Detailed Table of Contents
Summary
Résumé
List Of Abbreviations
List of tables
List of figures
Remerciements
1. Introduction
2. Review Of Literature
2.1. Monohalomethanes
2.1.1. Sources Of Ch
3
x
2.1.2. Biosynthesis Of Ch
3
x
2.1.2.1. Halide Methylation In Algae
2.1.2.2. Halide Methylation In Fungi
2.1.2.3. Halide Methylation In Higher Plants
2.1.3. Environmental And Physiological Significance Of Ch
3
x
2.2. Reduced Sulfur Gases
2.2.1. Origins Of Biogenic Ch
3
sh
2.2.2. Enzymatic Synthesis Of Ch
3
sh
2.2.2.1. Degradation Of Methionine
2.2.2.2. Degradation Of Ch
3
sch
3
And Dmsp
2.2.2.3. Direct Methylation Of Sulfide
2.2.2.3.1. Mammalian
s
-Methyltransferase
2.2.2.3.2. Microbial
s-
methyltransferases
2.2.2.3.3. Plant
s-
methyltransferases
2.2.3. Environmental And Physiological Significance Of Ch
3
sh Production
2.2.4. Sulfate Reduction In Higher Plants
2.2.5. Glucosinolates
3. Materials And Methods
3.1. Plant Material
3.3. Buffers
3.4. Measurement Of Halide/bisulfide Methyltransferase Activity
3.4.1. Gas Chromatographic Assay
3.5. Purification Of H/bmt
3.5.1. Anion Exchange Hplc
3.5.2. Analytical Gel Filtration Hplc
3.6. Purification Of H/bmt Isoforms
3.6.1. Standard Procedure For The Purification Of H/bmt Isoforms
3.6.1.1. Affinity Chromatography
3.6.1.2. First Anion Exchange Hplc Step
3.6.1.3. Preparative Gel Filtration Hplc
3.6.1.4. Second Anion Exchange Hplc Step
3.6.2. Alternative Procedure For H/bmt Purification
3.6.2.1. Extraction
3.6.2.2. Affinity Chromatography
3.6.2.3. Anion Exchange Hplc
3.7. Molecular Weight Determination
3.8. Protein Estimation
3.9. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (sds-Page)
3.10. Kinetic Studies
3.11. Subcellular Localization Of H/bmt
3.11.1. Isolation Of Intact Chloroplasts
3.11.2. Cell Fractionation
3.11.3. Measurement Of Marker Enzyme Activities And Chlorophyll
3.11.3.1. Triose Phosphate Isomerase
3.11.3.2. Succinate Dehydrogenase
3.11.3.3. Catalase
3.11.3.4. Nadph Cytochrome C Reductase
3.11.3.5. Photosynthetic Co
2
Fixation
3.11.3.6.
in Vitro
Lipid Synthesis
3.11.3.7. Chlorophyll Determination
3.11.3.8. Calculation Of Marker Enzyme Activities
3.12. Enzyme Extraction For The Survey Of H/bmt Activity
3.13. Immunological Methods
3.13.1. Production Of Polyclonal Antibodies For H/bmt
3.13.2. Western Blotting
3.13.3. Purification Of Antibodies From Immunoblots
3.14. Analysis Of H/bmt Amino Acid Sequence
3.15. Molecular Methods
3.15.1. Isolation And Purification Of Total And Poly(a)
+
Rna
3.15.2. Rna Electrophoresis
3.15.3. Rna Transfer
3.15.4. Northern Hybridization
4. Results
4.1. Purification And Characterization Of H/bmt
4.1.1 Enzyme Purification
4.1.2. Confirmation Of The Dual Activity Of H/bmt
4.1.3. Purification Of H/bmt Isoforms
4.1.4. Alternative Purification Of H/bmt Isoforms
4.1.5. Stability Of The Purified H/bmt
4.2. Characterization Of H/bmt
4.2.1. Determination Of The Molecular Mass Of H/bmt
4.2.2. Effect Of Sh Group Inhibitors On H/bmt Activity
4.2.3. Effect Of Ph On H/bmt Activity
4.2.4. Substrate Specificity Of H/bmt
4.2.4.1. Methyl-Group Acceptor Specificity Of H/bmt
4.2.4.2. Methyl-Group Donor Specificity Of H/bmt
4.3. Kinetic Analysis Of H/bmt
4.3.1. Substrate Interactions
4.3.2. Product Inhibition
4.4. Intracellular Localization Of H/bmt
4.5. Association Of H/bmt Activity With Glucosinolates
4.6. Molecular Studies
4.6.1. Amino Acid Sequence Analysis
4.6.2. Expression Of H/bmt In Cabbage Leaves
5. Discussion
5.1. Purification And Characterization Of H/bmt
5.2. H/bmt Is A Thiol Methyltransferase
5.3. H/bmt Is A Cytosolic Enzyme Involved In Glucosinolate Metabolism
5.4. Comparison Of H/bmt To Other
s
-Methyltransferases
5.5. Possibility Of Halide Methylation By H/bmt
5.6. Physiological Significance Of H/bmt
5.6.1. Methylation Of S-Containing Compounds And The Relationship With Glucosinolates
5.6.2. Functions Of S-Containing Volatiles From Plants
6. Conclusion And Future Prospects
7. Bibliography