Nitric Oxide part E

Cover Page......Page 1
METHODS IN ENZYMOLOGY......Page 2
Section I. Biochemical, Molecular, and Real-Time Detection of NO......Page 3
Contributors to Volume 396......Page 8
Preface......Page 16
Volumes in Series......Page 17
Volume 396. Nitric Oxide (Part E)......Page 39
Introduction......Page 40
Disproportionation and Autoxidation......Page 41
Example of Impurity Effects......Page 43
Preparing Solutions to Have a Specified NO Concentration......Page 44
The Vacuum Line and Purification Column......Page 45
Glassware......Page 47
Transferring Known Quantities of NO......Page 49
Distillation of NO......Page 50
Accounting for Impurities......Page 51
Sources of Equipment and Parts Described in This Chapter......Page 52
References......Page 53
Introduction......Page 54
NONOates as NO Donors in Biological Systems......Page 55
Synthesis of Diazen-1-ium-1,2-diolates Using the NOtizer......Page 57
Characteristics of Diazeniumdiolates to be Considered......Page 60
References......Page 61
3 Quantum Mechanical Determinations of Reaction Mechanisms, Acid Base, and Redox Properties of Nitrogen Oxides and Their Donors......Page 63
Introduction......Page 0
Computational Methodology......Page 64
N-Hydroxyguanidine Oxidative Decomposition......Page 68
S-N Bond Dissociation Energies of S-Nitrosothiols and Transnitrosation Reaction Energies......Page 70
NONOate Decomposition Mechanisms......Page 71
The Hydration Keq of HNO......Page 74
Computing the pKa of HNO and Reduction Potential of Nitric Oxide......Page 76
Theoretically Derived Reduction Potentials of HNO and Angeli's Salt......Page 77
References......Page 78
Chemical Properties......Page 82
Electrochemical Methods......Page 83
Materials and Methods......Page 84
Results and Discussion......Page 85
Spectrophotometric Method......Page 87
References......Page 90
Introduction......Page 91
Spectroscopic Detection of Nitric Oxide......Page 92
Signal Generation in LMRS......Page 94
Carbon Monoxide Laser-Based Faraday-LMRS......Page 95
Quantum Cascade Laser-Based Faraday-LMRS......Page 96
References......Page 97
Introduction......Page 98
Procedure......Page 99
Examples......Page 100
Interferences......Page 102
Conclusions......Page 104
Abstract......Page 105
Nitric Oxide in Blood......Page 106
New Experimental Approach......Page 107
Electrodes......Page 108
Selectivity of the Sensor for Nitric Oxide......Page 109
Responses to Changing Gas Mixtures......Page 110
Discussion......Page 111
Conclusions......Page 112
References......Page 113
Abstract......Page 114
Principal......Page 115
Procedure......Page 116
Impacts of Detergents to Detection of NO and ROS in Plants......Page 117
Conclusions......Page 119
References......Page 120
Introduction......Page 121
Principal......Page 122
Procedure......Page 123
Detection of NO in Plant (Wheat or Orchid) Tissue Extract......Page 124
Indirect Detection of NO in Live Plant (Wheat or Orchid)......Page 125
Direct Detection of NO in Live Plant (Wheat or Orchid)......Page 126
Conclusions......Page 127
References......Page 128
Section III. Peroxynitrite......Page 4
Introduction......Page 130
Inorganic Synthesis of Low-Mass SNOs in Acid......Page 133
General Comments......Page 134
Synthesis of SNO-Hemoglobin In Vitro......Page 135
Biochemical Mechanisms of SNO Formation In Vivo......Page 136
SNO Bioactivities......Page 137
References......Page 138
Abstract......Page 140
Introduction......Page 141
Principle of Assay System......Page 142
Preparation of Protein Samples......Page 144
In Vitro S-Nitrosylation......Page 145
Blockade of Protein Thiols......Page 146
Labeling of S-Nitrosothiols......Page 148
Detection and Purification of Labeled Proteins......Page 149
Negative Controls for In Vitro Nitrosylation......Page 150
References......Page 151
Abstract......Page 153
Introduction......Page 154
Principles and General Considerations......Page 155
General Protocol......Page 156
Cell-Associated Nitroso Species from Exogenous Exposure to NO......Page 157
