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Long-term exposure of Jurkat, L929 and J774 cells to exogenous NO inhibits cellular respiration at the level of complex IV that is reversed by oxyHb and at the level of complex I that is not reversed by oxyHb. Long-term exposure of NO to the cells causes a decrease in reduced glutathione concentration that precedes the inhibition at the level of complex I. This inhibition is reversed by addition of reduced glutathione or cold light, suggesting that S- nitrosylation of thiols is necessary for the activity of the enzyme. Furthermore, long-term exposure of the cells to NO decreases oxygen utilisation due to two distinct actions, one being the inhibition of cellular oxygen consumption and the other, the generation of a hypoxic microenvironment due to the oxygen consumption by the extracellular medium. Inhibition of basal NO, with a NO synthase inhibitor, increases cellular respiration in iNOS mutant, iNOS and eNOS wild type, but not in eNOS mutant mice. Administration of a low dose of endotoxin to the mice leads to a short-term generation of NO associated with a short-term inhibition of mitochondrial respiration resulting in non-lethal shock. Whereas administration of a high dose of endotoxin leads to a sustained generation of NO accompanied by a sustained inhibition of mitochondrial respiration, at the level (at least in part) of complex I, resulting in lethal shock. These results suggest that NO can be considered as a physiological regulator and a pathological inhibitor of tissue respiration. The basal release of small amounts of NO acts as a regulating mechanism, whereby cells respond to changes in their environment and regulate ATP production. By contrast the generation of high concentrations of NO over a sustained period decreases reduced glutathione concentration and pathologically inhibits cellular respiration leading to multiple organ failure and ultimately death.

Preliminary attempts were made to isolate hexokinase from human erythrocytes and placenta. The final purifications were made from heart, which contained more enzyme than other tissues. From 1.15 kg of heart, 25 mg of hexokinase was obtained of specific activity 58 units/rng. This represents a 1,700-fold purification and a yield of I+7% over six steps. A critical development in isolating " this enzyme was the use of affinity chromatography, using glucosamine linked to Sepharose". The material contained"a single protein component according to electrophoresis on starch gel, cellulose acetate and polyacrylamide gel. It gave a single peak on gel filtration, and on sedimentation in the ultracentrifuge. However, a heterogeneity in size was indicated by electrophoresis on SDS-polyacrylamide gels which gave two bands of approximately equal intensity. The apparent molecular weights of these components were 109,000 and 124,000. The small difference in size has not resolved by the other techniques used. Gel filtration on Sephadex G-200 suggested a molecular weight-of 106,000, as did the combination of the 'sedimentation coefficient of 5.5 S and diffusion coefficient of 47 μm2/s.

The aim of the work presented in this thesis is to establish a mathematical model for use in the study of blood glucose dynamics and their disorders in man. A large number of models have been developed that represent various aspects of blood glucose dynamics and of these the majority have been attempts to provide a mathematical description of glucose tolerance test curves. Most of these models have no immediate clinical application and have also failed to provide any real insight into the system that controls the flow of glucose into and from the blood glucose compartment. To do this a mathematical model must be formulated that approximates to an isomorphic description of the glucose regulatory system. Such a model must be based on sound biochemical and physiological knowledge. However, the complexity of the glucose system is such that complete simulation is not possible. In this study, a model has been developed and is described that places particular emphasis upon the role of the liver in controlling blood glucose dynamics. Compartments are provided for glucose, glucose-6-phosphate, and glycogen which are the dominant metabolic substrates. Mass balance equations have been written in terms of the enzymatic reactions that are involved in glucose transport and substrate kinetics. Insulin and glucagon hormonal controllers have also been incorporated. The model has been tested by inputs representing intravenous glucose infusion, intravenous glucose, insulin and glucagon injection. The model appears to simulate in general terms experimental data, and has yielded information about both the system structure and the enzyme dynamics involved. Of particular importance is the evidence that fine control of the glucose system is an intrinsic function of the enzyMe systems and that only coarse control of the system is provided by the hormonal environment.Little work has been done in this field, but the present model is unlike any previous model in that the inherent non-linearities of the metabolic system are developed from known enzymological data. The approach illustrates the possible value of using kinetic data obtained from -in -vitro, experiments in predicting physiological changes within the in vivo system. It is considered that mathematical models of this type can provide the biomedical scientist with insight into the functioning of metabolic systems and highlight areas of weak knowledge.