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Ribonucleases, Part A: Functional Roles and Mechanisms of Action - 1st Edition - ISBN: 9780121822422, 9780080496917

Ribonucleases, Part A: Functional Roles and Mechanisms of Action, Volume 341

1st Edition

Serial Volume Editor: Allen Nicholson
Hardcover ISBN: 9780121822422
eBook ISBN: 9780080496917
Paperback ISBN: 9780123917966
Imprint: Academic Press
Published Date: 18th September 2001
Page Count: 734
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Table of Contents

  • Contributors to volume 341
  • Preface
  • Volume in series
  • Section I: Ribonuclease Classification and Review
    • [1]: A Natural Classification of Ribonucleases
      • Introduction and Classification Approach
      • Evolutionary Classification of Ribonucleases
      • Functional Systems and Evolutionary History of Ribonucleases
    • [2]: The Ribonuclease T1 Family
      • Introduction
      • Similarities and Differences among RNase T1 Family Members
      • RNase Assay Method
      • Purification
    • [3]: Ribonuclease T2
      • Classification
      • Protein Structure
      • Mechanism of Action
      • Base Specificity
      • Physiological Roles
      • Enzyme Assay
      • Enzyme Units
      • Preparation of Fungal T2–like RNase
      • Preparation of RNase Trv from Commercial Enzyme Preparation “Cellulase T-AP” Produced from Trichoderma viride
      • Preparation of Oyster (Crassostrea gigas) RNase
      • Inhibitors
    • [4]: The Ribonuclease P Family
      • Introduction
      • RNase P Subunit Composition
      • RNase P Structure and Function
      • Universal Features of RNase P
      • Acknowledgments
  • Section II: Ribunucleases Assays
    • [5]: Fast, Facile, Hypersensitive Assays for Ribonucleolytic Activity
      • Assays for Ribonucleolytic Activity
      • Design and Synthesis of Fluorogenic Substrates
      • Preparation of Reagents
      • Assay Procedures and Data Analysis
      • Applications
    • [6]: Activity Staining for Detection of Ribonucleases Using Dried Agarose Film Overlay Method After Isoelectric Focusing
      • Introduction
      • Zymogram Method for Detection of RNase Activities after Isoelectric Focusing by Means of Dried Agarose Film Overlay Method
      • Materials and Analytical Methods
      • Specific Detection of Pancreatic-Type Ribonucleases Based on Polycytidylic Acid/Ethidium Bromide Fluorescence Following IEF
      • pH Gradient Electrophoresis of Basic Ribonucleases in Sealed Slab Polyacrylamide Gels Followed by DAFO Zymogram Detection
      • Acknowledgments
    • [7]: Gel Renaturation Assay for Ribonucleases
      • Introduction
      • Advantages and Limitations of Activity Gels Applied to Ribonucleases
      • General Guidelines for in Situ Recovery of Enzymatic Activity
      • Preparing, Casting (Pouring), and Running an Activity Gel
      • Processing of Gel for Renaturation, and in Situ Detection of Ribonuclease Activity
      • Interpretation of Results
    • [8]: Analysis of Ribonucleases following Gel Electrophoresis
      • Zymograms
      • One-Dimensional Zymograms
      • Notes
      • One-Dimensional Zymograms using p29 and RNase A
      • Two-Dimensional Zymograms
      • 2D Zymogram using p29
      • Elution of Proteins from SDS–Polyacrylamide Gels
      • Recovery of p29 following SDS–PAGE
      • Acknowledgments
    • [9]: Ribonuclease Assays Utilizing Toluidine Blue Indicator Plates, Methylene Blue, or Fluorescence Correlation Spectroscopy
      • Introduction
      • Toluidine Blue O Indicator Plates
      • Methylene Blue Assay
      • FCS Assay
      • Acknowledgments
    • [10]: Ribonuclease Activities of Trypanosome RNA Editing Complex Directed to Cleave Specifically at a Chosen Site
      • Background
