Kenyon, Susan Helene; (1997) Control and regulation of vitamin B12-dependent methionine synthase. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Vitamin B12-dependent motioning synthase activity is important for the functioning of the folate and sulphur amino acid pathways. Inappropriate motioning synthase activity has been associated with: macrocytic anaemia, neurological degeneration, psychological problems, cancer and atherosclerosis. This study reports the effects of various physiological and non-physiological compounds upon motioning synthase activity in vitro. Vitamin B12-dependent motioning synthase activity was purified from rat liver; its molecular weight was estimated to be 160 kDa by gel filtration. On a denaturing SDS PAGE two protein bands of approximately 95 kDa and 60 kDa were seen. Highly purified enzyme was shown to be irreversibly inhibited by nitric oxide; the IC50 was 3μM. Sodium nitroprusside, a nitric oxide donor also inhibited motioning synthase, the IC50 was 10 μM in vitro and 170 μM, for cytosolic motioning synthase from isolated rat hepatocytes. The viability of the hepatocytes, measured by ATP levels was not compromised during the experiment, suggesting that motioning synthase activity was more sensitive to the effects of NO. Cyanide was found not to affect enzyme activity at relevant concentrations. Polyamines stimulated vitamin B12-dependent motioning synthase activity. A rank order of potency was established in which spermine > spermidine > putrescine = cadaverine. The EC50's for spermine and spermidine were determined and found to be 8 μM and 40 μM respectively in hepes buffer, within reported physiological concentration ranges. The stimulation of the enzyme was found to be uncompetitive and reversible. A preliminary study examining the effect of protein kinases upon partially purified motioning synthase indicated that enzyme activity increased. In contrast some phospholipids were found to inhibit partially purified motioning synthase. Magnesium (but no other ion tested) reversibly and uncompetitively stimulated motioning synthase activity (similar to the effect of spermine). The EC50 was 260 μM, within reported physiological concentrations. Alcohol was reported to inhibit motioning synthase activity in vivo, but not in vitro, this was confirmed, and acetaldehyde discovered to inhibit enzyme activity in vitro. The IC 50 was 2.2 mM, higher than reported in vivo concentrations. A series of substrate analogues were tested as potential specific inhibitors of motioning synthase; the results failed to identify a potent inhibitor, but will form a basis for further design.

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