Monoamine Oxidase: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Purification of MAO A and MAO B from Mammalian Tissue Sources
1 Introduction
2 Materials
2.1 Mammalian Tissues
2.2 Solutions Used in the Preparation of Human Placental and Bovine Liver Mitochondria
2.3 Buffers and Reagents Used for Purification of Human Placental MAO A and Bovine Liver MAO B
2.4 Instruments and Appliances
3 Methods
3.1 Preparation of Human Placental Mitochondria
3.2 Preparation of Bovine Liver Mitochondria
3.3 Extraction and Purification of Human Placental MAO A
3.4 Preparation of Mitochondrial Outer Membranes and Purification of Bovine Liver MAO B
4 Notes
References
Chapter 2: Purification of Recombinant Eukaryotic MAO A and MAO B Utilizing the Pichia pastoris Expression System
1 Introduction
2 Materials
2.1 Plasmid Source
2.2 Media and Reagents for Pichia pastoris Cell Cultures
2.3 Buffers and Reagents for Purification of Recombinant MAOs
2.4 Instruments and Tools
3 Methods
3.1 Construct Preparation
3.2 Small-Scale Growth of Pichia pastoris Cultures
3.3 Large-Scale Growth of P. pastoris-Expressing Strains
3.4 Preparation of Membrane Particles Containing MAO Activity (See Note 5)
3.5 Extraction and Solubilization of MAO from Membrane Particles
3.6 Column Purification of Detergent-Solubilized MAO
3.6.1 Human and Rat MAO A
3.6.2 Human and Rat MAO B
3.6.3 Zebrafish MAO
4 Notes
References
Chapter 3: The Peroxidase-Coupled Assay to Measure MAO Enzymatic Activity
1 Introduction
1.1 HRP-Coupled Spectrophotometric Assay
1.2 HRP-Coupled Fluorometric Assay
2 Materials
2.1 Purified MAOs
2.2 Buffers, Substrates, and Other Reagents
2.3 Tools
2.4 Instrumentation and Software
3 Methods
3.1 HRP-Coupled Spectrophotometric Assay
3.2 Peroxidase-Coupled Fluorometric Assay
3.3 Determination of a Michaelis-Menten Curve on MAO B
3.4 Evaluation of MAO B Inhibition by a Compound
4 Notes
References
Chapter 4: Measurement of MAO Enzymatic Activity by Spectrophotometric Direct Assays
1 Introduction
1.1 Kynuramine Spectrophotometric Assay
1.2 Benzylamine Spectrophotometric Assay
1.3 MMTP Direct Assay
2 Materials
2.1 Purified MAOs
2.2 Buffers, Substrates, and Other Reagents
2.3 Tools
2.4 Instrumentation and Software
3 Methods
3.1 Enzymatic Activity Using the Kynuramine Spectrophotometric Assay
3.2 Enzymatic Activity Using the Benzylamine Spectrophotometric Assay
3.3 Enzymatic Activity Using the MMTP Direct Assay
3.4 Determination of a Michaelis-Menten Curve on MAO B
3.5 Evaluation of MAO B Inhibition by a Compound
4 Notes
References
Chapter 5: Radiochemical Assay of Monoamine Oxidase Activity
1 Introduction
2 Materials
2.1 Assay
2.2 Extraction
2.3 Counting
3 Methods
3.1 Incubation Step
3.2 Extraction and Counting
3.3 Calculations
4 Notes
References
Chapter 6: MAO Visible Spectroscopy for Ligand Interactions, Redox Chemistry, and Kinetics of Irreversible Inhibition
1 Introduction
2 Materials
2.1 Purified MAO
2.2 Buffers, Substrates, and Inhibitors for Spectral Analyses
2.3 Glassware
2.4 Spectrophotometers and Software
3 Methods
3.1 Analysis of MAO Spectral Perturbations in the Presence of Inhibitors
3.2 Reduction and Oxidation of MAO
3.2.1 Anaerobic Reduction
3.2.2 Redox Potential Determination by Chemical Reduction
3.3 Kinetic Studies Using the Spectral Changes
3.3.1 Kinetics of Substrate Reduction of MAO
3.3.2 Kinetics of Oxidation of MAO
3.4 Kinetics of Inactivation
4 Notes
References
Chapter 7: Conventional Receptor Radioligand Binding Techniques Applied to the Study of Monoamine Oxidase
1 Introduction
2 Materials
2.1 Assay
2.2 Separation
2.3 Counting
3 Methods
3.1 Incubation Step
3.2 Separation and Counting
3.3 Calculations
4 Notes
References
Chapter 8: Assay of MAO Inhibition by Chromatographic Techniques (HPLC/HPLC-MS)
1 Introduction
2 Materials
3 Methods
3.1 Assay of MAO A and MAO B Activity
3.2 Assay of Inhibition of MAO A and MAO B
3.3 Analysis of MAO Assays with RP-HPLC
3.4 Analysis and Identification of MAO Assays with HPLC-MS
3.5 Assay of MAO with HPLC Using MPTP
4 Notes
References
Chapter 9: Crystallization of Human Monoamine Oxidase B
1 Introduction
2 Materials
2.1 Purified Recombinant Human MAO B
2.2 Buffers and Reagents
2.3 Tools and Instrumentation
3 Methods
3.1 Sample Preparation
3.2 Size-Exclusion Chromatography Step
3.3 MAO B Crystallization
4 Notes
References
Chapter 10: Detecting Monoamine Oxidase A and B Proteins: A Western Blotting Protocol and Some Practical Considerations
