Methods in Neurosciences, Volume 6: Neuropeptide Technology: Synthesis, Assay, Purification, and Processing describes procedures and tools of assay useful for the identification, purification, and quantification of neuropeptides and their receptors. This volume is divided into four sections- chemical synthesis and biosynthesis; measurement of neuropeptides; purification and characterization; and neuropeptide degrading and processing enzymes. In these sections, this book specifically discusses the synthesis of peptide substrates for protein kinase C; synthesis of glycosyl neuropeptides; and ultrastructural localization of peptides. The measurement of neurokinin B by radioimmunoassay; purification and characterization of neuroendocrine peptides from rat brain; and preparation of glia maturation factor ß are also elaborated. This text likewise covers the assays for arginine/lysine carboxypeptidases and enzymes that metabolize atrial natriuretic peptide. This publication is beneficial to neuroscientists and students researching on the synthesis, assay, purification, and processing of neuropeptides.
Inhaltsverzeichnis
1;Front Cover;1 2;Neuropeptide Technology: Synthesis, Assay, Purification, and Processing;4 3;Copyright Page;5 4;Table of Contents;6 5;Contributors to Volume 6;10 6;Preface;14 7;Methods in Neurosciences;16 8;Section I: Chemical Synthesis and Biosynthesis;18 8.1;Chapter 1. Large-Scale Synthesis of Gonadotropin-Releasing Hormone Antagonists for Clinical Investigations;20 8.1.1;Introduction;20 8.1.2;Nal-Glu Antagonist;21 8.1.3;Chemicophysical Characterization of Nal-Glu Antagonist and Azaline;36 8.1.4;Acknowledgments;42 8.1.5;References;42 8.2;Chapter 2. Synthesis of Peptide Substrates for Protein Kinase C;44 8.2.1;Introduction;44 8.2.2;Preparation of Peptide Substrate;45 8.2.3;Assay Method;46 8.2.4;Application to Crude Extract;48 8.2.5;Acknowledgments;51 8.2.6;References;51 8.3;Chapter 3. Synthesis of Glycosyl Neuropeptides;52 8.3.1;Introduction;52 8.3.2;Glycosylation Methodologies;52 8.3.3;General Comments;64 8.3.4;Reagents and Solvents;65 8.3.5;Acknowledgments;66 8.3.6;References;66 8.4;Chapter 4. Assays for Biosynthesis of Thyrotropin-Releasing Hormone;68 8.4.1;Introduction;68 8.4.2;Transfection of AtT20 Cell Line with PreproTRH cDNA;68 8.4.3;Pulse-Chase Studies of ProTRH-Derived Peptides;74 8.4.4;Appendix;81 8.4.5;Acknowledgment;85 8.4.6;References;85 8.5;Chapter 5. Microsequence Analysis of Proteins Purified by Gel Electrophoresis;86 8.5.1;Introduction;86 8.5.2;Outline of Procedure;87 8.5.3;Discussion;92 8.5.4;Experimental Procedures;93 8.5.5;Acknowledgments;100 8.5.6;References;101 8.6;Chapter 6. Preparation of Site-Specific Peptide Immunogens Using Multiple Antigen Peptide Approach System;102 8.6.1;Introduction;102 8.6.2;Multiple Antigen Peptide Design;103 8.6.3;Direct Approach;103 8.6.4;Indirect Approach;104 8.6.5;Experimental Procedures;107 8.6.6;Copy Number of Dendritic Arms;119 8.6.7;Relationship of Orientation and Antigenicity;119 8.6.8;Conclusion;122 8.6.9;Acknowledgments;123 8.6.10;References;123 9;Section II: Measurement of Neuropeptides;126 9.1;Chapter
