PURIFICATION OF SOLUBLE CYTOCHROME B(5) AS A COMPONENT OF THE REDUCTIVE ACTIVATION OF PORCINE METHIONINE SYNTHASE

In mammals, methionine synthase plays a central role in the detoxifica tion of the rogue metabolite homocysteine. It catalyzes a transmethyla tion reaction in which a methyl group is transferred from methyltetrah ydrofolate to homocysteine to generate tetrahydrofolate and methionine . The vitamin B-12 cofactor cobalamin plays a direct role in this reac tion by alternately accepting and donating the methyl group that is in transit from one substrate (methyltetrahydrofolate) to another (homoc ysteine). The reactivity of the cofactor intermediate cob(I)alamin ren ders the enzyme susceptible to oxidative damage. The oxidized enzyme m ay be returned to the catalytic turnover cycle via a reductive methyla tion reaction that requires S-adenosylmethionine as a methyl group don or, and a source of electrons. In this study, we have characterized an NADPH-dependent pathway for the reductive activation of porcine methi onine synthase. Two proteins are required for the transfer of electron s from NADPH, one of which is microsomal and the other cytoplasmic. Th e cytoplasmic protein has been purified to homogeneity and is soluble cytochrome b(5). It supports methionine synthase activity in the prese nce of NADPH and the microsomal component in a saturable manner. In ad dition, purified microsomal cytochrome P450 reductase and soluble cyto chrome b(5) reconstitute the activity of the porcine methionine syntha se. Identification of soluble cytochrome b(5) as a member of the reduc tive activation system for methionine synthase describes a function fo r this protein in non-erythrocyte cells. In erythrocytes, soluble cyto chrome b(5) functions in methemoglobin reduction. In addition, it iden tifies an additional locus in which genetic polymorphisms may play a r ole in the etiology of hyperhomocysteinemia, which is correlated with cardiovascular diseases.