Cell Components

Cell-Wall-Isolation, General Growth Aspects.- 1 Introduction.- 2 Isolation Procedures.- 2.1 Cell Breakage.- 2.2 Cell-Wall Recovery.- 2.3 Removal of Contaminants.- 3 Composition and Ultrastructure of Plant Cell Walls.- 3.1 Chemical Composition of Plant Cell Walls.- 3.1.1 Standard Extraction Procedures.- 3.1.2 Analysis of Polysaccharide Fractions.- 3.1.2.1 Chemical Methods.- 3.1.2.2 Physical Methods.- 3.2 Supramolecular Organization of Plant Cell Walls.- 3.2.1 Morphological Observations.- 3.2.2 Selective Staining of Polysaccharides.- 3.2.2.1 Visualization of Esterified Carboxyl Groups.- 3.2.2.2 Detection of Acidic Functions.- 3.2.2.3 Periodic Oxidation of Glycol Groups.- 3.2.3 Visualization of Lignin.- 3.2.4 Identification of Wall Components by Means of Affinity Methods.- 3.2.5 Detection and Estimation of Cations.- 3.2.6 Ultracryotomy.- 4 Properties of Plant Cell Walls.- 4.1 Exchange Properties of Plant Cell Walls.- 4.2 Enzymatic Properties.- 4.2.1 Cytochemical Investigations.- 4.2.1.1 Cell-Wall Phosphatase Activities.- 4.2.1.2 Cell-Wall Peroxidase Activities.- 4.2.2 Biochemical Investigations.- 4.2.2.1 Properties of Immobilized and Solubilized Cell-Wall Enzymes.- 4.2.2.2 Biological Functions.- 4.3 Mechanical Properties.- 5 Growth Aspects.- 5.1 Cell-Wall Loosening.- 5.1.1 Wall-Loosening-Inducing Agents.- 5.1.2 Nature of the Broken Bonds.- 5.2 Deposition of Wall Material.- 5.3 Growth Direction.- References.- Cell-Wall Chemistry, Structure and Components.- 1 Introduction.- 2 Histochemical Analysis of Cell Walls.- 2.1 Specific Stainings.- 2.2 Staining with Fluorescent Brightener.- 2.3 Anisotropy Test.- 2.4 Selective Dissolution.- 2.4.1 Alkali Treatment.- 2.4.2 Cuprammonium Solution (Schweitzer's Reagent) Treatment.- 2.4.3 Enzymatic Digestion.- 3 Quantitative Analysis of Cell Walls.- 3.1 Plant Materials.- 3.1.1 Pure Culture.- 3.1.2 Synchronous Culture.- 3.1.3 Harvesting of Cells.- 3.2 Measurement of Cell Growth.- 3.3 Preparation and Fractionation of Cell Walls.- 3.3.1 Disruption of Cells.- 3.3.2 Separation and Purification of Cell Walls.- 3.3.3 Fractionation of Cell Walls.- 3.4 Quantitative Analysis of Whole Cell Walls.- 3.4.1 Gravimetry.- 3.4.2 Turbidimetry.- 3.4.3 Colorimetry.- 4 Qualitative Analysis of Cell-Wall Materials.- 4.1 Acid Hydrolysis.- 4.2 Enzymatic Hydrolysis.- 5 Chromatographic Analysis of Cell-Wall Constituents.- 5.1 Thin-Layer Chromatography.- 5.1.1 Neutral Sugars and Uronic Acids.- 5.1.2 Amino Acids and Amino Sugars.- 5.1.3 Thin-Layer Chromatographic Analyses of the Constituents of Chlorella Cell Walls.- 5.2 Liquid Chromatography.- 5.2.1 Amino Acids and Amino Sugars.- 5.2.2 Neutral Sugars.- References.- Protoplasts-for Compartmentation Studies.- 1 Introduction.- 2 Advantages of the Use of Protoplasts for Compartmentation Studies.- 3 Protoplast Isolation and Its Effect on Cellular Metabolism.- 3.1 Isolation Procedures.- 3.2 Effect of Isolation pH.- 3.3 Effect of Plasmolysis.- 3.4 Effect of Enzyme Contaminants.- 4 Protoplast Lysis.- 5 Protoplast Fractionation.