E-Book

Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding [electronic resource]

  • Gelb, Michael H.
  • Academic Press
  • 2017
Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding [electronic resource]
  • 자료유형
    단행본
  • 서명/저자사항
    Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding[electronic resource] / Michael H. Gelb.
  • 발행사항
    [Cambridge, MA] : Academic Press, 2017.
  • 개인저자
    Gelb, Michael H.
  • 형태사항
    1 online resource.
  • 총서명
    Methods in Enzymology ; 583
  • ISSN
    0076-6879
  • ISBN
    9780128094198
  • 언어
    영어

목차

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 Kdapp, KMSapp and VMAXapp of DGKθ
8 Vesicle Pulldown to Determine Interfacial Kd
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