Abstract
This chapter discusses methionine synthase from pig liver. Several methods are available for assaying methionine synthase. Mammalian methionine synthase serves two major metabolic functions. It recycles homocysteine—a toxic catabolite of S-adenosylmethionine (AdoMet)—and converts the circulating form of folic acid—5-CH3-H4folate—to H4folate, which can then support a variety of cellular reactions. The activity of the enzyme is dependent on the presence of the cofactor, cobalamin. Under in vitro assay conditions, the activity is additionally dependent on the presence of an electron source and AdoMet, which serves as a methylating agent. The chapter describes two methods that are employed for measuring methionine synthase activity from mammalian tissues or cell cultures. In both, transfer of the radiolabeled methyl group from the substrate, 5-14CH3-H4folate, to the product, methionine, is monitored. They differ in their source of reducing agents. The first employs dithiothreitol (DTT) and hydroxocobalamin (OH-B12) and is semianaerobic. The second employs titanium citrate and is anaerobic.
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