Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a mes senger molecule with multiple clinical implications. Understanding the acti vation of sGC is an important step for establishing new therapeutic princip les. We have now overexpressed sGC in a baculovirus/Sf9 system optimized fo r high protein yields to facilitate spectral and kinetic studies of the act ivation mechanisms of this enzyme. It was expressed in a belch fermenter us ing a defined mixture of viruses encoding the alpha(1) and beta(1) subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosol ic fraction by anion exchange chromatography, hydroxyapatite chromatography , and size exclusion chromatography. By use of this new method 2.5 1 cultur e yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. Th e enzyme did not contain stoichiometric amounts of copper. The basal activi ties of the purified enzyme were 153 and 1259 nmol min(-1) mg(-1) in the pr esence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-( N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fo ld with Mg2+, whereas the NO-independent activator 3-(5'-hydroxymethyl-2'-f uryl)-1-benzylindazole (YC-1) induced an increase in the activity of 101-fo ld at a concentration of 300 mu M. The combination of DEA/NO (10 mu M) and YC-1 (100 mu M) elicited a dose-dependent synergistic stimulation with a ma ximum of a 792-fold increase over the basal activity in the presence of Mg2 +, resulting in a specific activity of 121 mu mol min(-1) mg(-1). The syner gistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor I H(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 mu M) by 94%. In a diff erent experimental setup a saturated carbon monoxide solution in the absenc e of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme sp ectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 42 4 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme s pectra were not affected by YC-1 in the absence or in the presence of DEA/N O or of carbon monoxide, which reflects the fact that YC-1 does not interac t directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The de scribed method should help to facilitate the investigation of the new thera peutic principle of NO-independent guanylyl cyclase activators.