Ast, Tamara; (1995) Purification, characterisation and inhibition of methionine synthase from rat liver. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cobalamin-dependent methionine synthase catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Great attention has been focused on this enzyme because methionine synthase represents a key link between two important metabolic cycles. One regulates the synthesis of SAM and hence numerous methylation reactions, while the other cycle is involved in the regeneration of tetrahydrofolic acid and other folate cofactors. Insufficient methionine synthase activity is associated with various disorders observed in cobalamin and folate deficiency. This study reports the purification of mammalian methionine synthase from rat liver. The enzyme was purified ca 4000-fold and a 7.4 % yield was obtained. Several methods not previously used for characterisation of methionine synthase were employed in this research. These were: affinity chromatography on Vitamin B12-agarose, preparative isoelectrofocusing and capillary electrophoresis. The enzyme was eluted in two forms from anion-exchange columns. The molecular weight of the native enzyme was ca 160 kDa as determined by native polyacrylamide gel electrophoresis. However, the enzyme sometimes proved difficult to isolate in an intact form, resulting in different smaller, possibly proteolytic fragments. The 95 kDa fragment was most frequently encountered. Based on the analytical and preparative isoelectric focusing, the purified enzyme was found to have a pi of 5.2. The partially purified enzyme was shown to be inhibited in a time and dose- dependent manner by two different nitrogen oxides: anaesthetic gas, nitrous oxide and the ubiquitous signalling molecule, nitric oxide. The hypothesis is put forward, that NO and N₂O share some common molecular targets. This view is supported by their analogous effects on polyamine synthesis. Both gases were shown to inhibit polyamine synthesis in a time-dependent manner. The changes in polyamine levels took place in the first few minutes of incubation, thus supporting the hypothesis that these molecules are involved in cellular signalling. Various metabolic products of methionine biosynthesis were examined as potential allosteric regulators of methionine synthase: SAH, adenosine, phosphatidyl choline, arginine, taurine, ATP, dUMP, cAMP, cGMP and naturally occurring polyamines.

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