Murphy Mitochondrial Function, Part B

Mitochondrial Protein Kinases, Protein Phosphatases and Mitochondrial Diseases
1. Auflage 2009
ISBN: 978-0-08-092350-5
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 450 Seiten

In this second of two new volumes covering mitochondria, methods developed to assess the number and function of nuclear-encoded proteins in the mitochondrion are presented. Chapters focus on the regulation of mitochondrial function and mitochondrial diseases, with a section emphasizing the mitochondrial defects associated with type 2 diabetes.
The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. With more than 450 volumes published, each volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences.
New methods focusing on the examination of normal and abnormal mitochondrial function are presented in an easy-to-follow format by the researchers who developed them
Along with a companion volume covering topics including mitochondrial electron transport chain complexes and reactive oxygen species, provides a comprehensive overview of modern techniques in the study of mitochondrial malfunction
Provides a 'one-stop shop' for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods

Autoren/Hrsg.

Murphy, Anne

Inhaltsverzeichnis

1;Front Cover;1
2;Mitochondrial Function, Part B: Mitochondrial Protein Kinases, Protein Phosphatases and Mitochondrial Diseases;4
3;Copyright Page;5
4;Contents;6
5;Contributors;16
6;Preface;26
7;Methods in Enzymology;28
8;Section 1: Defining the Mitochondrial Proteome, Including Phosphorylated, Acetylated, and Palmitoylated Proteins;56
8.1;Chapter 1: The Mitochondrial Proteome Database: MitoP2;58
8.1.1;1. Introduction;59
8.1.2;2. Yeast, Human, and Mouse Proteome Information;61
8.1.3;3. Mitochondrial Reference Set, Candidates, and SVM Prediction Score;63
8.1.4;4. Integrated Genome-Wide Approaches;64
8.1.5;5. Other Search Options: Restrictions to Functional Categories, Gene Locus and Disease-Causing Proteins;67
8.1.6;6. Output Lists and Practical Examples;69
8.1.7;References;72
8.2;Chapter 2: Predicting Proteomes of Mitochondria and Related Organelles from Genomic and Expressed Sequence Tag Data;76
8.2.1;1. Introduction;77
8.2.2;2. Data Preparation;83
8.2.3;3. Screening Large Datasets for Mitochondrial Proteins;84
8.2.4;4. Detailed Analysis;88
8.2.5;5. Comparison of Methods;96
8.2.6;Acknowledgments;99
8.2.7;References;99
8.3;Chapter 3: Proteome Characterization of Mouse Brain Mitochondria Using Electrospray Ionization Tandem Mass Spectrometry;104
8.3.1;1. Introduction;105
8.3.2;2. Analysis of Mitochondrial Proteome by Two-Dimensional PAGE and LC-MS Techniques;106
8.3.3;3. Selective Proteome Enrichment by Capillary Isotachophoresis (CITP)-Based Separations;106
8.3.4;4. Analysis of Mouse Brain Mitochondria Using the CITP-Based Proteome Platform;107
8.3.5;5. Proteome Characterization of Mouse Brain Mitochondria;110
8.3.6;References;115
8.4;Chapter 4: 32P Labeling of Protein Phoshorylation and Metabolite Association in the Mitochondria Matrix;118
8.4.1;1. Introduction;119
8.4.2;2. Methods;120
8.4.3;3. Results and Discussion;127
8.4.4;4. Summary;133
8.4.5;References;134
8.5;Chapter 5: Selective Enrichment in Phosphopeptides for the Identification of Phosphorylated Mitochondrial Proteins;136
8.5.1;1. Introduction;137
