Gibbons, John M.; (2001) Purification of pig brain methionine synthase and its control and regulation by nitric oxide. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The vitamin B12 network links the folate and sulphur amino acid pathways with, the synthesis of polyamines and lipid biosynthesis. The role that this vitamin plays within human disease has been linked to a variety of haematological, developmental, neurological and pathological conditions such as cancer and heart disease. The role of nitric oxide within the body has also been shown to be extensive. The physiological effects range from cardiovascular function, peripheral nervous system, central nervous system, and the immune system. The pathophysiological actions include, septic shock, reperfusion injury, arteriosclerosis, hypertension, and neurotoxicity. In order to better study and identify neurological disorders linked to B12, the mammalian B12 dependent methionine synthase enzyme was purified. The enzyme appears under native conditions to be 155kd in weight composed of several subunits (94 and 36 Kd). The pi for the enzyme was determined to be 4.3 and 5.1. Concurrent with the purification work, studies were done to determine some of the biological effects of NO. NO inhibits B12 MS with an IC50 of 3 μM. A comparison between the effects of NO and N2O on B12 MS inhibition both of which inhibit B12 MS in a dose dependent manner and irreversibly. To contrast these studies a comparison of the effects of NO and N2O on cGMP synthesis. NO stimulates the production of cGMP while NO was found to have no significant effect. The production of NO was also studied in vivo and in vitro to determine the effect of changes in oxygen partial pressures. NO synthesis in vivo was measured using the production of nitrate and nitrite in plasma and measured by capillary electrophoresis. When human volunteers were exposed to 2.8 bar levels of nitrate increased 4.2 times above background.

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