      • Trypanosoma brucei RNA Editing Complex
      • gRNA-Directed Endonuclease
      • gRNAs for Cleavage at Natural A6 Editing Sites
      • Design of Minimal Anchor “gRNA” to Cleave Heterologous RNAs
      • U-Specific Exonuclease
      • Additional Relevant Assays
      • Conclusion
    • [11]: Ribonuclease YI*, RNA Structure Studies, and Variable Single-Strand Specificities of RNases
      • Introduction
      • Materials and Methods
      • Results
      • Discussion
  • Section III: Secreted Ribonucleases
    • [12]: Bovine Pancreatic Ribonuclease A: Oxidative and Conformational Folding Studies
      • Introduction
      • Preparative Methods
      • Native Structure of RNase A
      • Equilibrium Unfolding Transitions of RNase A
      • Reductive Unfolding and Oxidative Folding of RNase A
      • Effect of Proline cis/trans-Isomers in Disulfide-Intact Folding
      • Conformational Folding of RNase A
      • Methodology of Reductive Unfolding and Oxidative Folding Studies
    • [13]: Purification of Engineered Human Pancreatic Ribonuclease
      • Introduction
      • Heterologous Production of HP-RNase
      • Obtaining High Levels of HP-RNase
      • Native Conformation: From Solubilization to Purification
      • Characterization: Analysis of Protein Homogeneity and Structural Integrity
      • Future Prospects and Applications of HP-RNase Engineering
      • Acknowledgments
    • [14]: Degradation of Double-Stranded RNA by Mammalian Pancreatic-Type Ribonucleases
      • Introduction
      • Properties of Double-Stranded RNA
      • Mechanism of Double-Stranded RNA Degradation
      • Influence of Ionic Strength on dsRNA Degradation by Mammalian ptRNases
      • Experimental Procedure
      • Acknowledgment
    • [15]: Seminal Ribonuclease: Preparation of Natural and Recombinant Enzyme, Quaternary Isoforms, Isoenzymes, Monomeric Forms; Assay for Selective Cytotoxicity of the Enzyme
      • Introduction
      • Experimental Procedures
    • [16]: Angiogenin
      • Introduction
      • Methods of Assay
      • Isolation of Angiogenin
      • Biological Function
    • [17]: Eosinophil-Derived Neurotoxin
      • 1 Background
      • 2 GenBank Accession Numbers for EDN
      • 3 Recombinant EDN from Bacterial Expression Systems
      • 4 Recombinant EDN from Baculovirus
      • 5 Recombinant EDN from Eukaryotic Cell Culture
      • 6 Natural Forms of EDN
      • 7 Ribonuclease Assay
      • 8 Biological Assays: Antiviral Activity
    • [18]: Eosinophil Cationic Protein
      • Purification of ECP
      • RNase Activity
      • ECP Biological Properties
      • Quantification of ECP Levels in Biological Fluids
      • Acknowledgments
    • [19]: Deciphering the Mechanism of RNase T1
      • Introduction
      • Reactivity of Scissile Bond
      • Chemical Nature of Transition State
      • Experimental Basis for In-Line Acid–Base Catalysis
      • Thio Substitutions to Map Catalytic Interactions with Nonbridging Oxygens
      • Triester-Like Mechanism Involving Internal Proton Transfer
      • A Three-Centered Hydrogen Bond for Concerted Phosphoryl Transfer
      • Nucleophile Activation by Cooperative Hydrogen Bonding
      • Positioning of Proper Imidazole Tautomer Contributes to Leaving Group Activation
      • Transition State Stabilization by Specific Solvation/Desolvation
      • Subsite-Binding Effects on Turnover
      • Perfect Match between RNase T1 and Transition State
    • [20]: Mitogillin and Related Fungal Ribotoxins
      • Introduction
      • Assay Methods
      • Overexpression and Purification of Mitogillin
      • Conformational Analysis of Mitogillin
      • Immunological Properties
      • Procedures for Detection of IgE Antibodies against Asp f 1
      • Concluding Remarks and Future Directions