8.5.2;2. Off-Line Phosphopeptide Enrichment Methods;139
8.5.3;3. On-Line 2D-LC Phosphopeptide Enrichment Methods;145
8.5.4;4. Mass Spectrometry-Based Methods;147
8.5.5;5. Quantitative Phosphoproteomics;149
8.5.6;References;149
8.6;Chapter 6: Post-translational Modifications of Mitochondrial Outer Membrane Proteins;152
8.6.1;1. Introduction;153
8.6.2;2. Isolation of Mitochondria and Mitochondrial Membranes;154
8.6.3;3. Detection of Post-translational Modifications Using One-dimensional Gel Electrophoresis;156
8.6.4;4. Focused Proteomics Using Immunological Approaches;160
8.6.5;5. Shotgun Methods for Identification of Post-translational Modifications;164
8.6.6;6. Conclusion;166
8.6.7;Acknowledgments;166
8.6.8;References;167
8.7;Chapter 7: Analysis of Tyrosine-Phosphorylated Proteins in Rat Brain Mitochondria;172
8.7.1;1. Introduction;173
8.7.2;2. Identification of Src Tyrosine Kinase Substrates in Rat Brain Mitochondria;174
8.7.3;3. Mass Spectrometric Analysis of Tyrosine-Phosphorylated Peptides in Mitochondria;180
8.7.4;4. Bioinformatic Tools to Analyze the Potential Role of Tyrosine Phosphorylation;186
8.7.5;Acknowledgment;189
8.7.6;References;189
8.8;Chapter 8: Acetylation of Mitochondrial Proteins;192
8.8.1;1. Introduction;193
8.8.2;2. Purification of Enzymatically Active SIRT3;193
8.8.3;3. SIRT3 Enzymatic Deacetylation Assay;196
8.8.4;4. Detection of Acetylated Proteins in Mitochondria;199
8.8.5;5. Conclusion;201
8.8.6;References;202
8.9;Chapter 9: Non-radioactive Detection of Palmitoylated Mitochondrial Proteins Using an Azido-Palmitate Analogue;204
8.9.1;1. Introduction;205
8.9.2;2. Synthesis of 12-Azidododecanoate and 14-Azidotetradecanoate;208
8.9.3;3. Synthesis of Tagged Phosphine Probes;210
8.9.4;4. Preparation of Azido-Fatty Acids and Tagged Phosphine Stock Solutions;210
8.9.5;5. Production of 14-Azidotetradecanoyl-CoA Derivatives;211
8.9.6;6. In Vitro Labeling of Proteins with 14-Azidotetradecanoyl-CoA and Tagged Phosphines;211
8.9.7;7. Isolation of Intact Mitochondria from Livers of Sprague-Dawley Rats;212
8.9.8;8. Chromatography of Soluble Mitochondrial Proteins;213
8.9.9;9. Labeling of Anion Exchange Eluates with 14-Azidotetradecanoyl-CoA and Phosphine-Biotin;213
8.9.10;10. Identification of Labeled Proteins by Mass Spectrometry;214
8.9.11;11. Demonstration that Acylation of Cysteine Residues Occurs via a Thioester Bond;215
8.9.12;12. Concluding Remarks;217
8.9.13;References;218
9;Section 2: Characterization of Mitochondrial Kinases, Phosphatases, Dynamics, and Respiratory Function;222
9.1;Chapter 10: Detection of a Mitochondrial Kinase Complex That Mediates PKA-MEK-ERK-Dependent Phosphorylation of Mitochondrial Proteins Involved in the Regulation of Steroid Biosynthesis;224
9.1.1;1. Introduction;225
9.1.2;2. Steroidogenic Cells;226
9.1.3;3. Analysis of a Mitochondrial Kinase Complex;227
9.1.4;4. Analysis of a Mitochondrial Acyl-CoA Thioesterase, Acot2 as a Phosphoprotein;241
9.1.5;References;245
9.2;Chapter 11: Isolation of Regulatory-Competent, Phosphorylated Cytochrome c Oxidase;248
9.2.1;1. Introduction;249
9.2.2;2. Purification of Mitochondria Maintaining Protein Phosphorylation;250
9.2.3;3. Isolation of Cytochrome c Oxidase from Mitochondria;254
9.2.4;4. Analysis of Cytochrome c Oxidase Phosphorylation;259
9.2.5;5. Concluding Remarks;263
