Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding

Chapter One: A High-Throughput Fluorometric Assay for Lipid–Protein Binding

• Abstract
• 1 Introduction
• 2 Fluorescence Quenching Assay
• 3 Data Analysis
• Acknowledgments

Chapter Two: Fluorescence-Based In Situ Quantitative Imaging for Cellular Lipids

• Abstract
• 1 Introduction
• 2 Preparation of Tunable Orthogonal Lipid Sensors
• 3 In Situ Quantification of Cellular Lipids
• 4 Summary
• Acknowledgments

Chapter Three: Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A₂

• Abstract
• 1 Introduction
• 2 Strategies for Production of Recombinant sPLA₂s
• 3 Growth of Bacteria
• 4 Isolation of Inclusion Body
• 5 Preparation of Sulfonated Protein
• 6 Preparation of hGIB
• 7 Preparation of mGIB
• 8 Preparation of hGIIA
• 9 Preparation of mGIIA
• 10 Preparation of mGIIC
• 11 Preparation of hGIID
• 12 Preparation of mGIID
• 13 Preparation of hGIIE
• 14 Preparation of mGIIE
• 15 Preparation of hGIIF
• 16 Preparation of mGIIF
• 17 Preparation of hGIII sPLA₂ Domain
• 18 Preparation of mGIII sPLA₂ Domain
• 19 Preparation of hGV
• 20 Preparation of mGV
• 21 Preparation of hGX
• 22 Preparation of mGX
• 23 Preparation of hGXIIA
• 24 Preparation of mGXIIA, hGXIIB, and mGXIIB
• 25 Quality Control and Characterization of Refolded sPLA₂s by Mass Spectrometry
• 26 Enzymatic Assays

Chapter Four: Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA₂α to Membrane

• Abstract
• 1 Introduction
• 2 Constructs, Cell Types, and Transfection Protocols
• 3 Protocols to Study the Role of Calcium in Regulating the Translocation of FP-cPLA₂α and FP-C2 Domain
• 4 Protocol for Correlating Translocation of FP-cPLA₂α and Arachidonic Acid Release
• Acknowledgment

Chapter Five: Secreted Phospholipase A₂ Specificity on Natural Membrane Phospholipids

• Abstract
• 1 Introduction
• 2 Key Factors Determining the Substrate Specificity of sPLA₂s In Vivo
• 3 Methodologies of Lipidomics Analysis
• 4 Examples for Determination of the sPLA₂-Intrinsic Lipid Pathways
• 5 Concluding Remarks
• Acknowledgments

Chapter Six: Analyses of Calcium-Independent Phospholipase A₂beta (iPLA₂β) in Biological Systems

• Abstract
• 1 Introduction
• 2 Experimental Procedures
• 3 Summary
• Acknowledgments

Chapter Seven: Using Hydrogen–Deuterium Exchange Mass Spectrometry to Examine Protein–Membrane Interactions

• Abstract
• 1 Introduction
• 2 Mechanism of Hydrogen Exchange
• 3 Hydrogen–Deuterium Exchange to Study Protein–Membrane Interactions
• 4 Experimental Considerations for HDX-MS Membrane Experiments
• 5 Case Study of a Protein–Membrane HDX-MS SETUP experiment: PI3Kδ
• 6 Conclusions
• Acknowledgments

Chapter Eight: Reversible Chemical Dimerization by rCD1

• Abstract
• 1 Introduction
• 2 Methods
• 3 Limitations
• 4 Perspectives
• Acknowledgments

Chapter Nine: Interfacial Enzymes: Membrane Binding, Orientation, Membrane Insertion, and Activity

• Abstract
• 1 Introduction
• 2 Methods
• 3 Strengths and Limitations
• 4 Conclusions

Chapter Ten: Measuring Diacylglycerol Kinase-θ Activity and Binding

• Abstract
• 1 Theory
• 2 Equipment
• 3 Materials
• 4 Solutions and Buffers
• 5 Estimated Duration
• 6 DGKθ Activity Assay
• 7 Surface Dilution Experiment: Determining the K_d^app, K_MS^app and V_MAX^app of DGKθ
• 8 Vesicle Pulldown to Determine Interfacial K_d
• 9 Tips for Nonspecialists

Chapter Eleven: Studying Gastric Lipase Adsorption Onto Phospholipid Monolayers by Surface Tensiometry, Ellipsometry, and Atomic Force Microscopy

• Abstract
• 1 Introduction
• 2 Study of pH-Dependent Adsorption of Gastric Lipase Onto Phospholipid Monolayers Using Surface Tensiometry
• 3 Study of Gastric Lipase Adsorption Using Ellipsometry
• 4 Study of Gastric Lipase Adsorption by AFM
• 5 Conclusions

Chapter Twelve: Probing Conformational Changes and Interfacial Recognition Site of Lipases With Surfactants and Inhibitors

• Abstract
• 1 Introduction
• 2 Effects of Surfactants on Lipase Inhibition
• 3 X-Ray Crystallography for Studying Lipase Structures, Conformational Changes, and IRSs
• 4 Probing Lipase Lid Opening by SDSL and EPR Spectroscopy
• 5 Conclusions
• Acknowledgment

Chapter Thirteen: Measuring Phospholipase D Enzymatic Activity Through Biochemical and Imaging Methods

• Abstract
• 1 Introduction
• 2 Methods
• 3 Fluorescent-Based PLD Activity Assays
• 4 Summary
• Acknowledgments

Chapter Fourteen: PLD Protein–Protein Interactions With Signaling Molecules and Modulation by PA

• Abstract
• 1 Introduction
• 2 PLD2 and RAC2 Protein–Protein Interaction and the Effect of PA
• 3 Protocol
• 4 Conclusions and Outlook
• Acknowledgments

Chapter Fifteen: Analysis of Phosphatidic Acid Binding and Regulation of PIPKI In Vitro and in Intact Cells

• Abstract
• 1 Introduction
• 2 Bacterial Expression and Purification of PIPKIγ
• 3 Determination of the Direct Binding Between PA and PIPKIγ Using A Liposome Pulldown Assay
• 4 Measurement of PA-Stimulated PIPKIγ Activity Using Liposomes
• 5 Assessment of Actin Cytoskeletal Reorganization Induced by PIPKIγ Overexpression
• 6 Summary
• Acknowledgments