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中的一种周质底物结合蛋白。我们在2.3-A分辨率下测定了钾F蛋白与底物的络合物的晶体结构。钾氟分子的尺寸为54×42×30，由两个相似的球状结构域组成。腐胺通过12个氢键和36个van der Waals相互作用紧密结合在PotF的两个结构域之间的深裂缝中。通过对PotF和PotD结构的比较，可以深入了解这两种蛋白质在特异性上的差异。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。将YLLO28w基因转化酵母细胞，对其编码的膜蛋白的性质进行了检测。转化的细胞对Poll…产生了抗性。这种抗性可被液泡H~+-ATPase抑制剂巴非霉素A_1克服。尽管转化细胞对精胺的摄取活性与野生型细胞几乎相同，但转化细胞对液泡膜囊泡的摄取活性高于野生型细胞。转化细胞对MGBG和百草枯产生抗性，但对Ni^&lt；2+&gt；和Co^&lt；2+&gt；无抗性，提示YLLO28w编码的蛋白是多胺专一性的转运蛋白，当YLLO28w基因插入HIS3基因后，细胞对多胺变得敏感，液泡膜囊泡对精胺的摄取活性显著降低。YLL028w基因突变细胞的精胺积累量较野生型细胞显著减少，表明YLL02Sw(TP01)编码的膜蛋白是液泡膜上的多胺转运蛋白。用~1H核磁共振和~(31)H核磁共振研究了ATP在镁和精胺存在下的构象。在镁和精胺存在下，蛇床子素主要与ATP的β-和γ-磷酸盐相互作用。在没有镁离子的情况下，精胺不能观察到ATP与精胺的β-磷酸和γ-磷酸的构象变化。~1H核磁共振结果表明，精胺对ATP腺苷的构象没有明显的影响。精胺对镁离子与三磷酸腺苷的结合有轻微抑制作用，反之亦然。结果表明，精胺和镁离子在ATP上的结合部位只有部分重叠。POTA蛋白是一种依赖于三磷酸腺苷的酶，被用来作为模型系统来研究三磷酸腺苷-镁-精胺复合体的生物学作用。精胺能显著提高POTA的ATPase活性。在不同浓度的精胺激活剂下的双倒易图表明，精胺与ATP相互作用，而与POTA不相互作用。这一结果表明，在体内某些依赖于ATP的反应中，精胺-镁-精胺-精胺三元络合物可能起着重要作用。较少

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