- 5.1 Density Gradient Fractionation.- 5.2 Rapid Fractionation Procedures.- 6 Methods to Relate Protoplast Activity to That of Intact Tissue.- 7 Concluding Remarks.- References.- The Marker Concept in Cell Fractionation.- 1 Introduction.- 2 The Marker Concept.- 2.1 Basic Concepts.- 2.2 Types of Marker.- 2.2.1 Morphological.- 2.2.2 Cytochemical.- 2.2.3 Biochemical.- 3 Preservation of Marker Enzyme Activity During Cell Disruption.- 3.1 Choice of Material.- 3.2 Homogenization Procedure.- 3.3 Use of Additives in the Homogenization Medium.- 3.4 Gel Filtration to Remove Soluble Hydrolytic Activity.- 4 Methods Used to Separate Markers.- 4.1 General Approaches to Cell Fractionation.- 4.2 Differential Centrifugation.- 4.2.1 Preparative vs. Analytical Cell Fractionation.- 4.2.2 Need for Quantitation.- 4.2.3 Problems with Complete Quantitation and Interpretation of Data.- 4.3 Linear Density Gradient Centrifugation.- 4.3.1 Density Gradient Material.- 4.3.2 Pelleted vs. Unpelleted Overlays.- 4.3.3 Soluble Enzyme Contamination in Gradients.- 4.3.4 Equilibrium Density Centrifugation (Isopycnic Conditions).- 4.3.5 Other Factors Which Influence Marker Enzyme Profiles Across a Gradient.- 4.3.6 Need for Quantitation and Lack of Negative Marker Activity.- 5 Concluding Remarks.- References.- Plasma Membranes.- 1 Introduction.- 2 Theory of Phase Partition.- 2.1 The Phase System.- 2.2 Partitition of Membrane Particles.- 2.3 Effects of Polymer Concentrations.- 2.4 Effects of Salts.- 2.5 Multistep Procedures.- 3 Experimentals.- 3.1 Chemicals.- 3.2 Preparation Procedure.- 4 Purity of the Preparations.- 4.1 Specific Staining.- 4.2 K+-Stimulated, Mg2+-Dependent ATPase.- 4.3 Glucan Synthetase II.- 4.4 Light-Reducible b-Cytochrome.- 4.5 Markers for Contaminants.- 5 Protein and Lipid Composition.- 6 Surface Properties of the Isolated Vesicles.- References.- Vacuoles.- 1 Introduction.- 2 Methods of Isolation.- 2.1 Isolation of Vacuoles from Meristematic Tissues.- 2.2 Isolation of Vacuoles from Mature Plant Tissue.- 2.2.1 Isolation of Mature Vacuoles from Protoplasts-Methods pre 1981.- 2.2.2 Isolation of Mature Vacuoles from Protoplasts-Methods post 1981.- 2.2.3 Isolation of Mature Vacuoles Directly from Tissue-Methods pre 1981.- 2.2.4 Isolation of Mature Vacuoles Directly from Tissue-Methods post 1981.- 2.2.5 Preparation of Lutoids from Hevea Latex.- 2.2.6 Comments on Methods for Isolating Vacuoles from Higher Plants.- 2.2.7 Isolation of Proton-Pumping Vesicles.- 2.2.8 Preparation of Vacuoles from Yeast Neurospora.- 3 Isolation of Tonoplast and Tonoplast Markers.- 4 Comments on Physiological Functions.- 5 Concluding Remarks.- References.- Protein Bodies.- 1 Introduction.- 2 Special Consideration in Isolation of Protein Bodies.- 3 Nonaqueous Preparation in Glycerol.- 4 Nonaqueous Preparation in Hexane and Carbon Tetrachloride.- 5 Aqueous Preparation in Sources Gradients.- 6 Subfractionation of Isolated Protein Bodies.- 7 Analyses.- References.- Lipid Bodies.- 1 Introduction.- 2 Ontogeny.