Phase II Conjugation Enzymes and Transport Systems

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01 fm.pdf......Page 2
02 preface.pdf......Page 3
03 toc.pdf......Page 4
04 contributors.pdf......Page 8
Introduction......Page 14
Description of the Human UGT1 Complex Locus and Comparison to that in Rat and Mouse......Page 16
Prototypical Inducers of UGTs......Page 22
Impact of Mutations in Relationship to Structure of the UGT1 Locus......Page 26
Human and Rat UGT2 Loci......Page 29
References......Page 31
Identification and Characterization of Functional Hepatocyte Nuclear Factor 1-Binding Sites in UDP-Glucuronosyltransferase Ge......Page 36
HNF1 Transcription Factors......Page 37
HNF1alpha Serves a Dual Purpose in Gene Transcription......Page 38
HNF1 Regulation of Mammalian UDP-Glucuronosyltransferases......Page 40
Functional HNF1-Binding Sites Are Part of a Larger Regulatory Network......Page 42
Screening Genes for Potential HNF1-Binding Elements......Page 44
Luciferase Reporter Assays......Page 45
Site-Directed Mutagenesis of Promoter–Reporter Constructs......Page 46
Choosing Cell Lines for Transfection Assays and EMSA......Page 47
Preparation of Nuclear Extracts......Page 48
EMSA and Supershift Experiments......Page 49
Use of HNF1-Negative Cell Lines to Determine Relative Importance of HNF1 Elements......Page 50
Variation between Reporter Systems and Other Issues......Page 52
Electrophoretic Mobility-Shift Assays......Page 53
References......Page 55
Introduction......Page 61
UGT Preparations Used for Enzyme Assays......Page 63
Gradient HPLC Assay of UGTs Using [14C]UPDGA and Radiochemical Detection......Page 64
Sample Analysis by LCsolMS-MS......Page 65
Drug Glucuronide Quantification......Page 66
Determination of Substrate Specificity of UGTs......Page 67
Discussion......Page 69
References......Page 70
UDP-Glucuronosyltransferase 1A6: Structural, Functional, and Regulatory Aspects......Page 73
Structural Aspects......Page 74
Paracetamol Glucuronidation......Page 75
Serotonin as an Endogenous Substrate of UGT1A6......Page 76
Roles of UGT1A6 in Detoxification of Carcinogenic Arylamines and Aryl’Hydrocarbons......Page 77
Regulation of UGT1A6 Expression......Page 78
Homeodomain Protein HNF1 Is, in Part, Responsible for Tissue-Dependent UGT1A6 Expression......Page 79
Control of UGT1A6 by Nuclear Receptors PXR and CAR......Page 81
Ah Receptor Control of UGT1A6......Page 82
Key Role of Nrf2 in OxidativesolElectrophile Stress-Mediated UGT Induction......Page 83
Conclusions......Page 85
References......Page 86
The Role of Ah Receptor in Induction of Human UDP-Glucuronosyltransferase 1A1......Page 92
Introduction......Page 93
Cell Culture and Treatment......Page 94
Glucuronidation Activity by TLC Assay......Page 95
UGT1A1 RNA Transcripts......Page 96
Analysis of UGT1A1 Protein by Western Blot......Page 97
Identification and Genotyping of UGT1A Locus and Amplification of UGT1A1 Regulatory Fragments from a BAC Clone......Page 98
Transient Transfections with Reporter Constructs for Promoter Function Assay......Page 99
Generation of UGT1A1-Luciferase MH1A1L Cells......Page 100
Localization of cis-Acting Response Element by 5'/3' Deletion Analysis and Site-Directed Mutation Assay......Page 101
Analysis of Ah Receptor/Arnt Binding to UGT1A1 XRE Sequence by Electrophoretic Mobility-Shift Assay (EMSA) and Antibody Co......Page 104
Summary......Page 106
References......Page 107
Introduction......Page 109
Immunoblot Analysis......Page 111
Plasmids......Page 112
Transient Transfection and Luciferase Assays......Page 113
Deletion Assays......Page 114
Electrophoretic Mobility Shift Assays......Page 116
Concluding Remarks......Page 117
References......Page 119
Isoform-Selective Probe Substrates for In Vitro Studies of Human UDP-Glucuronosyltransferases......Page 122
Uses for UGT-Selective Probes......Page 123