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Buffers for Sample Preparation
2.3 Tools and Solutions for Gel Electrophoresis
3 Methods
3.1 Sample Preparation
3.1.1 Cell Culture
3.1.2 Cell Collection
3.1.3 Cell Lysis
3.1.4 Protein Determination
3.2 Gel Electrophoresis
3.2.1 Casting Your Gel
3.2.2 Loading Your Sample
3.2.3 Resolving the Samples by Gel Electrophoresis
3.2.4 Transferring Your Sample from the Gel to a Membrane
3.3 Immunodetecting MAO Proteins
3.3.1 Antibodies
3.3.2 Blocking the Membrane and Antibody Incubation
3.3.3 Detection
4 Notes
References
Chapter 11: An (Immuno) Fluorescence Protocol for Monitoring Monoamine Oxidase A/B Protein Distribution Within the Cell
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Buffers and Solutions
2.3 Materials and Equipment
3 Methods
3.1 Immunocytochemistry for Detection of Endogenous MAO Proteins
3.2 Imaging MAO Expression and Distribution in Cells Using N-Terminal GFP-Tagged MAO A and MAO B Constructs
4 Notes
References
Chapter 12: Expression and Function of MAO A in Cardiac Cells by Means of Adenovirus-Mediated Gene Transfer
1 Introduction
2 Materials
2.1 Recombinant Adenovirus with Human MAO A
2.2 Reagents and Tools for Cell Cultures
2.3 Reactive Oxygen Species (ROS) Detection
3 Methods
3.1 Adenoviral Transduction (Fig. 1)
3.2 Reactive Oxygen Species (ROS) Detection
4 Notes
References
Chapter 13: In Vitro and In Vivo Assays Characterizing MAO A Function in Cancers
1 Introduction
2 Materials
2.1 Perineural Invasion Assay
2.1.1 Dorsal Root Ganglion 50B11 Cell Culture
2.1.2 Induction of 50B11 Cell Differentiation and Neurite Outgrowth
2.1.3 Prostate Cancer Cell Culture
2.1.4 Cancer Cell Fluorescence Labeling
2.2 MTS Cell Proliferation Assay
2.2.1 Prostate Cancer Cell Culture
2.2.2 Cell Viability Assessment
2.3 In Vivo Tumorigenesis Assay
2.3.1 Prostate Cancer Cell Culture
2.3.2 Injection of Prostate Cancer Cells
3 Methods
3.1 Perineural Invasion Assay
3.1.1 Preparation of 50B11 Cells
3.1.2 Induction of 50B11 Cell Differentiation and Neurite Outgrowth
3.1.3 Preparation and Fluorescence Labeling of Prostate Cancer Cells
3.1.4 Setup of a Cancer-Nerve Cell Coculture
3.1.5 Analysis of MAO A-Induced Prostate Cancer Cell Perineural Invasion
3.2 MTS Cell Proliferation Assay
3.2.1 Preparation and Seeding of Prostate Cancer Cells
3.2.2 Measurement of Prostate Cancer Cell Viability
3.2.3 Analysis of MAO A-Induced Prostate Cancer Cell Proliferation
3.3 In Vivo Tumorigenesis Assay
3.3.1 Preparation of Prostate Cancer Cells Preinjection
3.3.2 Subcutaneous Injection of Cancer Cells into Mice
3.3.3 Tumor Growth Monitoring and Tumor Sample Collection
4 Notes
References
Chapter 14: In Vivo Study of Monoamine Oxidases Using Multisite Intracerebral Microdialysis
1 Introduction
2 Materials
2.1 Animals
2.2 Isoflurane Anesthesia
2.3 Surgery
2.4 Microdialysis
2.5 HPLC System and Electrochemical Detection
3 Methods
3.1 Surgeries and Microdialysis Probe Placement
3.2 Sample Collection and Pharmacological Treatment
3.3 Sample Analysis
4 Notes
References
Chapter 15: Informed Use of 3D-QSAR for the Rational Design of Coumarin Derivatives as Potent and Selective MAO B Inhibitors
1 Introduction
2 Materials
2.1 Protein and Ligand Dataset
2.2 Computational Studies
3 Methods
3.1 Protein and Ligand Preparation
3.2 3D-QSAR Model Preparation
3.3 Gaussian Field-Based 3D-QSAR Model
3.4 Gaussian Field-Based 3D-QSAR Analysis
4 Notes
References
Chapter 16: Hansch-Type QSAR Models for the Rational Design of MAO Inhibitors: Basic Principles and Methodology
1 Introduction
2 Materials
2.1 Datasets
2.2 Analysis Software
3 Methods
3.1 2D-QSAR by Stepwise Linear Regression: Case 1
3.2 2D-QSAR by Stepwise Linear Regression: Case 2
4 Notes
References
Chapter 17: Computational Chemistry and Molecular Modeling of Reversible MAO Inhibitors
1 Introduction
2 Materials
2.1 Molecular Docking
2.1.1 Visualization Tools
2.1.2 Ligands
2.1.3 Proteins
2.1.4 AutoDockTools (ADT)
2.1.5 AutoDock4.2.6
2.2 Compound Libraries for Virtual Screening
2.3 Materials for Molecular Dynamics
2.3.1 VMD (Visual Molecular Dynamics)
2.3.2 NAMD
3 Methods
3.1 Molecular Docking with AutoDock
3.1.1 Grid Parameter File (GPF) Preparation
3.1.2 Docking Parameter File (DPF) Preparation
3.1.3 Docking Process
3.1.4 Analyzing the Results
3.2 In Silico/Virtual Screening
3.3 Molecular Dynamics
3.3.1 General Procedure
3.3.2 Case Study for MAO B and Safinamide
4 Notes
References
Index