Dessauer C W, Gilman A G
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.
J Biol Chem. 1996 Jul 12;271(28):16967-74. doi: 10.1074/jbc.271.28.16967.
An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.
一种经过工程改造的可溶性哺乳动物腺苷酸环化酶已在大肠杆菌中表达，并通过三步色谱法进行纯化。该酶利用一分子ATP合成一分子环磷酸腺苷和焦磷酸，最大比活性为12.8微摩尔/分钟/毫克，对应于720分钟-1的周转数。尽管缺乏跨膜结构域，但该酶表现出哺乳动物腺苷酸环化酶共有的所有调节特性。它被Gsα和福斯可林协同激活，并被腺苷（P位点）类似物抑制，其动力学模式与天然酶相同。纯化后的酶也被G蛋白βγ亚基复合物直接抑制。在腺病毒介导下在缺乏腺苷酸环化酶的HC-1细胞中表达后，该酶可被Gs偶联受体和福斯可林协同刺激。