- 3 Isolation.- 4 Markers of Lipid Bodies.- 5 Assays.- 5.1 Fluorometric Assay.- 5.2 Colorimetrie Assay.- References.- Chloroplasts as a Whole.- 1 Introduction.- 2 Considerations of Integrity and Purity.- 3 Chloroplasts from Protoplasts.- 4 The Use of Silica Sols in Density Gradient Purification of Chloroplasts.- 5 General Notes on Isolation Procedures.- 6 Specific Isolation Protocols.- 6.1 Higher Plants.- 6.1.1 C3 Plants.- 6.1.2 C4 Plants.- 6.1.3 CAM Plants.- 6.2 Algae.- 6.2.1 Volvocales.- 6.2.2 (Ceramiaceae, Rhodophyta)-Griffithsiamonilis.- 6.2.3 Siphonales.- 6.2.4 (Xanthophyceae) Bumilleriopsisfiliformis.- 6.2.5 (Euglenophyceae) Euglenagracilis.- 7 Additional Comments on Chloroplast Isolation.- 8 Abbreviations.- References.- Purification of Inner and Outer Chloroplast Envelope Membranes.- 1 Introduction.- 2 General Considerations.- 3 The Procedure.- 3.1 Reagents and Equipment.- 3.1.1 Solutions.- 3.1.2 Materials.- 3.2 Growth of Peas and Purification of Intact Chloroplasts.- 3.3 Purification of Inner and Outer Envelope Membranes.- 4 Properties of the Isolated Membranes.- 4.1 Purity.- 4.1.1 Cross-Contamination by Envelope Membranes.- 4.1.2 Contamination by Thylakoids.- 4.1.3 Contamination by Stroma.- 4.2 Other Properties.- 5 Modifications of the Procedure.- 5.1 Alternate Methods of Chloroplast Rupture.- 5.2 Purification Subsequent to Rupture.- 5.3 Application to Other Tissues.- 6 Other Procedures.- References.- The Major Protein of Chloroplast Stroma, Ribulosebisphosphate Carboxylase.- 1 Introduction.- 2 Characteristics of RuBP Carboxylase.- 2.1 Molecular Arrangement and Physical Structure of Subunits.- 2.2 Molecular Structure.- 2.3 Biosynthesis and Assembly of Subunits.- 2.3.1 Large Subunit.- 2.3.2 Small Subunit.- 2.3.3 Subunit Heterogeneity.- 2.3.4 Coordinate Control of Subunit Synthesis.- 2.4 Catalytic Mechanism.- 2.4.1 Activation and Role of Mg2 +.- 2.4.2 Carboxylation of RuBP.- 2.4.3 Oxygenation of RuBP.- 2.4.4 Localization of Catalytic and Activator Site.- 3 Practical Aspects.- 3.1 Purification.- 3.1.1 Summary of Techniques.- 3.1.2 Interfering Compounds.- 3.1.3 Choice of Extraction Buffer and Grinding Procedures.- 3.1.4 Protein Determination.- 3.1.5 Example: RuBP Carboxylase from Soybean Leaves.- 3.2 Assay.- 3.2.1 Substrates.- 3.2.2 Activation of RuBP Carboxylase.- 3.2.3 Continuous Spectrophotometry Assay for RuBP Carboxylase Activity.- 3.2.4 Discontinuous Assays for RuBP Carboxylase Activity.- 3.2.4.1 Radiochemical Assay with [14C]NaHCO3.- 3.2.4.2 Radiochemical Assay Using [14C]NaHCO3 and [l-3H]RuBP.- 3.2.4.3 Discontinuous Assay Using Nonlabeled Substrates.- 3.2.5 Assays for RuBP Oxygenase.- 3.2.6 Kinetic Parameters of RuBP Carboxylase and RuBP Oxygenase.- 4 Conclusion.- References.- The Chloroplast Thylakoid Membrane-Isolation, Subfractionation and Purification of Its Supramolecular Complexes.- 1 Introduction.- 2 Function and Organization of the Thylakoid Membrane.- 3 Isolation of Thylakoid Membranes.- 4 Thylakoid Membrane Subfractionation.- 4.1 Photosystem I Stroma Lamellae Thylakoids.