      • Acknowledgments
    • [21]: RNase U2 and α-Sarcin: A Study of Relationships
      • Introduction
      • Acknowledgments
    • [22]: Secretory Acid Ribonucleases from Tomato, Lycopersicon esculentum Mill.
      • Introduction
      • Source of Enzymes
      • Assay Methods
      • Purification Procedures
      • Subcellular Localization of Intracellular Ribonucleases
      • Properties of Tomato Ribonucleases LE, LX, LV-1, LV-2, and LV-3
      • Acknowledgment
    • [23]: Leczyme
      • Introduction
      • Materials and Cells
      • Tumor Cell Agglutination and Growth Inhibition
      • Leczyme-Induced Apoptosis and Expression of Apoptosis-Related Antigens
      • Conclusion
      • Acknowledgments
  • Section IV: Ribonucleases H
    • [24]: Prokaryotic Type 2 RNases H
      • Introduction
      • Acknowledgment
    • [25]: RNase H1 of Saccharomyces cerevisiae: Methods and Nomenclature
      • Roles for RNases H in Cells
      • Solution-Based Assay
      • Gel-Renaturation Assay
      • Complementation of Temperature-Sensitive Growth Defect of rnhA Mutants of Escherichia coli
      • Northwestern Assay
      • Substrate Preparation
      • Northwestern Procedure
    • [26]: Ribonucleases H of the Budding Yeast, Saccharomyces cerevisiae
      • Introduction
      • Assays for Determining RNase H Activity
      • Enzyme Purification Procedures
      • Evolutionarily Related Proteins from Organisms Other than S. cerevisiae by Alignments of Predicted Amino Acid Sequences
      • Acknowledgments
    • [27]: Human RNases H
      • Introduction
      • Molecular Cloning and Expression of Human RNases H
      • Purification of Human RNase H Proteins
      • Preparation of Heteroduplex Substrate
      • RNase H1 Cleavage Assay
      • Determination of Binding Affinity
      • Conclusions
    • [28]: Assays for Retroviral RNase H
      • Assays for Retroviral RNase H
      • Nonspecific RNase H Assays
      • Specific RNase H Cleavages
  • Section V: Synthetic Ribonucleases
    • [29]: Sequence-Selective Artificial Ribonucleases
      • Significance of Artificial Ribonucleases
      • Molecular Design
      • Methods
      • Prospect
      • Acknowledgments
    • [30]: RNA Cleavage by 1,4-Diazabicyclo[2.2.2]octane–Imidazole Conjugates
      • Introduction
      • Design of RNA Cleaving Conjugates
      • Comparison of Ribonuclease Activities of the Designed Conjugates
      • Mechanism of RNA Cleavage by Imidazole Conjugates
      • RNA Cleavage by Conjugates ABL3Cm
      • Specificity of RNA Cleavage with Conjugates ABL3Cm; Comparison with RNase A
      • Specific Features of RNA Cleavage by Conjugates of 1,4-Diazabicyclo[2.2.2]octane and Imidazole
      • Materials and Methods
      • Structural Probing of RNA with Conjugates ABL3Cm
      • Analysis of Cleaved RNA and Quantitation of Data
      • Acknowledgment
    • [31]: Preparation and Use of ZFY-6 Zinc Finger Ribonuclease
      • Introduction
      • Synthesis and Purification of ZFY-6 Zinc Finger Peptide
      • Preparation of ZFY-6 Zinc Finger Homodimer
      • Formation of Homodimer
      • Preparation and Purification of Oligoribonucleotide Substrate
      • 32P Labeling of Oligoribonucleotide Substrate
      • Cleavage Assay
      • pH Dependence
      • Conclusions
  • Section VI: Ribonucleolytic Nucleic Acids
    • [32]: RNA Cleavage by the 10-23 DNA Enzyme
      • Introduction
      • 10-23 Motif
      • Kinetic Properties
      • Substrate Specificity
      • Target Site Selection
      • Delivery to Cells
      • Cellular Uptake
      • Conclusions
      • Acknowledgments
    • [33]: Leadzyme
      • Introduction
      • Active Structure Determination
      • Catalysis and Ions
      • Computational Studies
      • Conclusion
      • Acknowledgment
