-CoA Lyase
  - Purification of HMG-CoA Lyase
  - Characterization of Pseudomonas Mevalonii HMG-CoA Lyase
  - Acknowledgment
- [16]: Human 3-Hydroxy-3-methylglutaryl-CoA Lyase
  - Introduction
  - Assay Methods
  - Design of Expression Plasmid for Human Mitochondrial HMG-CoA Lyase
  - Expression of Mitochondrial HMG-CoA Lyase
  - Purification of Human HMG-CoA Lyase
  - Characterization of Human HMG-CoA Lyase
  - Acknowledgments
- [17]: Branched-Chain α-Keto Acid Dehydrogenase Kinase
  - Introduction
  - Principle for Purification of Branched-Chain α-Keto Acid Dehydrogenase Kinase
  - Assay Methods for Branched-Chain α-Keto Acid Dehydrogenase Complex
  - Assay Methods for Branched-Chain α-Keto Acid Dehydrogenase Kinase
  - Purification of Rat Liver Branched-Chain α-Keto Acid Dehydrogenase–Kinase Complex
  - Purification of Rat Heart Branched-Chain α-Keto Acid Dehydrogenase–Kinase Complex
  - Procedure for Concurrent Purification of Branched-Chain α-Keto Acid Dehydrogenase– and Pyruvate Dehydrogenase–Kinase Complexes from Rat Heart
  - Purification of Branched-Chain α-Keto Acid Dehydrogenase Kinase
  - Preparation of Kinase-Depleted Branched-Chain α-Keto Acid Dehydrogenase Complex
  - Branched-Chain α-Keto Acid Dehydrogenase Kinase cDNA
  - Expression of Recombinant Branched-Chain α -Keto Acid Dehydrogenase Kinase in Escherichia coli
  - Properties of Native Branched-Chain α-Keto Acid Dehydrogenase Kinase, cDNA Encoding Branched-Chain α-Keto Acid Dehydrogenase Kinase, and Wild-Type Recombinant Branched- Chain α-Keto Acid Dehydrogenase Kinase
  - Acknowledgments
- [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
  - Acknowledgments
- [21]: Mammalian Methylmalonate-Semialdehyde Dehydrogenase
  - Introduction
  - 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
  - Acknowledgments
- [22]: Mammalian 3-Hydroxyisobutyrate Dehydrogenase
  - Introduction
  - Assay of 3-Hydroxyisobutyrate Dehydrogenase Activity
  - Native Rabbit Liver 3-Hydroxyisobutyrate Dehydrogenase
  - Recombinant Rat 3-Hydroxyisobutyrate Dehydrogenase
  - Characteristics of Native, Wild-type Recombinant, and Mutant 3-Hydroxyisobutyrate Dehydrogenase
  - Acknowledgments
- [23]: 3-Hydroxyisobutyryl-CoA Hydrolase
  - Introduction
  - Assay Methods for 3-Hydroxyisobutyryl-CoA Hydrolase
  - Purification of 3-Hydroxyisobutyryl-CoA Hydrolase from Rat Liver
  - Generation of Recombinant 3-Hydroxyisobutyryl-CoA Hydrolase
  - Properties of Native and Recombinant 3-Hydroxyisobutyryl-CoA Hydrolase
  - Acknowledgments
- [24]: Mammalian Branched-Chain Acyl-CoA Dehydrogenases: Molecular Cloning and Characterization of Recombinant Enzymes
  - Cloning of Mammalian Isovaleryl-CoA Dehydrogenases and Short/Branched-Chain Acyl-CoA Dehydrogenases
  - Assay Methods
  - Expression and Purification of Recombinant Human Isovaleryl-CoA Dehydrogenase
  - Characteristics of Recombinant Human Isovaleryl-CoA Dehydrogenase
  - Expression and Characterization of Rat and Human Short/Branched-Chain Acyl-CoA Dehydrogenase
- [25]: 3-Hydroxy-3-methylglutaryl-CoA Reductase
  - 1 Reactions Catalyzed by 3-Hydroxy-3-methylglutaryl-CoA Reductase*
  - 2 Preparation of Substrates*
  - 3 Assay of 3-Hydroxy-3-methylglutaryl-CoA Reductase Activity*
  - 4 Syrian Hamster 3-Hydroxy-3-methylglutaryl-CoA Reductase*
  - 5 Pseudomonas mevalonii 3-Hydroxy-3-methylglutaryl-CoA Reductase*
  - 6 Haloferax volcanii 3-Hydroxy-3-methylglutaryl-CoA Reductase*
  - 7 Sulfolobus solfataricus 3-Hydroxy-3-methylglutaryl-CoA Reductase*
- [26]: Characterization of 3-Methylcrotonyl-CoA Carboxylase from Plants
  - Introduction
  - Purification and Assay Methods
  - Characterization of 3-Methylcrotonyl-CoA Carboxylase
- [27]: Purification of D-Hydroxyisovalerate Dehydrogenase from Fusarium sambucinum
  - Growth of Organisms
  - Assay Methods
  - Purification of D-2-Hydroxyisovalerate Dehydrogenase
  - Properties of D-2-Hydroxyisovalerate Dehydrogenase
- [28]: Purification and Characterization of Recombinant 3-Isopropylmalate Dehydrogenases from Thermus thermophilus Other Microorganisms
  - Introduction
  - Assay Methods
  - Purification of Recombinant Thermus thermophilus 3-Isopropylmalate Dehydrogenase
  - Purification Procedures for 3-Isopropylmalate Dehydrogenase from Other Sources
  - Storage
  - Properties of 3-Isopropylmalate Dehydrogenase
  - Thermostability
  - Acknowledgments
- [29]: Wild-Type and Hexahistidine-Tagged Derivatives of Leucine-Responsive Regulatory Protein from Escherichia coli
  - Purification of Wild-Type Leucine-Responsive Regulatory Protein
  - Purification of Hexahistidine–Leucine-Responsive Regulatory Protein
  - Remarks
  - Acknowledgments
- [30]: Purification of Branched-Chain Keto Acid Dehydrogenase Regulator from Pseudomonas putida
  - Introduction
  - Materials and Reagents
  - Methods
  - Discussion
  - Summary
- [31]: Mitochondrial Import of Mammalian Branched-Chain α-Keto Acid Dehydrogenase Complex Subunits
  - General Methods
  - 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