- 4.2 Photosystem II Oxygen Evolving Thylakoid Preparations.- 4.2.1 Isolation by Press Treatment and Phase Partition.- 4.2.2 Isolation by Detergent Fractionation.- 4.2.3 Choice of Preparation.- 4.3 Separation of Inside-Out and Right-Side-Out Thylakoid Vesicles with the Same Composition.- 5 Isolation of Thylakoid Supramolecular Complexes.- 5.1 The Photosystem I Complex and the Light-Harvesting Complex of Photosystem II (LHC II).- 5.2 The Light-Harvesting Complex of Photosystem I (LHC I).- 5.3 The Inner Core Complex of Photosystem II (CC II).- 5.4 The Cytochrome b/f Complex.- 5.5 The ATP Synthase (CF0-CF1).- References.- The Isolation and Characterization of Nongreen Plastids.- 1 Introduction.- 2 The Terminology of Nongreen Plastids.- 2.1 Proplastids.- 2.2 Etioplasts.- 2.3 Chromoplasts.- 2.4 Amyloplasts.- 2.5 Leucoplasts.- 2.6 Other Nongreen Plastids.- 3 Basics of Plastid Isolation and Separation.- 3.1 Experimental Design.- 3.2 Isolation Medium.- 3.3 Tissue Disruption.- 3.4 General Methods of Chloroplast Isolation.- 4 Isolation of Nongreen Plastids from Developing Ricinus Endosperm.- 4.1 Rate-Zonal Sedimentation.- 4.1.1 Protocol.- 4.1.2 Analysis.- 4.1.3 Comments.- 4.2 Isopycnic Banding on Linear Sucrose Gradients.- 4.2.1 Protocol.- 4.2.2 Comments.- 4.3 Rate-Zonal Sedimentation on Linear Sucrose-Magnesium Co-Gradients.- 4.3.1 Protocol.- 4.3.2 Comments.- 4.4 Rate-Zonal Sedimentation on Discontinuous Sucrose Gradients.- 4.4.1 Protocol.- 4.4.2 Comments.- 4.5 Rate-Zonal Sedimentation on Discontinuous Percoll Gradients.- 4.5.1 Protocol.- 4.5.2 Comments.- 4.6 Nonaqueous Methods.- 4.6.1 Isopycnic Banding on Linear Hexane-CCl Gradients.- 4.6.2 Silicon Oil Centrifuged Filtration.- 4.7 Noncentrifugal Methods.- 4.7.1 Gel Permeation.- 4.7.1.1 Materials.- 4.7.1.2 Protocol.- 4.7.1.3 Comments.- 4.7.2 Phase Partition.- 4.7.3 Unit-Gravity Sedimentation.- 5 Metabolic Capabilities of Ricinus Endosperm Plastids.- 5.1 Glycolysis, the Pentose-Phosphate Pathway and Fatty Acid Synthesis.- 5.2 The Calvin Cycle.- 5.3 Nitrogen Metabolism.- 5.4 Terpenoid Metabolism.- 6 Composition and Biochemical Properties.- 6.1 Structure.- 6.2 Protein Composition.- 6.3 Membranes.- 6.4 Nucleic Acids.- 7 Future Prospects.- References.- Mitochondria.- 1 Introduction.- 2 Preparation for DNA Analysis.- 2.1 Cytoplasmic Male Sterility and Structure ot Mitochondrial DNA.- 2.2 Isolation of Mitochondria for DNA Preparation.- 2.3 Preparation of Mitochondrial DNA.- 2.4 Electrophoresis of Mitochondrial DNA.- 2.5 Restriction Analysis of Mitochondrial DNA.- 2.6 Notes on Mitochondrial DNA Studies.- 3 Preparation of Intact Mitochondria for Oxidative Studies.- 3.1 Introduction.- 3.2 Mitochondrial Preparation and Purification.- 3.3 Tests for Integrity of Mitochondria.- 3.3.1 Succinate: Cytochrome c Reductase.- 3.4 Tests for Integrity.- 3.4.1 Measurement for Oxygen Consumption for Respiratory Control and P/O Ratios.- 3.5 Notes on the Methods.- References.- Endoplasmic Reticulum.- 1 Introduction.- 2 Structure and Organization of the ER.