A guanylyl cyclase from Paramecium with 22 transmembrane spans -: Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases
A guanylyl cyclase from Paramecium with 22 transmembrane spans -: Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases
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DOI：
10.1074/jbc.275.15.11235
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发表时间：
2000-04-14
影响因子：
4.8
通讯作者：
Schultz, JE
中科院分区：
文献类型：
--
作者：
Linder, JU;Hoffmann, T;Schultz, JE
草履虫具有 280 kDa 鸟苷酸环化酶。 N 末端类似于 P 型 ATP 酶，C 末端是鸟苷酸环化酶，具有典型哺乳动物腺苷酸环化酶的膜拓扑结构，但胞质环 C1 和 C2 与哺乳动物顺序相反。我们在大肠杆菌中表达了原生动物鸟苷酸环化酶的细胞质结构域，独立地并通过肽连接，作为可溶性蛋白质。 His(6) 标记的蛋白质通过亲和层析富集并通过免疫印迹分析。重组 C1a 和 C2 定位结构域混合后可重建鸟苷酸环化酶活性，并且在连接的 C1a-C2 构建体中，腺苷酸环化酶活性最小。当 C1 位置结构域中定义氨基酸 Glu(1681) 和 Ser(1748) 的底物突变为腺苷酸环化酶特异性氨基酸 Lys 和 Asp 时，核苷酸底物特异性从 GTP 转换为 ATP。使用哺乳动物腺苷酸环化酶 II 型或 IX 型的 C2 结构域以及草履虫鸟苷酸环化酶的 C2 定位结构域，我们重建了可溶性全 C2 腺苷酸环化酶。所有含有原生动物结构域的酶均不受 G α(s)/GTP 或毛喉素的影响，P 位点抑制剂仅略微有效。
Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct, Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase, All enzymes containing protozoan domains were not affected by G alpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.