Lipidomics and bioactive lipids : lipids and cell signaling / edited by H. Alex Brown.

Format:

Book

Contributor:

Brown, H. Alex (Hugh Alex), 1960-

Series:

Methods in enzymology ; v. 434.

Methods in enzymology ; v. 434

Language:

English

Subjects (All):

Lipids--Analysis.

Lipids.

Lipids--Spectra.

Mass spectrometry.

Physical Description:

1 online resource (387 p.)

Place of Publication:

Amsterdam ; London : Academic Press, c2007.

Language Note:

English

Summary:

This volume in the well-established Methods in Enzymology series features methods for the study of lipids using mass spectrometry techniques. Articles in this volume cover topics such as Phospholipase A1 assays using a radio-labeled substrate and mass spectrometry; Real-time Cell Assays of Phospholipases A2 Using Fluorogenic Phospholipids; Analysis and Pharmacological Targeting of Phospholipase C â interactions with G proteins; Biochemical Analysis of Phospholipase D.; Measurement of Autotaxin/Lysophospholipase D Activity; Platelet-Activating Factor; Quantitative measurement of PtdIns(3

Contents:

Cover; Contents; Contributors; Preface; Volumes in Series; Chapter 1: Phospholipase A1 Assays Using a Radiolabeled Substrate and Mass Spectrometry; 1. Introduction; 2. Types of PLA1; 3. Conventional PLA1 Assay Using Radiolabeled Substrates; 3.1. Materials; 3.2. Preparation of radiolabeled phospholipid substrates; 3.3. Assays; 4. Novel PLA1 Assay Using ESI-MS; 4.1. Materials; 4.2. Preparation of recombinant PLA1; 4.3. Phospholipase A1 assay; 4.4. PLA1 activity of intracellular PLA1s; 5. Perspective; Acknowledgments; References

Chapter 2: Real-Time Cell Assays of Phospholipase A2s Using Fluorogenic Phospholipids1. Introduction; 2. Fluorogenic PLA2 Substrates; 2.1. Substrate specificity of PLA2s; 2.2. Selection of fluorophores and design of fluorogenic PLA2 substrates; 2.3. Real-Time cellular PLA2 assay using fluorogenic phospholipids; 3. Measuring Cellular sPLA2 Activity Using PED6 and Red-PED6; 3.1. Materials; 3.2. Labeling of cell membranes with PED6 and monitoring cellular sPLA2 activities; 4. Measuring Cellular cPLA2alpha Activity Using DAPC; 4.1. Synthesis of DAPC

4.2. Labeling of cell membranes with DAPC and monitoring cellular cPLA2alpha activityReferences; Chapter 3: Analysis and Pharmacological Targeting of Phospholipase C beta Interactions with G Proteins; 1. Introduction; 2. Methods; 2.1. Protein expression and purification; 2.2. PLCbeta assay; 2.3. PLC assay optimization; 2.4. Application of the PLC assay to evaluate peptide/small molecule modulation of Gbetagamma-dependent PLC activity; 2.5. Evaluation of Gbetagamma-PLC binding; 3. Concluding Remarks; Acknowledgment; References; Chapter 4: Biochemical Analysis of Phospholipase D

1. Introduction2. Assay of Recombinant PLD In Vitro; 2.1. Expression of recombinant PLD1; 2.2. Purification of PLD1; 2.3. Chromatography (6His); 2.4. Size-exclusion chromatography (Superdex 200); 2.5. Anion-exchange chromatography (Q-Sepharose); 2.6. Characteristics of purified, full-length PLD1; 2.7. Purification of N-terminally truncated PLD1; 2.8. Assay of phospholipase D activity in vitro; 3. Regulated PLD1 Activity; 4. Preparation of Activators of PLD1; 4.1. Protein kinase Calpha (PKCalpha); 4.2. Myristoyl-ADP ribosylation factor 1

4.3. Purification of geranylgeranylated RhoA, Rac1, and Cdc425. Effects of Activators on PLD1 Activity; 6. Synergy between PLD1 Activators; 7. Binding of PLD1 to Phospholipid Vesicles; 8. Kinetic Parameters of PLD1 Catalytic Activity; 9. Kinetic Analyses of Synergistic Responses; 10. Phosphatidylinositol 4,5-Bisphosphate is an Essential PLD1 Activator; 11. In Vivo PLD Assay Using Radioisotopes; 12. In Vivo PLD Assay Using Deuterated 1-Butanol; 12.1. Materials and methods; 12.2. Application; 13. Fluorescent In Vitro PLD Assay; 13.1. Preparation of lipid substrates; 13.2. Phospholipase D assay

14. Real-Time Diacylglycerol Lipase Assay

Notes:

Description based upon print version of record.

Includes bibliographical references and index.

ISBN:

9786611070731

9781281070739

1281070734

9780080554099

0080554091

OCLC:

476114694