7. Subcellular Distribution of Neuropeptides and Measurement of Their in Vitro Release;128 9.1.1;General Introduction;128 9.1.2;Subcellular Fractionation;128 9.1.3;Neuropeptide Release;131 9.1.4;References;135 9.2;Chapter 8. Analysis of Tachykinin Peptide Family Gene Expression Patterns by Combined High-Performance Liquid Chromatography-Radioimmunoassay;136 9.2.1;Preparation of Synthetic Tachykinin Peptides and Precursor Fragments;137 9.2.2;Extraction of Tachykinin Peptides from Tissues and Cells;142 9.2.3;Acknowledgments;146 9.2.4;References;146 9.3;Chapter 9. Ultrastructural Localization of Peptides: Comparison of Methods;147 9.3.1;Introduction;148 9.3.2;Methods;149 9.3.3;Results;154 9.3.4;Discussion;161 9.3.5;Acknowledgments;163 9.3.6;References;163 9.4;Chapter 10. Measurement of Immune System in Response to Peptides in Central Nervous System;164 9.4.1;Introduction;164 9.4.2;Methods;166 9.4.3;Results and Discussion;170 9.4.4;Acknowledgments;173 9.4.5;References;174 9.5;Chapter 11. Identification of Functionally Significant Phosphorylation Sites on Neuronal Proteins and Preparation of Antibodies That Recognize Them;175 9.5.1;Introduction;175 9.5.2;Identification of Functionally Significant Phosphorylation Sites;176 9.5.3;Preparation of Antibodies That Recognize Individual Phosphorylated Sites;184 9.5.4;Conclusions;191 9.5.5;Acknowledgments;192 9.5.6;References;192 9.6;Chapter 12. Measurement of Brain Peptides: Angiotensin and Atrial Natriuretic Peptide in Tissue and Cell Culture;194 9.6.1;Introduction;194 9.6.2;Methods;195 9.6.3;Cell Culture Methods;214 9.6.4;Acknowledgments;222 9.6.5;References;222 9.7;Chapter 13. Regionally Specific Antisera to Human ß-Preprotachykinin;224 9.7.1;Introduction;224 9.7.2;Radioimmunoassay for N-Terminal Flanking Peptide of Human /3-Preprotachykinin;226 9.7.3;Radioimmunoassay for Substance P Using Antiserum Directed against COOH-Terminal Residues;229 9.7.4;Radioimmunoassay for Neuropeptide K Using NH2-Terminally Directed Antiserum;2
31 9.7.5;Radioimmunoassay for Neuropeptide K(1-24) Peptide Using COOH-Terminally Directed Antiserum;232 9.7.6;Radioimmunoassay for Neurokinin A Using COOH-Terminally Directed Antiserum;234 9.7.7;Radioimmunoassay for COOH-Terminal Flanking Peptide of ß-Preprotachykinin;235 9.7.8;References;237 9.8;Chapter 14. Measurement of Neurokinin B by Radioimmunoassay;238 9.8.1;Introduction;238 9.8.2;Measurement of Neurokinin B by Radioimmunoassay Using NH2-Terminally Directed Antiserum;239 9.8.3;Measurement of Neurokinin B Radioimmunoassay Using COOH-Terminally Directed Antiserum;244 9.8.4;Comparison of Radioimmunoassay Methods for Measurement of Neurokinin B;247 9.8.5;References;248 9.9;Chapter 15. Radioimmunoassay of Tachykinins;249 9.9.1;Introduction;249 9.9.2;Preparation of Immunogens;249 9.9.3;Immunization;250 9.9.4;Labeling with Bolton-Hunter Reagent;251 9.9.5;Tissue Extraction;257 9.9.6;Radioimmunoassay;258 9.9.7;References;263 9.10;Chapter 16. Radioimmunoassay of a-Melanocyte-Stimulating Hormone;264 9.10.1;Introduction: Biochemistry and Physiology;264 9.10.2;Radioimmunoassay;266 9.10.3;References;273 9.11;Chapter 17. Radioimmunoassay of Atrial Natriuretic Peptide;277 9.11.1;Introduction;277 9.11.2;Materials and Methods;278 9.11.3;Results;280 9.11.4;Discussion;286 9.11.5;References ;287 9.12;Chapter 18. Radioimmunoassay of Cholecystokinin;288 9.12.1;Introduction;288 9.12.2;Radiolabeling and Tracer Purification;289 9.12.3;Radioimmunoassay Procedures;290 9.12.4;Measurement of Plasma Cholecystokinin;291 9.12.5;Measurement of Tissue Cholecystokinin;294 9.12.6;Summary;295 9.12.7;Acknowledgments;296 9.12.8;References;296 9.13;Chapter 19. Secretion and Radioimmunoassay of Somatostatin: In Vitro System;298 9.13.1;Introduction;298 9.13.2;General Aspects of Radioimmunoassay of Somatostatin;299 9.13.3;Somatostatin Secretion from Isolated Pancreatic Islets;302 9.13.4;Secretion of Somatostatin from Isolated Pancreatic Islets;305 9.13.5;References;309 10;Section III: Purification and