Evaluation of UGT Probe Selectivity......Page 127
Incubation Protocol......Page 128
Data Analysis......Page 130
Acknowledgments......Page 132
References......Page 133
Structure of UDP-Glucuronosyltransferases in Membranes......Page 135
Introduction: Characterization of Membrane-Bound UDP-Glucuronosyltransferase (UGT) Proteins......Page 136
Methodological Approach......Page 137
UGTs Are Associated with ER Membranes......Page 138
Homo/heterooligomerization of UGTs......Page 140
Functional Relevance of UGT–UG T Interactions......Page 142
Structural Determinants of UGT for Membrane Association, Residency, and Activity......Page 143
Architecture and Organization of the Active Site within the Membrane......Page 146
Crucial Amino Acids of UGTs......Page 147
Application of Photoaffinity Labeling to the Characterization of Membrane-Bound and Purified Recombinant UGTs......Page 150
Expression......Page 152
5-Azido-UDP-glucuronic acid (5N3UDP-GlcUA)......Page 153
Photoaffinity Labeling with 5N3UDP-GlcUA......Page 155
Photoaffinity Labeling with AzMC......Page 156
Direct Photoaffinity Labeling with Retinoids......Page 157
Photoaffinity Labeling of Recombinant Proteins......Page 158
Protein Identification by Matrix-Assisted Laser Desorption Ionization (MALDI)......Page 160
Concluding Remarks......Page 161
References......Page 162
Human SULT1A Genes: Cloning and Activity Assays of the SULT1A Promoters......Page 167
Introduction......Page 168
Properties of Human SULT1A Genes......Page 169
Cloning of Human SULT1A Promoters......Page 171
Cell-Based Transfection of SULT1A Promoter Constructs......Page 176
SULT1A Promoter Luciferase Activity Assays......Page 177
Activity of SULT1A Promoters......Page 180
References......Page 183
Vitamin D Receptor Regulation of the Steroid/Bile Acid Sulfotransferase SULT2A1......Page 186
Introduction......Page 187
Sulfotransferases and the SULT2 Family......Page 188
SULT2A1 Enzymology......Page 189
Expression of Recombinant Enzyme......Page 192
Critical Points......Page 193
Nuclear Receptors as Regulators of SULT2A1......Page 194
VDR Regulation of SULT2A1......Page 195
Hormone Treatment and Endogenous SULT2A1 Expression......Page 197
Promoter Isolation, Promoter–Reporter Constructs, and Mutagenesis.......Page 198
Assay for Promoter Activity in Transfected Cells.......Page 199
Critical Points......Page 200
Regulatory Elements Directing VDR Regulation......Page 201
DNase I Footprinting......Page 204
Procedure......Page 205
Critical Points......Page 206
Critical Points......Page 207
Conclusion......Page 208
References......Page 209
NAT Enzymes......Page 213
NAT and Bioactivation of Mutagens......Page 214
Screening for Variants of the Human NAT2 Sequence with Altered Activity......Page 215
Solutions and Materials......Page 217
lacZ Assay Protocol I: Initial Screening......Page 218
Solutions and Materials......Page 219
Modeling the Three-Dimensional Structures of Human NAT Enzymes......Page 220
Overview......Page 221
Specific Variants......Page 223
Acknowledgment......Page 231
References......Page 232
Introduction......Page 237
NAT-Mediated Catalysis......Page 238
Oxidative Inhibition of NATs......Page 239
NAT1 Assays......Page 241
Third Method......Page 242
Effect of H2O2 on Recombinant NAT1......Page 243
Effect of Peroxynitrite (PN) on Cellular NAT1......Page 245
References......Page 247
Introduction......Page 252
Natural Expression of SULTs and NATs in Target Cells of Test Systems......Page 255
SULT and NAT Activity in Subcellular Activating Systems......Page 256
Historical Background......Page 258
Vectors......Page 260
Characteristics and Levels of Expressed Proteins......Page 261
Cofactor Supply......Page 264
Positive Controls......Page 265
References......Page 268
A Comparative Molecular Field Analysis-Based Approach to Prediction of Sulfotransferase Catalytic Specificity......Page 272
Introduction......Page 273