Isolation of Nitrosated Proteins Using Gas-Phase Chemiluminescence as the Detection Assay......Page 160
Biotin Switch Assay......Page 161
Summary and Conclusion......Page 163
References......Page 165
Abstract......Page 166
The Biotin Switch Method......Page 167
Treatment of Cells with S-nitroso-l-cysteine and Extract Preparation......Page 170
Biotin Switch Treatment......Page 171
Avidin Capture......Page 172
References......Page 173
Abstract......Page 174
Nitrosative Stress and Parkinson's Disease......Page 175
Photolysis Chemiluminescence......Page 176
Mass Spectrometry......Page 178
Colorimetric and Fluorometric Methods......Page 179
Biotin Switch Method......Page 180
S-Nitrosylation and PD......Page 182
References......Page 183
Introduction......Page 186
Synthesis of Probe: Deuterium-Labeled PHPA......Page 190
Measurement of Plasma Concentrations of NHPA and PHPA......Page 191
Conclusion......Page 192
References......Page 194
Introduction......Page 195
2D-Gel Electrophoresis......Page 197
Affinity Enrichment after Covalent Attachment of a Biotin-Affinity Tag......Page 198
Combination of HPLC and SDS-PAGE......Page 199
Solution Isoelectrofocusing......Page 200
Mass Spectrometry......Page 201
MALDI and ESI Ionization Techniques......Page 202
Proteomic Studies Providing MS/MS Characterization of 3-NT-Containing Peptides......Page 203
References......Page 204
Abstract......Page 206
Principles of Electrochemical Array Detection......Page 207
5-NO2-gamma-Tocopherol as an Indicator for Lipid-Phase Nitration......Page 209
Materials......Page 211
Instrumental Analysis......Page 212
Discussion......Page 214
References......Page 215
Abstract......Page 217
Peptide synthesis and purification......Page 219
3-Nitro N-t-BOC L -tyrosine t-butyl ester (NO2-BTBE):......Page 221
Extraction of BTBE:......Page 223
Results and Discussion......Page 224
Characterization of Hydrophobic Tyrosyl Analogue and Transmembrane Peptides......Page 225
Location of BTBE and Tyrosyl Peptides in the Bilayer: Fluorescence and’Fluorescence Quenching Experiments......Page 226
Analysis of Reaction Products by HPLC......Page 229
Studies of Nitration/Oxidation Reaction in Membrane Using BTBE and Tyrosyl Transmembrane Peptides......Page 230
Slow infusion of peroxynitrite :......Page 232
SIN-1-induced nitration......Page 234
The effect of bicarbonate:......Page 235
Nitration induced by MPO/H2O2/NO-2......Page 236
References......Page 237
Introduction......Page 240
The Chemistry of the Reaction......Page 241
Synthesis of Peroxynitrite......Page 242
Manganese Dioxide Synthesis......Page 244
Working with Peroxynitrite......Page 245
References......Page 246
Introduction......Page 248
src Tyrosine Kinase Structure and Regulation......Page 249
src Kinase Domains......Page 250
Regulation of src Activity by Phosphorylation/Dephosphorylation of Tyrosine Residues......Page 251
Activation of Tyrosine Phosphorylation Signal in Erythrocytes by Peroxynitrite......Page 252
Activation of src Kinases......Page 253
Regulation through the Nitration of Peptides Mimicking Protein Domains with SH2 Affinity......Page 254
Peroxynitrite Preparation......Page 255
Determination of PTP Activity in the Membrane Fraction......Page 256
Immunoprecipitation Protocol......Page 257
Kinase Assay......Page 258
Solid-Phase Binding Assay......Page 259
References......Page 260
Abstract......Page 262
Introduction......Page 263
Oxyhemoglobin and Peroxynitrite......Page 264
Purification of Human Oxyhemoglobin and Spectroscopic Analysis......Page 266
Determination of Nitrite and Nitrate......Page 267
Detection of a Ferrylhb Intermediate......Page 268
Oxygen and Hydrogen Peroxide Yields......Page 269
Analysis of Protein Radical Formation......Page 270
Red Blood Cells and Peroxynitrite......Page 271
Oxidation of Intracellular oxyHb by Peroxynitrite in the Presence of Extracellular Targets......Page 272
Estimation of Peroxynitrite Diffusion Distances......Page 273
