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Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding - 1st Edition - ISBN: 9780128094198, 9780128096956

Enzymology at the Membrane Interface: Interfacial Enzymology and Protein-Membrane Binding, Volume 583

1st Edition

Serial Volume Editor: Michael Gelb
Hardcover ISBN: 9780128094198
eBook ISBN: 9780128096956
Imprint: Academic Press
Published Date: 13th January 2017
Page Count: 430
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Table of Contents

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


Enzymology at the Membrane Interface, the latest volume in the Methods in Enzymology series, covers a subset of enzymes that work in the environment of the biological cell membrane. This field, called interfacial enzymology, involves a special series of experimental approaches for the isolation and study of these enzymes.

Key Features

  • Covers a subset of enzymes that work in the environment of the biological cell membrane
  • Offers a series of experimental approaches for the isolation and study of enzymes


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


No. of pages:
© Academic Press 2017
13th January 2017
Academic Press
Hardcover ISBN:
eBook ISBN:

Ratings and Reviews

About the Serial Volume Editor

Michael Gelb

Michael H. Gelb studied chemistry and biochemistry as an undergraduate at the University of California at Davis. His Ph.D. studies with Stephen G. Sligar at Yale University led to a better understanding of the catalytic mechanism of cytochrome P450. As an American Cancer Society Postdoctoral Fellow in the laboratory of the late Robert H. Abeles at Brandeis University, Gelb studied a variety of mechanism-based inactivators of serine proteases and developed fluorinated ketones as tight-binding inhibitors of several classes of proteases. In 1985 Gelb became a faculty member in the Departments of Chemistry and Biochemistry at the University of Washington. Major breakthroughs in the group include the development of methods to properly analyze the action of enzymes on membrane surfaces, the discovery of protein prenylation (farnesylation and geranylgeranylation) in mammalian cells (together with John A. Glomset), the development of Isotope-Coded Affinity Tags (ICAT reagents) for proteomic applications (together with Ruedi Aebersold), and the development of newborn screening for lysosomal storage diseases by mass spectrometry.

Affiliations and Expertise

Dept. of Chemistry, University of Washington, Seattle, USA