- 3 Interactions Between Tubular and Cisternal ER.- 3.1 Role in Protein Transport.- 3.2 Role in Cell Division.- 4 Synthesis and Degradation of ER.- 4.1 Membrane Proteins.- 4.2 Membrane Lipids.- 5 Isolation and Characterization of ER.- 5.1 Isolation Media.- 5.2 Tissue Homogenization.- 5.3 Organelle Isolation.- 5.3.1 Molecular Sieve Chromatography.- 5.3.2 Differential Centrifugation.- 5.3.3 Density Gradient Centrifugation.- 5.4 Identification of ER Membranes.- 5.4.1 Magnesium Shift.- 5.4.2 Marker Enzymes.- 5.4.3 Auxin Binding.- 5.4.4 Calcium Transport.- 5.4.5 Structural Proteins.- 5.5 Concluding Remarks.- References.- Polyribosomes.- 1 Introduction.- 2 Isolation of Polysomes from Plant Cells.- 2.1 Factors that Affect the Stability and Recovery of Polyribosomes.- 2.2 Tissue Preparation.- 2.3 Subcellular Fractionation and Polysome Isolation.- 3 Purification and Analysis of Polyribosomes.- 3.1 Sucrose Gradient Centrifugation.- 3.2 Purification of Polysomes with Discontinuous Sucrose Gradients.- 3.3 Analysis of Polyribosome Profiles.- 4 Polyribosome Extraction Buffers.- 4.1 pH.- 4.2 Potassium Chloride.- 4.3 Magnesium Chloride.- 4.4 Reducing Agents.- 4.5 Chelation of Divalent Metals.- 4.6 Proteinase K.- 4.7 Other Ribonuclease Inhibitors.- 5 Uses of Purified Polyribosomes.- 5.1 Changes in Protein Synthetic Activity.- 5.2 In Vitro Protein Synthesis.- 5.3 Purification of mRNA's.- 5.4 Subcellular Distribution of mRNA's.- References.- The Nucleus-Cytological Methods and Isolation for Biochemical Studies.- 1 Introduction.- 2 Structure of the Plant Nucleus and Implications for Nuclear Isolation and Staining.- 3 Cytology.- 3.1 Nuclei of Whole Cells.- 3.1.1 Feulgen Microspectrophotometric Methods.- 3.1.2 Microfluorometry DNA Determination.- 3.2 Staining Nuclei During Isolation.- 3.3 Nucleolus Staining.- 3.4 Chromosome Staining.- 3.5 Other Nuclear Stains.- 4 Isolation of Plant Nuclei-General.- 4.1 Isolation of Plant Nuclei-Methods.- 4.1.1 Nuclei from Tobacco Callus Cultures for RNA Synthesis Studies.- 4.1.2 Nuclei from Tobacco Cells in Culture-for General Purpose Studies.- 4.1.3 Nuclei from Soybean Cells-for DNA Studies.- 4.1.4 Plant-Root Nuclei-for DNA Analysis.- 5 Summary.- References.- Microtubules.- 1 Introduction.- 2 Extraction of Microtubule Proteins.- 3 Purification of Tubulin and MAP's.- 3.1 DEAE-Sephadex Ion Exchange Chromatography.- 3.2 Phosphocellulose Chromatography.- 3.3 Affinity Chromatography.- 3.4 Cycles of Polymerisation and Depolymerisation.- 3.4.1 Pre-Conditions for Microtubule Assembly.- 3.4.2 Microtubule Assembly in the Presence or Absence of Glycerol.- 3.4.3 The Dynamics of Polymerisation and the Use of Taxol.- 3.4.4 Co-Polymerisation.- 4 Fractionation and Identification of Tubulin by SDS-PAGE.- 5 Colchicine-Binding Assay for Tubulin.- 6 Immunochemical Methods of Analysis.- 6.1 Radioimmunoassay.- 6.2 Enzyme-Linked Immunosorbent Assay (ELISA).- 6.3 Antibody Purification of Antigen-Affinity Column.- 6.4 Western Blots.- 7 Concluding Remarks.- References.