Application of CoMFA to Studies on Sulfotransferases......Page 275
CoMFA for rSULT1A1 Substrates......Page 276
Homology Modeling of the Catalytically Competent Complex of rSULT2A3......Page 278
Application of the Alignment Rule and Construction of the CoMFA Model......Page 280
Conclusion......Page 282
References......Page 284
Glucuronidase Deconjugation in Inflammation......Page 287
Introduction......Page 288
Inflammation and Plasma beta-Glucuronidase Activity in Mice......Page 289
beta-Glucuronidase Activity in Various Human Cells......Page 290
Activity of beta-Glucuronidase Released from Human Neutrophils......Page 292
Inflammation and HPLC Plasma Profile of Luteolin-Administered Rats......Page 293
Discussion......Page 294
References......Page 295
Introduction......Page 297
Overall Structure of Sulfatases......Page 299
Conservation of Catalytic Machinery......Page 304
Substrate-Binding Modes in ES......Page 306
The Catalytic Mechanism......Page 308
Does Membrane Anchoring Have a Functional Role?......Page 310
Oligomeric States of Sulfatases......Page 312
Structural Explanation for Steroid Sulfatase Deficiency......Page 313
References......Page 315
Sulfoconjugates of Estrogens......Page 318
Procedure for Determination of Estrogen Sulfatase Activity......Page 320
Activity of Estrogen Sulfatase......Page 322
Estrogen Sulfatase and Hormonal Activity......Page 323
References......Page 325
Introduction......Page 328
Immunohistochemistry......Page 329
mRNA In Situ Hybridization......Page 332
Laser Capture Microdissection/Reverse Transcription-Polymerase Chain Reaction......Page 335
References......Page 339
Metabolism of Phytoestrogen Conjugates......Page 342
Introduction......Page 343
Phytoestrogen Conjugates in Soybeans, Other Foods, and Dietary Supplements......Page 344
Phytoestrogen Standards......Page 346
Extraction of Phytoestrogens from Foods......Page 347
Acidified Solvent Extraction of Phytoestrogens......Page 349
Extraction Followed by Enzymatic Hydrolysis......Page 351
Extraction of Lignans......Page 352
Reversed-Phase HPLC Analysis of Phytoestrogens in Foods......Page 353
Deconjugation by Intestinal Lactase Phlorizin Hydrolase (LPH)......Page 354
Metabolism in the Cannulated Everted Sac Model......Page 355
Microsomal Hydroxylation of Isoflavones......Page 357
Analysis of Biliary Phytoestrogen Glucuronides......Page 358
Isolation of Biliary Phytoestrogen beta-Glucuronides......Page 359
Sulfation of Phytoestrogens......Page 360
Metabolism Studies of Isoflavones in Cells from the Mammary......Page 361
Synthesis of Chlorinated and Nitrated Phytoestrogens......Page 362
Formation of Chlorinated and Nitrated Isoflavones by Inflammatory Cells......Page 364
References......Page 365
Sulfation and Glucuronidation of Phenols: Implications in Coenyzme Q Metabolism......Page 369
Glucuronidation of Phenols......Page 370
Sulfation of Phenols......Page 372
Quinones in Biology......Page 377
Sulfate and Glucuronic acid Conjugatory Activity Toward Coenyzme Q (CoQ) Hydroquinone Metabolites......Page 379
References......Page 382
Physiological Importance and History......Page 387
Standards for Analysis of Bile Acid CoA Thioesters......Page 388
Bile Acid CoA Thioester Synthesis......Page 389
Microsome Preparations......Page 391
Isolation and Purification of BAL Activity......Page 392
Cloning and Expression of rBAL......Page 393
Recombinant Expression of rBAL......Page 394
rBAL Amino Acid Sequence and Relationship to Other Enzymes......Page 395
Generation of Rabbit Anti-rBAL Polyclonal Antibody......Page 397
Identification of Products Generated by rBAL......Page 398
References......Page 399
Introduction......Page 401
Relationships to Amino Acid Conjugation of Fatty Acids and Other Carboxylic Acids......Page 402
Standards for Analysis of Bile Acid Amino Acid Conjugating Enzymes......Page 403
Chemical Synthesis of Bile Acid Amino Acid Conjugates......Page 404
