Krishna G, Krishnan N
J Cyclic Nucleotide Res. 1975;1(6):293-302.
An extremely rapid and sensitive assay for guanylate cyclase utilizing [alpha-32P]-GTP has been developed. It involves incubation of 5-100 mug of enzyme protein with 1 mM [alpha-32P]-GTP in 40 mM Tris HC1 buffer (pH 7.4) containing 3-3 mM MnSO2, 10 mM theophylline and 1 mM cyclic GMP. The reaction is terminated by addition of EDTA, and [32P]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H+ and alumina. Recovery of 75-85% of [3H]-cyclic GMP and a blank of 0.001-0.003% of added [32P]-GTP was routinely obtained. The [32P] radioactivity isolated was shown to be cyclic GMP by a variety of techniques. The assay has also been shown to be applicable for a variety of tissues.
已开发出一种利用[α-32P]-GTP对鸟苷酸环化酶进行极其快速且灵敏的检测方法。该方法包括将5 - 100微克酶蛋白与1 mM [α-32P]-GTP在含有3 - 3 mM MnSO2、10 mM茶碱和1 mM环鸟苷酸的40 mM Tris HCl缓冲液（pH 7.4）中孵育。通过加入EDTA终止反应，然后通过在Dowex - 50 - H+和氧化铝上的顺序色谱法分离形成的[32P]-环鸟苷酸。常规情况下，[3H]-环鸟苷酸的回收率为75 - 85%，空白值为添加的[32P]-GTP的0.001 - 0.003%。通过多种技术表明，分离出的[32P]放射性物质为环鸟苷酸。该检测方法也已证明适用于多种组织。
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