Regulatory Protein Modification: Techniques and Protocols / Edition 1

Regulatory Protein Modification: Techniques and Protocols / Edition 1

by Hugh C. Hemmings
Pub. Date:
Springer-Verlag New York, LLC
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Regulatory Protein Modification: Techniques and Protocols / Edition 1

Distinguished international experts offer critical reviews of the major categories of posttranslational protein modification in neurons, as well as a comprehensive collection of state-of-the-art techniques for their analysis. They provide detailed and specific methods for studying protein phosphorylation/dephospohorylation, methylation, long chain fatty acylation, ADP-ribosylation, glycosylation, and glycosylphosphatidylinositol anchors. Using both conventional analytical and novel molecular biological approaches, these experts present important techniques and reagents and cite numerous examples from the neuroscience literature in a way that allows investigators to determine the best experimental approaches for their own systems. The book offers the most complete review of protein kinase and phosphatase inhibitor applications available.

Product Details

ISBN-13: 9780896034150
Publisher: Springer-Verlag New York, LLC
Publication date: 12/01/1996
Series: Neuromethods Series , #30
Edition description: 1997
Pages: 446
Product dimensions: 6.10(w) x 9.25(h) x 0.04(d)

Table of Contents

Analysis of Protein Phosphorylation in Intact Cells and Extracts, S. Ivar Walaas and Anne Carine Østvold. Introduction. Investigating Protein Phosphorylation Systems. General Considerations. Experimental Pitfalls. Phosphorylation of Proteins in Intact Preparations. General Considerations. Intact Brain. Brain Slices. Isolated Cells and Nerve Terminals. Phosphorylation of Proteins in Cell-Free Preparations. General Considerations. Labeling and Stimulation of Cell-Free Preparations. Analysis of Phosphoproteins. General Considerations. Analysis of Protein Kinases. General Considerations. Analysis of Protein Kinase Activity In Vitro. Analysis of Specific Protein Kinases In Vitro. Analysis of Protein Kinase Activity in Intact Cells. Analysis of Protein Kinase Activity by Renaturation In Vitro. References. Analysis of Protein Dephosphorylation in Intact Cells and Extracts, Shelley Halpain. Introduction. Serine/Threonine Protein Phosphatases. Regulation of Phosphatase Activity. Measurement of Phosphatase Activity in Neural Tissue. Prools. Measurement of Protein Phosphatase Activity in Extracts. Assaying the Phosphorylation State of Phosphoprotein Substrates in Tissue. Pharmacological Tools for Identifying Phosphatases that Target Proteins in Intact Cells. Dephosphorylation vs Proteolysis: A Caveat. Acknowledgments. References. Protein Phosphorylation and Dephosphorylation in Isolated Nerve Terminals (Synaptosomes), Talvinder S. Sirha. Introduction. Isolated Nerve Terminals (Synaptosomes). Background (Historical). Preparation of Synaptosomes and Purification by Percoll Gradients. Protein Phosphorylation/Dephosphorylation in Synaptosomes. Background. Prelabeling Synaptosomes with [32P] Orthophosphate. Modulators of Protein Kinases and Phosphatases. Analysis of Synaptosomal Phosphoproteins. Materials. Final Remarks. Acknowledgments. References. Protein Kinase and Phosphatase Inhibitors: Applications in Neuroscience, Hugh C. Hemmings, Jr. Introduction. Protein Kinase Inhibitors. Cyclic Nucleotide-Dependent Protein Kinase Inhibitors. Ca2+/Calmodulin-Dependent Protein Kinase Inhibitors. Nonselective Protein Kinase Inhibitors (Staurosporine and Analogs). PKC Inhibitors. Cyclin-Dependent Kinase Inhibitors. Mitogen-Activated Protein Kinase (MAPK) and MAPK Kinase (MEK) Inhibitors. Protein-Tyrosine Kinase Inhibitors. Protein Phosphatase Inhibitors. Protein-Serine/Threonine Phosphatase Inhibitors . Protein-Tyrosine Phosphatase Inhibitors. Acknowledgments. Appendix I: Sources of Protein Kinase Inhibitors. Appendix II: Sources of Protein Phosphatase Inhibitors. References. Phosphorylation State-Specific Antibodies, Andrew J. Czernik, Jeffrey Mathers, and Sheenah M. Mische. Introduction. Phosphorylation State-Specific Antibody Production. Design of Phosphopeptides. Preparation of Phosphopeptides. Phosphopeptide Conjugation and Immunization. Primary Screening of Antiserum for Phosphorylation State-Specificity. Affinity Purification of Antisera. Advantages, Limitations, and Applications