Regulation of polyamine contents in cells

细胞内多胺含量的调节

• 批准号: 07680645
• 负责人: KASHIWAGI Keiko
• 金额: $ 0.38万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680645/
• 关键词: Polyamine transport; Putrescine; Spermidine; PotA; PotD; PotE; PotD; ポリアミン; 輸送; ATPase; ABC transporter

1. PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system. The crystal structure of PotD in complex with spermidine has been solved at 2.5-* resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. Spermidine binding sites on PotD were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. It was found that Trp-34, Thr-35, Clu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine, and that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids … More listed above.2. The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential. The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 muM,respectively. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein. Both the NH_2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE and the function of these three residues could not be replaced by other amino acids. Less

1.PotD蛋白是一种周质结合蛋白，是多胺转运系统的主要受体。亚精胺与PotD形成的配合物的晶体结构已在2.5*分辨率下解析。PotD蛋白由两个结构域组成，具有交替的β-α-β拓扑结构。多胺结合位点位于结构域之间界面的中心裂隙中。用定点突变的PotD基因制备了突变的PotD蛋白，通过测定膜泡的多胺转运活性和这些突变的PotD蛋白的多胺结合活性，研究了PotD上的亚精胺结合位点。研究发现，PotD蛋白的Trp-34、Thr-35、Clu-36、Tyr-37、Ser83、Tyr-85、Asp-168、Glu-171、Trp-229、Trp-255、Asp-257、Tyr-293和Gln-327参与了亚精胺与亚精胺的结合，其中Glu-171、Trp-255和Asp-257参与亚精胺与PotD蛋白结合的能力强于其他氨基酸…上面列出的更多。对多胺转运蛋白POTE的结构和功能进行了研究。POTE对腐胺的摄取依赖于膜电位。相反，用内向外膜囊泡研究POTE的腐胺-鸟氨酸逆向转运蛋白活性不依赖于膜电位。腐胺摄取的Km值为1.8um，腐胺-鸟氨酸逆向转运蛋白活性的Km值为73um。因此，POTE不仅可以作为腐胺-鸟氨酸逆向转运蛋白排泄腐胺，还可以作为腐胺摄取蛋白发挥作用。POTE的NH_2和COOH末端都位于细胞质中，这是由不同融合后蛋白对碱性磷酸酶和β-半乳糖苷酶的激活所决定的。利用突变的POTE蛋白研究其对腐胺的摄取和排泄活性。研究发现，谷氨酸207是POTE蛋白摄取和排泄腐胺所必需的，谷氨酸77和433也参与了这两种活性。这三种谷氨酸位于POTE的胞质侧，其功能是其他氨基酸所不能替代的。较少

项目成果

K.Kashiwagi et al.: "Spermidine-preferential uptake system in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

K.Kashiwagi 等人：“大肠杆菌中的亚精胺优先摄取系统。PotD 蛋白中参与多胺结合的氨基酸的鉴定。”

Sugiyama, S., Matsuo, Y., Maenaka, K., Vassylyev, D.G., Matsushima, M., Kashiwagi, K., Igarashi, K., and Morikawa, K.: "The 1.8* X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protei

Sugiyama, S.、Matsuo, Y.、Maenaka, K.、Vassylyev, D.G.、Matsushima, M.、Kashiwagi, K.、Igarashi, K. 和 Morikawa, K.：“1.8* X 射线结构的

J.Fukuchi et al.: "Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli." J. Biol. Chem.270. 18831-18835 (1995)

J.Fukuchi 等人：“由于亚精胺乙酰转移酶缺陷型大肠杆菌突变体中亚精胺的积累，导致细胞活力下降。”

K.Kashiwagi et al.: "Spermidine-preferential uptake system in Escherichia coli.Identifi-cation of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

K.Kashiwagi 等人：“大肠杆菌中的亚精胺优先摄取系统。PotD 蛋白中参与多胺结合的氨基酸的鉴定。”

K. Williams et al.: "An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation." Mol. Pharmacol.48. 1087-1098 (1995)

K. Williams 等人：“N-甲基-D-天冬氨酸受体中的一种酸性氨基酸，对精胺刺激很重要。”