Russwurm Michael, Koesling Doris
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Germany.
Methods Enzymol. 2005;396:492-501. doi: 10.1016/S0076-6879(05)96041-2.
Nitric oxide (NO)-sensitive guanylyl cyclase (GC) represents the receptor for the signaling molecule NO in mammals. The enzyme consists of two different subunits and contains a prosthetic heme group acting as an NO acceptor. Binding of NO to the heme accelerates the catalytic conversion from guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) by 2.0-2.5 orders of magnitude. NO-sensitive GC represents a well-established drug target because the NO-releasing drugs used in the therapy of coronary heart disease act by stimulation of the enzyme. Furthermore, new NO-independent GC activators are under development. Characterization of the molecular mechanisms by which NO-releasing and NO-independent drugs control enzyme activity often requires very large amounts of enzyme, as do attempts to resolve the structure of GC. Because heterologous expression systems turned out to yield only small amounts of the enzyme, the purification from bovine lungs is still a convenient way to obtain the enzyme in large quantities. In this chapter, a fast method for purification of the enzyme from bovine lungs is described, and basic methods for characterization of the enzyme are summarized.
一氧化氮（NO）敏感型鸟苷酸环化酶（GC）是哺乳动物中信号分子NO的受体。该酶由两个不同的亚基组成，并含有一个作为NO受体的辅基血红素基团。NO与血红素的结合使鸟苷三磷酸（GTP）向环鸟苷单磷酸（cGMP）的催化转化加速2.0 - 2.5个数量级。NO敏感型GC是一个成熟的药物靶点，因为用于冠心病治疗的NO释放药物通过刺激该酶起作用。此外，新型非NO依赖性GC激活剂也在研发中。与解析GC结构的尝试一样，表征NO释放药物和非NO依赖性药物控制酶活性的分子机制通常需要大量的酶。由于异源表达系统只能产生少量的酶，从牛肺中纯化仍然是大量获取该酶的便捷方法。本章描述了一种从牛肺中快速纯化该酶的方法，并总结了表征该酶的基本方法。
