Katayama M, Wada Y, Ohmori M
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan.
J Bacteriol. 1995 Jul;177(13):3873-8. doi: 10.1128/jb.177.13.3873-3878.1995.
Molecular cloning of the structural gene for adenylate cyclase (cya) of the cyanobacterium Anabaena cylindrica was carried out by complementation of an Escherichia coli strain defective in the cya gene. The cya-defective strain produced significant amounts of cyclic AMP when it was transformed with the cya gene isolated from A. cylindrica. This gene encodes a polypeptide consisting of 502 amino acid residues (molecular weight, 55,300). The deduced primary protein structure showed that the carboxyl-terminal region of the adenylate cyclase of A. cylindrica shows strong structural similarity to the conserved regions of the adenylate cyclases of various eukaryotes. No similarity was found between the amino acid sequences of the cya gene of A. cylindrica and that of E. coli. A hydropathy plot suggests that this protein has two hydrophobic regions, a transmembrane span and a signal peptide. An antiserum specific to this adenylate cyclase was prepared by immunizing a rabbit with a glutathione S-transferase-adenylate cyclase fusion protein expressed in E. coli. This antiserum recognized a 55-kDa protein in Anabaena cell lysates. Subcellular fractionation analysis showed that A. cylindrica adenylate cyclase localized in the thylakoid membrane.
通过对缺乏腺苷酸环化酶（cya）基因的大肠杆菌菌株进行互补，实现了对圆柱鱼腥藻腺苷酸环化酶（cya）结构基因的分子克隆。当用从圆柱鱼腥藻分离的cya基因转化该cya缺陷型菌株时，它产生了大量的环磷酸腺苷。该基因编码一个由502个氨基酸残基组成的多肽（分子量为55,300）。推导的一级蛋白质结构表明，圆柱鱼腥藻腺苷酸环化酶的羧基末端区域与各种真核生物腺苷酸环化酶的保守区域具有很强的结构相似性。在圆柱鱼腥藻的cya基因与大肠杆菌的cya基因的氨基酸序列之间未发现相似性。亲水性图谱表明该蛋白质有两个疏水区、一个跨膜区和一个信号肽。通过用在大肠杆菌中表达的谷胱甘肽S-转移酶-腺苷酸环化酶融合蛋白免疫兔子，制备了针对这种腺苷酸环化酶的抗血清。该抗血清在鱼腥藻细胞裂解物中识别出一种55 kDa的蛋白质。亚细胞分级分离分析表明，圆柱鱼腥藻腺苷酸环化酶定位于类囊体膜中。