9.2.6;References;264
9.3;Chapter 12: Using Functional Genomics to Study PINK1 and Metabolic Physiology;266
9.3.1;1. Introduction;267
9.3.2;2. Studying Mitochondrial Modulation in Humans In Vivo;269
9.3.3;3. Cellular Mitochondrial Models;271
9.3.4;4. Considerations When Using Short Interfering RNAs;273
9.3.5;5. Ex Vivo Gene Expression Analysis;277
9.3.6;References;281
9.4;Chapter 13: Functional Characterization of Phosphorylation Sites in Dynamin-Related Protein 1;286
9.4.1;1. Introduction;287
9.4.2;2. Replacement of Endogenous with Phosphorylation-Site Mutant Drp1;289
9.4.3;3. Analysis of Drp1 Phosphorylation;292
9.4.4;4. Production and Assays of Recombinant Drp1;295
9.4.5;5. Cell Death Assays;298
9.4.6;6. Mitochondrial Shape Analysis with ImageJ;300
9.4.7;7. Appendix: Morphometry Macro;305
9.4.8;References;306
9.5;Chapter 14: Functional Characterization of a Mitochondrial Ser/Thr Protein Phosphatase in Cell Death Regulation;310
9.5.1;1. Introduction;311
9.5.2;2. Identification of Protein Phosphatases in Mitochondria;313
9.5.3;3. Characterization of Mitochondrial Localization of PP2Cm in Mammalian Cells;315
9.5.4;4. Functional Characterization of PP2Cm in Cultured Cells;319
9.5.5;5. Functional Characterization of PP2Cm in Cell Death and Mitochondrial Regulation;322
9.5.6;6. Mitochondrial Phosphatase in Cell Death Regulation;325
9.5.7;Acknowledgments;326
9.5.8;References;326
9.6;Chapter 15: Distinguishing Mitochondrial Inner Membrane Orientation of Dual Specific Phosphatase 18 and 21;330
9.6.1;1. Introduction;331
9.6.2;2. Analysis of Phosphatase Activity of DSP18;332
9.6.3;3. Isolation of Highly Purified Rat Kidney Mitochondria and Subfractionation;334
9.6.4;4. Mitochondrial Inner Membrane Association of DSP18 and DSP21;338
9.6.5;Acknowledgments;341
9.6.6;References;342
9.7;Chapter 16: Monitoring Mitochondrial Dynamics with Photoactivateable Green Fluorescent Protein;344
9.7.1;1. Mitochondrial Dynamics;345
9.7.2;2. PAGFPmt;347
9.7.3;3. Tracking Individual Fusion and Fission Events;348
9.7.4;4. Quantifying Networking Activity in Whole Cells: Whole Cell Mitochondrial Dynamics Assay;351
9.7.5;5. Potential Artifacts and Important Controls;356
9.7.6;6. Comparison with an Alternative Method;357
9.7.7;Acknowledgments;358
9.7.8;References;358
9.8;Chapter 17: Determination of Yeast Mitochondrial KHE Activity, Osmotic Swelling and Mitophagy;360
9.8.1;1. Introduction;361
9.8.2;2. Disturbance of Mitochondrial K+ Homeostasis by dox-Regulated Mdm38 Expression;362
9.8.3;3. Morphological Changes in Mitochondria in Mdm38-Depleted Cells: Confocal Microscopy and Electron Microscopy;364
9.8.4;4. Measurement of KHE Activity and Other Bioenergetic Parameters;365
9.8.5;5. Following Mitophagy Using a Novel Biosensor and Fluorescence Microscopy;368
9.8.6;6. Growth and Preparation of Cells for Fluorescence Microscopy Observations of Mitophagy;369
9.8.7;7. Fluorescence Microscopy;370
9.8.8;Acknowledgments;371
9.8.9;References;372
9.9;Chapter 18: Imaging Axonal Transport of Mitochondria;374
9.9.1;1. Introduction;375
9.9.2;2. Protocols to Image Axonal Transport of Mitochondria in Rat Hippocampal Neurons;379
9.9.3;3. Protocols to Image Axonal Transport of Mitochondria in Drosophila Larval Neurons;383
9.9.4;4. Conclusion;386
9.9.5;Acknowledgments;387
9.9.6;References;387
