| Budget Amount *help |
¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2015: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2013: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2012: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Outline of Final Research Achievements |
Tetrahymena pyriformis contains two arginine kinases (AKs), a typical 40-kDa AK (AK1) and a two-domain 80-kDa enzyme (AK2). Interestingly, the amino acid sequence of T. pyriformis AK1, had a distinct myristoylation signal sequence at the N-terminus. In this work, we isolated and cloned the N-myristoyltransferase (NMT) cDNA from T. pyriformis. Then the NMT assay were performed, using the recombinant NMT and the N-terminal peptide GCSNSRSG of T. pyriformis AK1 or the peptide CSNSRSG which lacks N-terminal G of the former peptide. The use of the former peptide gave a typical Michaelis-Menten curve, while that for the latter gave no activity. A sufficient amount of modified AK1 enzyme was synthesized using an insect cell-free protein synthesis system. Then peptide mass fingerprinting (PMF) analyses were performed. The target N-myristoylated peptide was clearly observed with an experimental mass (m/z-H+) of 832.4747, indicating that the synthesized T. pyriformis AK1 is myristoylated.
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