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周质底物结合蛋白。我们已经确定的晶体结构的PotF蛋白质在复杂的基板在2.3-A的分辨率。PotF分子的尺寸为54 × 42 × 30 - 1，由两个相似的球状结构域组成。腐胺通过12个氢键和36个货车德瓦尔斯相互作用紧密结合在PotF的两个结构域之间的深裂缝中。PotF结构与PotD结构的比较提供了对两种蛋白质之间特异性差异的洞察。PotF结构与突变分析相结合，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）以及水分子对腐胺降解的重要性.使用用该基因转化的酵母细胞检查由YLLO 28 w编码的膜蛋白的性质。转化细胞对pol ...更多信息 用液泡膜H^+-ATPase抑制剂巴弗洛霉素A_1可克服抗性。虽然转化细胞的精胺摄取活性几乎与野生型细胞相同，但转化细胞的液泡膜囊泡的摄取活性高于野生型细胞。转化细胞对MGBG（甲基乙二醛双（胍基腙））和百草枯产生了抗性，但对Ni^2+和Co^2+没有抗性，表明YLLO 28 w编码的蛋白是一种多胺特异性转运蛋白。当插入HIS 3基因破坏YLLO 28 w基因时，细胞对多胺变得敏感，液泡膜小泡对精胺的摄取活性显著降低。与野生型相比，YLL 028 w基因敲除细胞中精胺的积累量显著降低，表明YLL 02 Sw（TP 01）编码的膜蛋白是一种位于液泡膜上的多胺转运蛋白.用~ 1 P和~ 1H NMR研究了在Mg^&lt;2+&gt;和/或精胺存在下ATP的构象<31>，发现在Mg^&lt;2+&gt;存在下精胺主要与ATP的β-和γ-磷酸相互作用。在无Mg^2+存在的条件下，用~ 1H NMR观察到精胺对ATP的β-和γ-磷酸的构象没有影响<31>，用~ 1H NMR观察到精胺对ATP的腺苷部分的构象没有明显影响。精胺对Mg^&lt;2+&gt;与ATP的结合有轻微的抑制作用，反之亦然。结果表明，Mg^&lt;2+&gt;与精胺在ATP上的结合位点仅部分重叠. PotA蛋白是一种ATP依赖性酶，被用作研究ATP-Mg^&lt;2+&gt; -精胺复合物生物学作用的模型系统。精胺能显著提高PotA的ATP酶活性。在不同浓度的精胺激活剂作用下的双倒数图表明精胺与ATP相互作用，而与PotA不相互作用。结果提示ATP-Mg ^&lt;2+&gt; -精胺三元复合物在体内某些ATP依赖性反应中可能起重要作用。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