INDEX
Abstract 1
List of abbreviations 2
Chapter 1: INTRODUCTION
31.1 Lens Structure and function 3
1.2 Glutathione 4
1.3 GSH metabolism and catabolism: the γ-glutamyl-cycle 5 1.4 Sulfur containing amino acids and transsulfuration pathway 6
1.5 Cystathionine γ-lyase in mammals 8 1.6 Disorders in transsulfuration pathway and related pathologies 9 1.7 The role of transsulfuration pathway in maintaining GSH pool 10 1.8 The redox regulation of transsulfuration pathway 11 1.9 CGL and the production of H2S: physiological role 12
and possible pathological implications
1.10 Role of CysteinylGlycine in GSH homeostasis in cultured bovine lens 14
Chapter 2: MATERIALS AND METHODS
152.1 Materials 15 2.2 Calf kidney crude extract preparation 17
2.3 Calf lens crude extract preparation 17 2.4 Primary culture of bovine lens epithelial cells 17 2.5 BLEc crude extract preparation 17 2.6 βB2 crystallin purification 17 2.7 Determination of protein content 18 2.8 Determination of Cystathionine -γ-lyase activity 18
-2.8 a Ninhydrin assay 18
-2.8 b Bindschedler’s Green assay 19
2.9 Determination of γ-GCS and of GSH synthase activities 19
2.10 SDS –PAGE 20
2.11 Silver staining 20
2.12 Western blot: transfer 20
-2.12a Classical immunostaining 20 -2.12b Immunostaining with Snap system device (Millipore) 21
Chapter 3: RESULTS
22
3.1 Isolation of CGL from Calf Kidney 22
-3.1.1 Ammonium sulphate fractionation 22 -3.1.2 Hydrophobic interaction chromatography on Phenyl sepharose 22 -3.1.3 Anionic exchange chromatography on DE-52 23 -3.1.4 Molecular exclusion chromatography on Sephacryl S-300 24
3.2 Isolation of CGL from Calf Lenses 25
-3.2.2 Western blot of lens CE and 60% pellet sample 25 -3.2.3 Separation of the two different molecular weight species 25 through Phenyl sepharose and western blot analysis.
-3.2.4 Molecular exclusion chromatography on Sephacryl S-300 26
-3.2.5 On the nature of 25 kDa band 29
-3.2.6 Analysis of distribution of γ-GC synthase and GSH synthase 31 in calf lens
3.3 Isolation of CGL from calf cultured epithelial cells (BLEc) 32
-3.3.1 CGL activity in BLEc and western blot analysis 32 -3.3.2 Ammonium sulphate fractionation 32 -3.3.3 Hydrophobic interaction chromatography on Phenyl sepharose 32 -3.3.4 Anionic exchange chromatography on DE-52 32 -3.3.5 Molecular exclusion chromatography on Sephacryl S-300 33 -3.3.6 Assessment of the potential inhibitory effect of CysGly on 36 partially purified of BLEc CGL.