    • [34]: Hammerhead Ribozyme Structure and Function in Plant RNA Replication
      • Introduction
      • Structure of Hammerhead Ribozymes
      • Function of Hammerhead Ribozymes in Their Natural Habitat
      • In Vitro Assays to Test Hammerhead-Mediated RNA Cleavage
      • Conclusions and Perspectives
      • Acknowledgments
    • [35]: Kinetic Analysis of Bimolecular Hepatitis delta Ribozyme
      • Design of Bimolecular delta Ribozyme System
      • Materials and Methods
      • Results and Discussion
      • Perspectives
      • Conclusion
      • Acknowledgment
    • [36]: Catalytic and Structural Assays for the Hairpin Ribozyme
      • Introduction
      • Ribozyme Reaction Chemistry
      • Structure
      • Catalytic Assays
      • Structural Analysis
    • [37]: Intracellular Applications of Ribozymes
      • Catalytic RNAs
      • Target Accessibility
      • Intracellular Ribozyme Expression
      • Colocalization of Ribozyme and RNA Target
      • Ribozyme Delivery
      • Intracellular in Vivo Ribozyme Activity
      • Conclusions
      • Acknowledgments
  • Section VII: Ribonuclease Inhibitor
    • [38]: Barnase–Barstar Interaction
      • Introduction
      • Barnase–Inhibitor Equilibria
      • Simultaneous Randomization of Groups of Structurally Related Residues of Barstar and Selection for Activity
    • [39]: Cytoplasmic Ribonuclease Inhibitor
      • Introduction
      • Physicochemical Properties and Primary Structure
      • Specificity
      • Structural Basis for Tight Binding
      • Biological Role of RI
      • Isolation of RI
      • Stability of RI
      • Quantification of RI
      • Kinetic Characterization of RI Complexes
      • Acknowledgments
    • [40]: Small Molecule Inhibitors of RNase A and Related Enzymes
      • Introduction
      • Binding Subsites on RNase A
      • Pancreatic RNase Superfamily
      • Inhibitors of RNase A
      • Inhibitors of Human RNase-2, RNase-4, and Angiogenin
      • Practical Considerations for Use or Assay of RNase Inhibitors
      • Preparation and Characterization of ppA-3′-p, ppA-2′-p, pdUppA-3′-p, and pTppA-3′-p
      • Structures of Complexes of RNase A with Adenosine 5′-Pyrophosphate Derivatives
      • Acknowledgments
    • [41]: Ribonuclease-Resistant RNA Controls and Standards
      • Introduction
      • Ribonuclease-Resistant RNA Controls and Standards
      • In Vitro Transcription using Modified NTPs
  • Section VIII: Nonenzymatic Cleavage of RNA
    • [42]: Nonenzymatic Cleavage of Oligoribonucleotides
      • Introduction
      • Materials and Methods
      • Features of Oligoribonucleotide Cleavage
      • Effects of Polyamines on Cleavage of Oligoribonucleotides
      • Effect of Oligoribonucleotide Structure on Cleavage
      • Mechanism of Cleavage, and External Factors Affecting Cleavage
      • Stability of Biologically Active RNA Molecules
      • Biological Implication of Nonenzymatic RNA Cleavage
      • Conclusions
  • Author Index
  • Subject Index


This first of two volumes provides up-to-date, methods-related information on ribonuclease functions, assays, and applications. Chapter topics include the identification of, characterization of, and assays for secreted ribonucleases; viral ribonucleases, artificial and engineered ribonucleases, and ribozymes.
The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.


Biochemists, Molecular Biologists, Microbiologists, Cell Biologists, and Biomedical Researchers.


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18th September 2001
Academic Press
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About the Serial Volume Editor

Allen Nicholson

Allen Nicholson

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Wayne State University, Detroit, U.S.A.