Concluding Remarks......Page 275
References......Page 276
Abstract......Page 278
Introduction......Page 279
Materials......Page 280
Preparation of Ring-Labeled Nitrotyrosine Standards......Page 281
Protein Hydrolysis of Samples......Page 283
LC/ESI/MS Quantification of Tyrosine and 3-Nitrotyrosine......Page 284
Adjusting for Artificial Nitrotyrosine Generation......Page 285
Monitoring 3-Nitrotyrosine in Solid Tissue......Page 288
Standard Curves of 3-Nitrotyrosine and Tyrosine......Page 289
Example: LC/MS Quantification of 3-Nitrotyrosine in EPO Knockout Mice following Ovalbumin Challenge Model of Asthma......Page 291
LC/MS Analysis of Free 3-Nitrotyrosine in Plasma......Page 293
General Comments......Page 294
Acknowledgments......Page 296
References......Page 297
Abstract......Page 299
Introduction......Page 300
Chemicals......Page 301
MALDI-MS Analysis of Nitrated Angiotensin II......Page 302
MALDI-MS Pattern for Nitrotyrosine in Proteins......Page 304
Concluding Remarks......Page 305
References......Page 306
Abstract......Page 309
Introduction......Page 310
Animals......Page 311
Chemicals......Page 312
Immunoblotting......Page 313
18F-DOPA Synthesis......Page 314
MPTP-Induced Striatal Release of Zinc Inhibits Peroxynitrite......Page 315
MTs Prevent MPP+ Apoptosis in SK-N-SH Neurons......Page 316
Attenuation of Peroxynitrite Apoptosis and alpha-Syn Nitration by MTs......Page 317
SIN-1 Accentuates 6-OHDA Hemiparkinsonism......Page 319
MTs Inhibit Peroxynitrite Neurotoxicity by Donating Zinc......Page 321
Inhibition of MPTP- and THIQ-Induced Peroxynitrite by MTs......Page 322
cDNA Microarray Analysis of Oxidative and Nitrative Stress......Page 323
MT Neuroprotection in Peroxynitrite Neuroinflammations......Page 324
MT Neuroprotection in Peroxynitrite Apoptosis......Page 325
Peroxynitrite and MTtrans Stem Cell Therapy......Page 326
Conclusion......Page 328
References......Page 330
Section IV. Signaling and Gene Expression......Page 5
Introduction......Page 332
General Methods of Yeast Culture......Page 336
beta-Galactosidase Reporter System......Page 337
Effects of NO and HNO on the Transcription Factor Ace1......Page 338
Toxicological Effects of HNO on Different Growth Phases of Yeast......Page 340
NO as an Antioxidant Against Paraquat Toxicity......Page 341
References......Page 344
Introduction......Page 348
Cultured Sheep Pulmonary Artery Endothelial Cells.......Page 349
FRET Detection......Page 350
Zinc Detection......Page 351
Results and Discussion......Page 352
Concluding Remarks......Page 355
References......Page 356
Abstract......Page 357
Nitric Oxide Biochemistry......Page 358
Nitric Oxide Signaling......Page 359
Nitric Oxide and Gene Expression......Page 363
References......Page 367
Chemicals......Page 372
Luciferase Reporter Gene Assay......Page 373
Sulfur Amino Acid-Deficient Culture Medium......Page 374
Preparation of Nuclear and Cytoplasmic Fractions......Page 375
Immunoblot Analysis of Nrf2......Page 376
Immunocytochemistry of Nrf2......Page 377
Gel Shift Assay......Page 378
References......Page 379
Overview on the Participation of Protein Tyrosine Phosphorylation in Nitric Oxide-Mediated Signaling Events......Page 381
Immunoblotting for Detection of Protein Tyrosine Phosphorylation......Page 384
Immunoblotting for Detection of ERK1/2 and Phospho-ERK1/2 MAP Kinases......Page 386
Assay for p21Ras Activation......Page 387
References......Page 388
Introduction......Page 390
Cell Culture......Page 392
cDNA Library Construction......Page 394
Hybridization......Page 395
Immunological Detection......Page 396
Microarray Construction and Analysis......Page 397
QPCR (SYBR Green Method ).......Page 398
Abstract......Page 399
Introduction......Page 400
Reverse Transcription Polymerase Chain Reaction......Page 403
Western Blot Analysis......Page 404
Preparation of Rat Liver Cytosol to be Used as the Source of Biliverdin Reductase (Yet et al., 2002)......Page 406