of Phosphorylation State-Specific Antibodies. Acknowledgments. References. Protein Tyrosine Phosphorylation, Pascal Derkinderen and Jean-Antoine Girault. Introduction. Overview of Tyrosine Phosphorylation in the Nervous System. Protein-Tyrosine Kinases. Protein-Tyrosine Phosphatases (PTPs). Biochemical Consequences of Tyrosine Phosphorylation. Neuronal Proteins Phosphorylated on Tyrosine. Regulation of Protein Tyrosine Phosphorylation in the Nervous Tissue. Methods to Study Protein Tyrosine Phosphorylation and Dephosphorylation in Nervous Tissue. Overview of the Methods Available. Model Systems for Studying the Regulation of Tyrosine Phosphorylation. Antiphosphotyrosine Antibodies. Immunoblotting with Antiphosphotyrosine Antibodies. Immunoprecipitation. In Vitro Protein Kinase Assays Using Immunoprecipitates. References. Identification of Posttranslational Modification Sites by Site-Directed Mutagenesis, James A. Bibb and Edgar F. da Cruz e Silva. Introduction. General Considerations. Choice of Target Amino Acids. Choice of Oligonucleotide. Performing Mutagenesis. Choice of Vector. Specific Methods. Uracil Incorporation. Thionucleotide Incorporation. Methyl-DNA Method. Restriction Site Elimination. Screening on the Basis of Antibiotic Resistance. Gapped-Heteroduplex Formation Method. PCR-Based Methods of Site-Directed Mutagenesis. Screening via Automated Sequencing. Expression Systems. Bacterial Expression. Eukaryotic Expression. Examples. Uracil Incorporation. Restriction Site Elimination. Thionucleotide Incorporation. Antibody Resistance. PCR. Conclusions. Appendix: Sources of Materials Cited. References. Protein Methylation in the Nervous System, Darin J. Weber and Philip N. McFadden. Introduction. Methodological Differences in Studies of Protein N-Methylation and Carboxyl O-Methylation. Hydrolytic Lability of Methyl Groups Incorporated by Protein Carboxyl O-Methyltransferases. Hydrolytically Stable Protein Methylation. Radiochemical Methyl Groups and Their Incorporation into Proteins of the Nervous System . Radiochemical Characteristics of Methyl Group Donor Compounds. Radiolabeling Procedures in the Methylation of Proteins. Methylation of Altered Aspartyl Residues in Proteins. Methylation Procedures. Quantitation of Sites Methylated by PIMT. Electrophoretic Separation of Proteins Containing Altered Aspartyl Residues. Localization of Sites of Altered Aspartyl Residues in Protein Sequences. References. Long-Chain Fatty Acylation of Proteins, Sean I. Patterson and J. H. Pate Skene. Introduction. Reagents and Equipment. Sources of Specific Reagents. General Equipment Requirements. Handling of Lipids and Solvents. Methodological Considerations. Approaches for Measuring Different Aspects of the Acylation Cycle. Criteria for the Identification of Thioester-Linked Long-Chain Acylation. Measurements of Lipid Radiolabeling. The Use of Protein Synthesis Inhibitors and Tunicamycin. Incorporation of [3H] Palmitate into Proteins. S-Palmitoylation with [3H] Palmitoyl-CoA. S-Palmitoylation with [3H] Palmitate. Perspectives. Acknowledgments. References. Protein ADP-Ribosylation, Keith D. Philibert and Henk Zwiers. Introduction. Poly-ADP-Ribosylation. Mono-ADP-Ribosylation. ADP-Ribosylation Reactions. Assay Conditions. Detection and Quantification. Characterization of ADP-Ribosylated Proteins. ADP-Ribosylation vs Phosphorylation. The ADP-Ribosylation Reaction. Generation and Purification of ADP-Ribose. ADP-Ribosyltransferase Activity. ADP-Ribosylhydrolase Activity. Functional Implications. Acknowledgements. References. Glycosylation and Glycosylphosphatidylinositol Membrane Anchors, Anthony J. Turner, Edward T. Parkin, and Nigel M. Hooper. Introduction. Protein Glycosylation. Identification of Glycoproteins. Protein Deglycosylation. Structure and Functions of GPI Membrane Anchors. GPI-Anchored Proteins in the Nervous System. Techniques for the Identification of GPI-Anchored Proteins. Phospholipase Release. Identification of the Crossreacting Determinant (CRD). Detergent Solubilization and Phase Separation in Triton X-114. Metabolic Labeling Studies. Membrane Microdomains and Caveolae. Isolation of Detergent-Insoluble Membrane Microdomains. Protein Components of Detergent-Insoluble Membrane Microdomains. Lipid Analysis. Glycolipid-Enriched Membrane Microdomains. Conclusions. Acknowledgments. References. Index.

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