Characterization;312 10.1;Chapter 20. Purification and Characterization of Immunoregulatory Peptides from Neuroendocrine Tissues: Suppressin as a Model;314 10.1.1;Introduction;314 10.1.2;Bioassay;314 10.1.3;Extraction and Purification of Suppressin;315 10.1.4;Verification of the Novelty of Suppressin;320 10.1.5;Summary;322 10.1.6;References;322 10.2;Chapter 21. Purification and Characterization of Neuroendocrine Peptides from Rat Brain: Prosomatostatin Isolation;323 10.2.1;Introduction;323 10.2.2;Methods Applied for Extraction and Purification of Prosomatostatin;330 10.2.3;Characterization of Peptides and Propeptides: General Considerations;333 10.2.4;Methods Applied in Characterization of Prosomatostatin;335 10.2.5;References;337 10.3;Chapter 22. Preparation of Glia Maturation Factor ß;338 10.3.1;Purification of Natural GMF-ß by Conventional Method;338 10.3.2;Purification of Natural GMF-ß by Immunoaffinity;343 10.3.3;Monitoring Purification Process;348 10.3.4;Activation and Bioassay of Pure GMF-ß;349 10.3.5;Characterization of Purified Products;350 10.3.6;References;354 10.4;Chapter 23. Detection and Purification of Thyrotropin-Releasing Hormone Precursor Peptides Using Antisera Generated against Synthetic Peptides;354 10.4.1;Introduction;354 10.4.2;Antibody Production;355 10.4.3;Radioimmunoassay;358 10.4.4;Assay;359 10.4.5;Peptide Purification;364 10.4.6;Acknowledgment;370 10.4.7;References;370 10.5;Chapter 24. Physiologic Roles of Peptides as Central Nervous System Transmitters;371 10.5.1;Introduction;371 10.5.2;Demonstrating Presence of Authentic Peptide within Relevant CNS Areas;373 10.5.3;Characterizing the Biological Actions of Peptides;375 10.5.4;Identifying CNS Site(s) of Action of Peptides;376 10.5.5;Evaluating Neuroanatomical Relationships between Peptide Immunoreactivity, Peptide Receptors, and Sites of Action;381 10.5.6;Monitoring Peptide Release from Neurons;381 10.5.7;Development and Application of Methods for Altering Synthesis, Release, and Receptor
Actions of Peptides;383 10.5.8;Additional Considerations;384 10.5.9;Acknowledgments;385 10.5.10;References;385 11;Section IV: Neuropeptide Degrading and Processing Enzymes;388 11.1;Chapter 25. Assays for Arginine/Lysine Carboxypeptidases: Carboxypeptidases H (E; Enkephalin Convertase), M, and N;390 11.1.1;Introduction;390 11.1.2;Assay Methods for Arginine/Lysine Carboxypeptidases;390 11.1.3;Assay with Dns-Ala-Arg;396 11.1.4;Acknowledgment;401 11.1.5;References;401 11.2;Chapter 26. Assay of Peptidase That Removes Carboxy-Terminal Tripeptide from 125I-Labeled Atrial Natriuretic Factor;403 11.2.1;Introduction;403 11.2.2;Material and Methods;404 11.2.3;Discussion;412 11.2.4;References;413 11.3;Chapter 27. Assays for Enzymes That Metabolize Atrial Natriuretic Peptide;414 11.3.1;In Vitro Assay;415 11.3.2;Results and Discussion;416 11.3.3;Results and Discussion;421 11.3.4;References;425 11.4;Index;428
