| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Since the mammalian tissues contain the enzyme in an extremely low level, we established a method for high-level expression of rat methionine synthase cDNA in insect cells using a baculovirus expression system. The expressed enzyme was chiefly as apoenzyme and very unstable. After complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K_m values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob (II) alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium (III) and S-adenosylmethionine. The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective o
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r effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme.
Severe B_<12>-deficient rats were produced by breeding with B_<12>-deficient diet. The status of B_<12>-deficiency was confirmed by increase in urinary methylmalonate excretion and decreases in liver B_<12> contents and cobalamin-dependent methionine synthase activity. It was demonstrated that rat liver methionine synthase exists almost exclusively as holoenzyme. Upon B_<12>-deficiency, the level of methionine synthase protein decreased, whereas the mRNA level did not change. When methylcobalamin was administered to the B_<12>-deficient rats, growth, liver B_<12> levels, and urinary excretion of methylmalonate reversed to the control (B_<12>-sufficient) levels. During this recovery process, methionine synthase activity and protein levels increased, while the mRNA level remained essentially unchanged. We have mentioned above that rat apomethionine synthase is very unstable and markedly stabilized by complexation with methylcobalamin. Thus, the decrease of mammalian methionine synthase activity in B_<12>-deficient animals can be interpreted in terms of not "coenzyme induction" but stabilization of the enzyme by cobalamin binding ("coenzyme stabilization"). Less
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