| Project/Area Number |
05671811
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Biological pharmacy
|
|---|
| Research Institution | Chiba University |
|---|
Principal Investigator |
KASHIWAGI Keiko Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80169424)
|
|---|
| Project Period (FY) |
1993 – 1994
|
| Project Status |
Completed (Fiscal Year 1994)
|
| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1993: ¥1,200,000 (Direct Cost: ¥1,200,000)
|
|---|
| Keywords | Polyamine / Putrescine / Spermidine / Spermine / Polyamine transport / Gene regulation / 輸送系 / ATPase / アンチポーター / RNaseIII |
|---|
| Research Abstract |
The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes in Escherichia coli.
Two of them were spermidine-preferential and putrescine-specific uptake systems, respectively. Spermidine-preferential uptake system consisted of four kinds of proteins : periplasmic substrate-binding protein (potD) , membrane-associated ATP binding protein (potA) and two transmembrane proteins (potB and C). Putrescine uptake system also consisted of four kinds of proteins : those similar to proteins of the spermidine-preferential uptake system.
From activity measurement and cellular localization of mutated proteins obtained by site-directed mutagenesis, it was found that ATP binding domain and active center of ATPase activity were located in the NH_2-terminal of potA protein and COOH-terminal of the protein stimulated association between potA and potB,C transmembrane proteins. Spermidine binding site of potD protein was also determined by X-ray crystal analysis of the purified protein and activity measurement of mutated proteins. It was found that Glu36 and Glu171 were important for the recognition of primary amines and Asp257 was important for the recognition of secondary amine of spermidine.
The third transport system was the excretion system of putrescine. This was catalyzed by potE protein, which has putrescine-ornithine antiporter activity. PotE protein consisted of 439 amino acids. There are 16 acidic amino acids existed in the hydrophilic region of potE protein. By changing those to neutral amino acids, we looked for the catalytic site of the protein. It was found that Glu207 was essential for the activity. We also found that RNase III was involved in the enhancement of the expression of potE gene.
|
