Purification and characterization of NO-sensitive guanylyl cyclase.
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.
Russwurm
Michael
M
Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum, Germany.
Koesling
Doris
D
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006162:Guanylate Cyclase / Q000302:isolation & purification* / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase