KASHIWAGI Keiko Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80169424)
KAKINUMA Yoshimi Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80134394)
|Budget Amount *help
¥11,400,000 (Direct Cost : ¥11,400,000)
Fiscal Year 1998 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1997 : ¥8,600,000 (Direct Cost : ¥8,600,000)
1. PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 _ and consists of two similar globular domains. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.
2. Properties of a membrane protein encoded by YLLO28w were examined using yeast cells transformed with the gene. The transformed cells became resistant to pol
yamine toxicity, and the resistance was overcome by bafilomycin A_1, an inhibitor of vacuolar H^+-ATPase. Although spermine uptake activity of the transformed cells was almost the same as that of wild type cells, the uptake activity of vacuolar membrane vesicles from the transformed cells was higher than that from wild type cells. The transformed cells became resistant to MGBG (methylglyoxal bis(guanylhydrazone)) and paraquat, but not Ni^<2+> and Co^<2+>, suggesting that the protein encoded by YLLO28w is a transport protein specific for polyamines, When the YLLO28w gene was disrupted by inserting the HIS3 gene, the cells became sensitive to polyamines, and spermine uptake activity of the vacuolar membrane vesicles decreased significantly. The accumulated spermine in YLL028w gene disrupted cells decreased greatly compared with that in wild type cells, The results indicate that a membrane protein encoded by YLL02Sw (TP01) is a polyamine transport protein on the vacuolar membrane.
3. The conformation of ATP in the presence of Mg^<2+> and/or spermine was studied by ^<31>P and ^1H NMR.Spermine predominantly interacted with the beta- and gamma -phosphates of ATP in the presence of Mg^<2+>. A conformational change of the beta- and gamma -phosphate of ATP with spermine could not be observed in the absence of Mg^<2+> by ^<31>P NMR.It was found by ^1 H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg^<2+> to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg^<2+> and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg^<2+> - spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA, The results suggest that a ternary complex of ATP-Mg^<2+> -spermine may play an important role in some ATP-dependent reactions in vivo.
4. ポリアミン、特にスペルミンがATP-Mg^<2+>と三者複合体を形成することをNMRを用いて証明した。この三者複合体形成により、ポリアミン輸送系のPotAのATPase活性が強く促進を受けることを見出した。また、protein kinase Aによる蛋白質のリン酸化も三者複合体形成により約2倍の促進を受けた。
5. 大腸菌でポリアミンにより最も強く合成促進を受ける蛋白質はオリゴペプチド結合蛋白質(OppA)である。ポリアミンはOppA mRNAのSD配列と開始コドンAUGの立体構造を変化させることにより、OppA合成を促進することを明らかにした。 Less