Assay for BAT Activity......Page 405
BAT Isolation and Purification......Page 406
Cloning and Expression of hBAT......Page 407
Expression of BAT......Page 410
Expression and Recovery of Biotinylatable BAT......Page 411
Relationship to Other Enzymes......Page 413
Metabolism of Glycine-Conjugated Bile Acids......Page 414
Analysis of Bile Acid Amino Acid Conjugates in Rat and Mouse Bile......Page 415
LC-MS Analysis of the Metabolism of Glycine-Conjugated Bile Acids by PAM......Page 416
Acknowledgments......Page 419
References......Page 420
Introduction......Page 422
Reagents and Materials......Page 424
Cell Cultures......Page 425
Quantification of Extracellular H2O2......Page 426
Incubation of Cells to Test for GSH Export......Page 427
Incubation of Cells with the H2O2-Generating System to Test for GSSG Export......Page 429
References......Page 432
ATP-Binding Cassette (ABC) Gene Organization and Protein Structure......Page 437
Human ABC Genes......Page 438
Cystic Fibrosis / CFTR......Page 440
Adrenoleukodystrophy / ABCD1......Page 442
Macular Degeneration / ABCA4......Page 443
Tangier Disease / ABCA1......Page 444
Lamellar Ichthyosis Type 2 (LI2) / ABCA12......Page 445
Cholestasis / ABCB4 / ABCB11 / ABCC2......Page 446
Sideroblastic Anemia / ABCB7......Page 447
Perspectives......Page 448
References......Page 450
Functional Analysis of Detergent-Solubilized and Membrane-Reconstituted ATP-Binding Cassette Transporter......Page 458
Introduction......Page 459
Chromatography......Page 462
Imidazole......Page 463
Binding of Substrates to SBPs and Translocator Subunits......Page 464
Procedure 3......Page 465
Procedure 4......Page 466
Binding of Nucleotides and Fluorescent Nucleotide Analogues to NBDs......Page 469
Procedure 5......Page 470
Strategy and Procedure......Page 471
Procedure 6......Page 474
ATP versus ATP-Regenerating System......Page 475
Choice of Lipids......Page 476
Substrate Import versus Export......Page 477
ATP/Substrate Stoichiometry......Page 478
Fluorescent Substrates to Monitor Transport......Page 480
Giant Unilamellar Vesicles......Page 481
Fluorescence Correlation Spectroscopy......Page 482
Concluding Remarks......Page 484
References......Page 485
Introduction......Page 489
Pleiotropic Drug Resistance in Yeast......Page 491
The First Defense Line: Plasma Membrane Pumps Mediating PDR......Page 492
Looking Out My Back Door: Vacuolar Sequestration......Page 494
Genetic Analysis and Phenotypic Characterization......Page 495
Substrate Specificity and Structure-Function Analysis of PDR Transporters......Page 497
Life of PDR Pumps: Where Do They Come From- Where Are They Going?......Page 499
Functional Assays for Yeast ABC Pumps Mediating Drug Resistance......Page 501
Overexpression and Purification of Yeast ABC Pumps......Page 504
Conclusions and Perspectives......Page 506
References......Page 507
Introduction......Page 514
Construction of Expression Vector......Page 516
Amplification of Recombinant Virus and Titer Check......Page 518
Plasma Membrane Vesicles from Insect Cells......Page 519
Immunological Detection of ABCG2 in Plasma Membrane Vesicles......Page 520
Development of a High-Speed Screening System......Page 522
Functional Screening of Genetic Polymorphisms of ABCG2......Page 523
Human ABC Transporter ABCB1......Page 525
High-Speed Screening System to Measure ABCB1 ATPase Activity......Page 527
Quantitative Structure-Activity Relationship (SAR) Analysis to Evaluate the Substrate Specificity of ABCB1......Page 529
SNP Array to Detect Genetic Polymorphisms of ABC Transporters......Page 534
Concluding Remarks......Page 536
References......Page 537
Overview of Transport and Metabolism of Bile Acids, Drugs, and’Xenobiotics......Page 540
Metabolic Nuclear Receptors as Transcription Factors......Page 541
Role of FXR in Bile Acid Homeostasis......Page 543
Positive Transcriptional Regulation by FXR......Page 544