9.10;Chapter 19: Generation of mtDNA-Exchanged Cybrids for Determination of the Effects of mtDNA Mutations on Tumor Phenotypes;390
9.10.1;1. Introduction;391
9.10.2;2. Establishment of rho0 Cells of Mice;392
9.10.3;3. Preparation and Enucleation of mtDNA-Donor Cells;395
9.10.4;4. Cell Fusion between rho0 Cells and Cytoplasts;396
9.10.5;5. Concluding Remarks;398
9.10.6;References;399
10;Section 3: Mitochondrial Function in Pancreatic Beta Cells and Insulin-Responsive Tissues;402
10.1;Chapter 20: Functional Assessment of Isolated Mitochondria In Vitro;404
10.1.1;1. Introduction;405
10.1.2;2. Mitochondrial Isolation Procedures;409
10.1.3;3. Mitochondrial ATP Production;415
10.1.4;4. Mitochondrial Respiration;420
10.1.5;5. Summary;424
10.1.6;References;424
10.2;Chapter 21: Assessment of In Vivo Mitochondrial Metabolism by Magnetic Resonance Spectroscopy;428
10.2.1;1. Introduction;429
10.2.2;2. In Vivo Magnetic Resonance Spectroscopy;430
10.2.3;3. Insulin Resistance and Resting Mitochondrial Metabolism;441
10.2.4;4. Conclusions;444
10.2.5;References;444
10.3;Chapter 22: Methods for Assessing and Modulating UCP2 Expression and Function;450
10.3.1;1. Introduction;451
10.3.2;2. Analysis of UCP2 Expression by Immunoblot;452
10.3.3;3. Analysis of UCP2 Expression by Real-Time PCR;454
10.3.4;4. Mitochondria Respiration Assay;455
10.3.5;5. Modulating UCP2 Expression by Cold Exposure;455
10.3.6;6. Inhibiting UCP2 Expression by Antisense Oligonucleotide;456
10.3.7;7. Inhibiting PGC-1alpha as an Indirect Means of Controlling UCP2 Expression;457
10.3.8;References;458
10.4;Chapter 23: Measuring Mitochondrial Bioenergetics in INS-1E Insulinoma Cells;460
10.4.1;1. Introduction;461
10.4.2;2. Quantification of Cellular Bioenergetics-Theoretical Aspects;462
10.4.3;3. INS-1E Cells-A Valuable Pancreatic Beta Cell Model;466
10.4.4;4. Measurement of Coupling Efficiency in Trypsinized INS-1E Cells;471
10.4.5;5. Noninvasive Measurement of Cellular Bioenergetics;473
10.4.6;Acknowledgment;477
10.4.7;References;478
10.5;Chapter 24: Investigating the Roles of Mitochondrial and Cytosolic Malic Enzyme in Insulin Secretion;480
10.5.1;1. Introduction;481
10.5.2;2. Malic Enzyme mRNA Expression in Rat Insulinoma INS-1 832/13 Cells, Rat Islets, and Mouse Islets;483
10.5.3;3. siRNA Knock-Down of ME1 and ME2 in INS-1 832/13 beta-Cells;487
10.5.4;4. Enzymatic Assays to Determine Activity of Cytosolic and Mitochondrial Malic Enzymes;490
10.5.5;5. Calculating Relative Rates of Anaplerotic Pathways from 13C-Glutamate Isotopomer Distribution;497
10.5.6;6. Discussion;503
10.5.7;References;504
10.6;Chapter 25: Insulin Secretion from beta-Cells is Affected by Deletion of Nicotinamide Nucleotide Transhydrogenase;506
10.6.1;1. Introduction;507
10.6.2;2. Gene-Driven ENU Screens;508
10.6.3;3. Generating BAC Transgenics;512
10.6.4;4. Isolation of Islets of Langerhans from Mouse Pancreas;515
10.6.5;5. RNA Interference (RNAi) Methodologies on Insulinoma Cell Lines;519
10.6.6;6. Measuring Intracellular Calcium;522
10.6.7;7. Measuring Hydrogen Peroxide in Mitochondria;523
10.6.8;8. Imaging of Mitochondrial Membrane Potential Changes by Confocal Microscopy;526
10.6.9;9. Measuring NNT Activity;530
10.6.10;10. Conclusions;532
10.6.11;References;533
11;Author Index;536
12;Subiect Index;564
13;Colour Plates;575