| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
1. To examine whether Erythroid specific 5-aminolevulinate synthase (ALAS-E) and ATP-specific succinyl CoA synthethase (A-SCS) form functional enzyme complex in vitro, we have expressed recombinant proteins of components of each enzyme (ALAS-E, SCS-α, SCS-βA and SCS-βG) using Baclo-virus based expression system. In this experiment, we have successfully purified enzymatically active ALAS-E recombinant protein, however, enzymatic activity of SCS could not be detected in any combination of purified component (SCS-α and SCS-βA = A-SCS, or SCS-α and SCS-βG = G-SCS). These results suggest that phosphorilation or glycosilation SCS might be needed for enzymatically active SCS, which specifically occur in only mammalian cells. Alternatively, mammalian SCS may request specific co-factor(s) for its catalytic activity. If such co-factor(s) exist, decrease amount of the co-factor may cause sideroblastic anemia.
2. To determine the specific region for interaction of ALAS-E and SCS-βA, several deletion mutants of ALAS-E protein were made. Then, the interaction of such mutant ALAS-E proteins and SCS-βA protein were determined using yeast two hybrid system. As a results, 147 amino acid deletion of N-terminal or 50 amino acid deletion of C-terminal of ALAS-E disrupt the interaction of ALAS-E and SCS-βA protein. Since these deletion mutant of ALAS-E results the disruption of homo-dimer formation of ALAS-E, homodimer formation of ALAS-E might be important for enzyme complex formation of ALAS-E and A-SCS in mitochondria.
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