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by Chan Yee Man Elaine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 130-143). / Abstract --- p.I / Acknowledgements --- p.III / Abbreviations --- p.IV / Objectives of the study --- p.VII / Table of Contents --- p.VIII / Chapter Section 1 --- Introduction --- p.1 / Chapter 1.1 --- Necrosis vs Apoptosis --- p.2 / Chapter 1.2 --- Cell Death by Apoptosis --- p.4 / Chapter 1.3 --- The Biochemistry of Nitric Oxide --- p.9 / Chapter 1.4 --- Mechanisms of NO Action --- p.14 / Chapter 1.5 --- Signal Transduction Pathways to Apoptosis --- p.17 / Chapter 1.5.1 --- Regulation by Ca2+ --- p.17 / Chapter 1.5.2 --- Protein Kinase C --- p.20 / Chapter 1.5.3 --- cAMP --- p.21 / Chapter 1.5.4 --- Protein tyrosine kinase --- p.21 / Chapter 1.5.5 --- Ceramide --- p.22 / Chapter 1.5.6 --- pH --- p.23 / Chapter 1.5.7 --- Oxygen Radicals --- p.23 / Chapter 1.5.8 --- Anchorage Dependence and Extracellular Matrix --- p.24 / Chapter Section 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Animal --- p.28 / Chapter 2.1.2 --- Cell line --- p.28 / Chapter 2.1.3 --- "Culture media, buffers and chemicals" --- p.28 / Chapter 2.1.4 --- Dye solutions --- p.30 / Chapter 2.1.5 --- Reagents and buffers for polyacrylamide gel electrophoresis (PAGE) --- p.31 / Chapter 2.1.6 --- Reagents and buffers for Western blotting --- p.32 / Chapter 2.1.7 --- Reagents and buffers for agarose gel electrophoresis --- p.33 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Cell culture --- p.35 / Chapter 2.2.2 --- [3H]-Thymidine incorporation --- p.35 / Chapter 2.2.3 --- MTT assay --- p.36 / Chapter 2.2.4 --- Determination of NO by Griess assay --- p.36 / Chapter 2.2.5 --- Observation of apoptotic morphology of cells by confocal laser scanning microscopy (CLSM) --- p.37 / Chapter 2.2.6 --- Determination of cell death induced by NO producing drugs --- p.38 / Chapter 2.2.7 --- Determination of cell death induced by Concanavalin A --- p.38 / Chapter 2.2.8 --- Determination of effect of nitric oxide synthase (NOS) inhibitor on cell death induced by Con A --- p.39 / Chapter 2.2.9 --- Determination of the requirement of Ca2+ in cell death induced by NO producing drugs --- p.39 / Chapter 2.2.10 --- Determination of the requirement of cGMP in cell death induced by NO producing drugs --- p.40 / Chapter 2.2.11 --- Determination of cell death induced by PKC activation and depletion --- p.40 / Chapter 2.2.12 --- Determination of effect of PKC depletion on cell death induced by NO producing drugs and Con A --- p.41 / Chapter 2.2.13 --- Observation of immunofluorescence by confocal laser scanning microscopy --- p.41 / Chapter 2.2.14 --- Preparation of protein samples for PAGE --- p.42 / Chapter 2.2.15 --- Polyacrylamide gel electrophoresis --- p.43 / Chapter 2.2.16 --- Western blotting of PKC --- p.44 / Chapter 2.2.17 --- Preparation of DNA samples from cells --- p.46 / Chapter 2.2.18 --- Agarose gel electrophoresis of DNA --- p.47 / Chapter 2.2.19 --- Statistical analysis --- p.48 / Chapter Section 3 --- Results --- p.49 / Chapter 3.1 --- Induction of apoptosis in macrophages by NO producing drugs --- p.50 / Chapter 3.2 --- Apoptosis induced by NO producing drugs was caused by NO --- p.54 / Chapter 3.3 --- Signal transduction pathways to the NO-induced cell death in macrophages --- p.66 / Chapter 3.3.1 --- Calcium ion --- p.66 / Chapter 3.3.2 --- cGMP --- p.68 / Chapter 3.3.3 --- Protein kinase C --- p.71 / Chapter 3.4 --- Induction of apoptosis by Con A --- p.89 / Chapter 3.5 --- Tubulin structure in Con A-treated cells --- p.94 / Chapter 3.6 --- Nitric oxide and Con A-induced cell death in macrophages --- p.96 / Chapter 3.7 --- Effect ofNOS inhibitor on cell death induced by Con A --- p.102 / Chapter 3.8 --- Involvement of PKC in the Con A-induced cell death in macrophages --- p.106 / Chapter Section 4 --- Discussion --- p.114 / Chapter 4.1 --- Induction of apoptosis in macrophages by NO producing drugs --- p.118 / Chapter 4.2 --- Signal transduction pathways to the NO-induced cell death in macrophages --- p.120 / Chapter 4.2.1 --- Ca2+ ion --- p.120 / Chapter 4.2.2 --- cGMP --- p.120 / Chapter 4.2.3 --- Protein kinase C --- p.121 / Chapter 4.3 --- Induction of apoptosis by Con A --- p.124 / Chapter 4.4 --- Tubulin structure in Con A-treated cells --- p.124 / Chapter 4.5 --- Nitric oxide and Con A-induced cell death --- p.125 / Chapter 4.6 --- Effect ofNOS inhibitor on cell death induced by Con A --- p.125 / Chapter 4.7 --- Involvement of PKC in the Con A-induced cell death in macrophages --- p.127 / Chapter Section 5 --- Bibliography --- p.129 / References --- p.130

Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L-citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)-5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.