References......Page 407
Abstract......Page 408
Introduction......Page 409
Experimental Protocols Related to G-Protein Research......Page 410
Transient Transfection......Page 411
G-Protein Immunoblotting......Page 412
[35S]GTPgammaS Binding Assay......Page 413
Immunoblot Analysis......Page 414
Gel Retardation Assay......Page 415
Cell Signaling for iNOS Induction by Thrombin......Page 416
References......Page 417
Abstract......Page 418
Introduction......Page 419
Preparation of Tissue Samples for Gene Array Analysis (Tran et.al., 2003)......Page 420
Capture of Low-Density Gene Array Data......Page 421
Statistical Treatment of Gene Array Data......Page 422
Results and Discussion......Page 424
References......Page 425
Section V. Cell Biology and Physiology......Page 6
O2- and H2O2 as a Function of Oxygen Concentration......Page 427
Mitochondrial NO Utilization Conducts to O2- Formation......Page 428
mtNOS, H2O2, and Peroxynitrite......Page 430
The Calculation of Cell H2O2 Steady-State Concentration......Page 431
Measurement of d[H2O2]/dt......Page 432
Total Oxidants......Page 433
Intracellular and Intramitochondrial Superoxide Anion......Page 434
Cell Proliferation and H2O2......Page 435
Proliferation Assays......Page 437
Redox Activation of MAPKs and Phosphatases in Mitochondria......Page 438
Gel Electrophoresis and Detection of PTPs......Page 439
References......Page 440
Abstract......Page 442
Introduction......Page 443
Cell Survival by MTT Assay......Page 448
Total glutathione assay :......Page 449
References......Page 450
Abstract......Page 452
Targets for NO......Page 453
Mitochondrial Targets of NO......Page 454
mtNOS and Mitochondrial Bioenergetics......Page 456
mtNOS, Apoptosis, and Oxidative Stress......Page 457
What Isozyme Is mtNOS?......Page 458
Isolation of Mitochondria......Page 461
Oxyhemoglobin Assay......Page 462
Preparation of Broken Mitochondria......Page 463
Procedure......Page 464
Fluorophotometric Determination of mtNOS Activity......Page 465
Amperometric mtNOS Assay......Page 466
mtNOS and Mitochondrial Respiration......Page 467
Procedure......Page 468
References......Page 469
Abstract......Page 472
Mitochondrial Nitric Oxide Production......Page 473
Mitochondrial NOS Functional Activity: Inhibition of O2 Consumption......Page 474
Mitochondrial NOS Functional Activity: Enhancement of H2O2 Production......Page 476
Mitochondrial NO Release......Page 478
Mitochondrial NOS Functional Activity in Pathological Situations and after Pharmacological Treatments......Page 480
References......Page 482
Nitric Oxide Synthesis......Page 484
The Role of Tetrahydrobiopterin......Page 485
Sample Preparation for Cryogenic EPR Experiments......Page 486
Identification of BH4 as an Obligate Electron Donor to Fe(II).O2......Page 487
Putative Role of BH4 as an Obligate Proton Donor in NO Synthesis......Page 488
Cryogenic CO/O2 Exchange Studies......Page 490
Comparison with Rapid Kinetic Methods......Page 491
References......Page 492
Design of Oligonucleotides......Page 495
Procedure for ODN Incorporation......Page 496
Control for ODN Uptake......Page 501
Analyses of mRNA Formation, Protein Expression, and Nitrite Production......Page 502
Antisense Inhibition on the mRNA, Protein, and Activity Level......Page 503
References......Page 504
Abstract......Page 506
Introduction......Page 507
cDNA Isolation and Baculovirus Construction......Page 508
Protein Purification......Page 510
Binding of NO and CO to sGC......Page 513
EPR Analysis of sGC Heme......Page 514
Monitoring the Kinetics of the Pentacoordinated Nitrosyl Heme Formation......Page 516
Time-Resolved Measurement of sGC NO-Heme Complex Formation......Page 517
References......Page 519
Abstract......Page 520
Introduction......Page 521
Assay for cGMP-Forming Activity......Page 522
Preparation of a Guanylyl Cyclase Antibody Affinity Column......Page 523
Preparation of a Cleared Homogenate......Page 524
Immunoaffinity Chromatography......Page 525
