Regulation of polyamine contents in cells and their physiological functions

1. PotF蛋白是大肠杆菌腐胺转运系统的一种质周底物结合蛋白。我们在2.3-A分辨率下测定了与底物复合物中的PotF蛋白的晶体结构。PotF分子的尺寸为54 x 42 x 30，由两个相似的球状结构域组成。腐胺通过12个氢键和36个范德华相互作用紧密结合在PotF两个结构域之间的深间隙中。PotF和PotD结构的比较提供了对两种蛋白特异性差异的洞察。结合突变分析，PotF结构揭示了腐胺结合的关键残基（Trp-37、Ser-85、Glu-185、Trp-244、Asp-247和Asp-278）以及水分子对腐胺识别的重要性。利用经ylo28w基因转化的酵母细胞检测了ylo28w编码的膜蛋白的特性。转化后的细胞对多亚胺毒性产生抗性，并被液泡H +- atp酶抑制剂巴菲霉素A_1所克服。转化细胞对精胺的摄取活性与野生型细胞基本相同，但其液泡膜泡的摄取活性高于野生型细胞。转化后的细胞对MGBG （methylglyoxal bis(guanylhydrazone)）和百草草有抗性，但对Ni^<2+>和Co^<2+>没有抗性，表明YLLO28w编码的蛋白是一种多胺特异性转运蛋白。当插入HIS3基因破坏YLLO28w基因后，细胞对多胺敏感，液泡膜囊泡对精胺的摄取活性显著降低。2 .与野生型细胞相比，YLL028w基因破坏细胞中积累的精胺明显减少，表明YLL028w基因编码的膜蛋白（TP01）是液泡膜上的多胺转运蛋白。用^<31>P和^1H NMR研究了Mg^<2+>和/或精胺存在下ATP的构象。在Mg^<2+>存在的情况下，精胺主要与ATP的β -磷酸和γ -磷酸相互作用。在没有Mg^<2+>的情况下，通过^<31 >p NMR无法观察到ATP的β -磷酸和γ -磷酸与精胺的构象变化。通过^ 1h核磁共振发现，精胺对ATP中腺苷部分的构象没有明显影响。精胺能轻微抑制Mg^<2+>与ATP的结合，反之亦然。结果表明，Mg^<2+>与精胺在ATP上的结合位点仅部分重叠。以atp依赖性酶PotA蛋白为模型系统，研究了ATP-Mg^<2+> -精胺复合物的生物学作用。精胺能显著增强PotA的atp酶活性。双互易图显示，精胺与ATP相互作用，但与PotA不相互作用。结果表明，ATP- mg ^<2+> -精胺三元配合物可能在体内某些ATP依赖性反应中起重要作用。少

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 … More 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. Less