Small Heterodimer Partner (SHP) as a Mediator of Negative Transcriptional Regulation by FXR......Page 545
FXR Negatively Regulates Bile Acid Uptake Systems......Page 546
Role of PXR and CAR in Drug Transport and Metabolism......Page 547
Intertwined Transcriptional Regulation of Bile Acid and Drug Metabolism by FXR, PXR, and CAR......Page 549
Role of HNF-4alpha in the Regulation of Bile Acid and Drug Metabolism......Page 550
References......Page 553
Introduction......Page 560
Transport Assays......Page 564
Vectorial Transport by Double-Transfected Cells......Page 565
Substrates and Inhibitors for Vectorial Transport by Double-Transfected Cells......Page 567
References......Page 568
Biliary Transport Systems: Short-Term Regulation......Page 572
Canalicular and Sinusoidal Transport Systems......Page 573
Methods Used to Study Subcellular Transporter Distribution......Page 576
Determination of Subcellular Localisation of Transporters by Confocal Laser-Scanning Microscopy......Page 577
Determination of Distribution of Mrp2 by Differential Centrifugation......Page 578
Determination of Distribution of Mrp2 or Ntcp by Sucrose Gradient’Centrifugation......Page 580
Determination of Distribution of Ntcp by FLAG-Tag and Flow Cytometry......Page 582
References......Page 584
Progressive Familial Intrahepatic Cholestasis......Page 588
Dubin-Johnson Syndrome......Page 591
Genetic Abnormalities in Sinusoidal Transporter Proteins......Page 592
Diagnostic Procedures for Progressive Familial Intrahepatic Cholestasis......Page 593
References......Page 595
Epoxide Hydrolases: Structure, Function, Mechanism, and Assay......Page 600
Introduction......Page 601
Detoxification......Page 602
Processing of Signal Molecules......Page 603
alpha/beta Hydrolase Fold EHs......Page 604
Limonene Epoxide Hydrolase......Page 606
Leukotriene A4 Hydrolase......Page 607
Recombinant Expression of EH......Page 608
Refolding Procedure......Page 609
Assay Procedure......Page 610
Calculation......Page 611
Assay Procedure......Page 612
Important Considerations......Page 613
Important Considerations......Page 614
References......Page 615
Pregnane X Receptor-Mediated Transcription......Page 620
Tissue Distribution, Ontogeny, and Interindividual Expression......Page 621
Species Specificity of PXR Activation......Page 622
Determination of PXR Transcriptional Activity......Page 623
Cell Culture......Page 624
Treatment of Transfected Cells with PXR Ligand......Page 625
Data Analysis......Page 626
References......Page 627
Animal Models of Xenobiotic Receptors in Drug Metabolism and Diseases......Page 631
Drug Metabolizing Enzymes and Transporters......Page 632
Nuclear Receptor (NR) and Xenobiotic NRs......Page 633
Generation of Chimeric Mice, Germ Line Transmission, and Confirmation of Loss of PXR Expression......Page 634
Construction of Transgene......Page 635
Generation of Transgenic Mice and Confirmation of Transgene Expression......Page 636
Generation of hPXR Humanized Mice......Page 637
Humanized Drug Response Profile in "Human ized" Mice......Page 638
Northern Blot Analysis......Page 639
Mouse Liver Microsome Preparation......Page 640
Glucuronidation assay using HPLC fluorescence detection......Page 641
Sulfotransferase Assay......Page 642
Use of Xenobiotic Receptor Mouse Models for In Vivo Drug Metabolism’Studies......Page 643
Prevention of Cholestasis by PXR Activation......Page 644
Hyperbilirubinemia......Page 645
Differential Effect of Individual and Combined Loss of PXR and CAR on Hyperbilirubinemia......Page 646
References......Page 647
Introduction......Page 652
Glutathione S-transferase......Page 653
UDP-Glucuronosyltransferase......Page 655
Sulfotransferase......Page 656
N-Acetyltransferase......Page 658
References......Page 659
x subject index.pdf......Page 662
xx author index.pdf......Page 675
xxx mie index.pdf......Page 728