Electrophoresis......Page 526
UV-Vis Spectroscopy......Page 527
Concluding Remarks......Page 528
References......Page 529
Chemiluminescence Analysis of EC NO Production......Page 530
Application to Cultured EC......Page 531
Electron Spin Resonance Techniques for Measuring EC NO Production......Page 534
Application to Cultured ECs......Page 535
Electrochemical Detection of EC NO Production......Page 538
Electrode Fabrication......Page 539
Application to Cultured ECs......Page 540
References......Page 541
Abstract......Page 542
Principles of Tissue Microdialysis and Potential for Measurements of Extracellular ROS......Page 543
Placement of the Probe......Page 545
Procedure......Page 546
Procedure......Page 548
Comment and Limitations......Page 549
Comment and Limitations......Page 550
Procedure......Page 551
References......Page 552
Nitric Oxide Stimulation of Proteasome Function: A New Antioxidant Mechanism......Page 554
Upregulation of Proteasomal Activity by bullNO......Page 555
Reversal of Antioxidant Effects of bullNO by Proteasomal Inhibitors......Page 557
Implications in Age-Related Neurodegenerative Diseases......Page 560
References......Page 561
Introduction......Page 563
Primary Cell Isolation......Page 564
Flow Cytometry......Page 565
Acknowledgments......Page 568
Abstract......Page 569
Invasive Studies......Page 570
Invasive Studies......Page 572
Noninvasive Studies: Pulse Amplitude Tonometry......Page 573
Noninvasive Studies: Extent of Reactive Hyperemia......Page 575
Conclusions......Page 578
References......Page 579
Abstract......Page 581
Hypoxia and Vascular Functions of NO......Page 582
Basic Principle of Vessel Bioassays......Page 583
Controlling Oxygen Partial Pressure......Page 584
Oxygen Sensor Calibration......Page 586
"Closed" Systems......Page 587
Simultaneous Measurement of Vessel Tension and Oxygen Partial Pressure......Page 588
Critical pO2......Page 590
Hemolysis......Page 591
Oxygen Offloading from Red Cells......Page 592
Order of Preconstriction and Hypoxia......Page 593
References......Page 594
Abstract......Page 596
Decision for an Applicative UVA Source......Page 597
Preparation of Human Skin Samples......Page 598
Preparation of Human Skin Homogenate Supernatants......Page 599
S-Nitrosothiols......Page 600
Chemiluminescence for Detection of Nitrite, RSNO, and RNNO......Page 601
UVA-Induced NO Formation from Human Skin......Page 602
Detection of UVA-Induced NO Release from Skin Homogenate Supernatants......Page 603
Detection of S-Nitroso Proteins by Immunohistochemistry......Page 604
References......Page 605
Abstract......Page 607
Alternative Methods to Evaluate Apoptosis in Intact Animals......Page 608
Tissue Preparation, Immunohistochemistry, and Determination of Apoptosis......Page 609
T2 Measurements......Page 610
NO-Dependent Apoptosis and Involvement of Caspases......Page 611
Abstract......Page 612
Electrochemical Detection Principle......Page 613
A Sample Experiment: Electrode Construction and Application for In Vivo Real-Time Detection......Page 614
Preparation of Solutions......Page 615
Electrode Fabrication......Page 616
NO Detection......Page 617
Commercial Electrochemical NO Measurement Systems......Page 618
Abstract......Page 620
Respiratory Mass Spectrometry: Animal Studies (14NO)......Page 621
Laser Magnetic Resonance Spectroscopy: Human Studies (15NO)......Page 623
References......Page 624
Abstract......Page 625
Introduction......Page 626
Preparation of Blood for Measurements of HbNO......Page 628
Incubation of Aorta with Colloid Fe(DETC)2......Page 629
Effect of Dietary Supplementation of Nitrates......Page 630
Detection of NO in Aorta......Page 632
Association of Blood HbNO with Vascular NObull Production......Page 633
References......Page 636
A,B,C,D......Page 639
E......Page 640
F,G......Page 641
H......Page 642
I......Page 643
L,M......Page 644
N......Page 646
P......Page 650
Q......Page 651
S......Page 652
T,U,V,W......Page 653
Y......Page 654