Purifieation of a Soluble, Sodium‐Nitroprusside‐Stimulated Guanylate Cyclase from Bovine Lung

Abstract

A soluble, sodium‐nitroprusside‐stimulated guanylate cyclase has been purified from bovine lung by DEAE cellulose chromatography, ammonium sulfate precipitation, chromatography on Blue Sepharose CL‐6B and preparative gel electrophoresis. Apparent homogeneity was obtained after at least 7000‐fold purification with a yield of 3%. A single stained band (Mr 72000) was observed after gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme migrated as one band also under non‐denaturing conditions in acrylamide gels (5–12%). The mobility of this band corresponded to an Mr of 145000. The enzyme sedimented on sucrose gradients with an S20.w of 7.0 S. Gel filtration yielded a Stokes' radius of 4.6 nm. These data suggest that the enzyme has an Mr of approximately 150000 and consists of two, presumably identical, subunits of Mr 72000. Sodium nitroprusside stimulated the purified enzyme 15‐fold and 140‐fold to specific activities of 8.5 and 15.7 pmol cGMP formed min−1 mg−1 in the presence of Mn2+ and Mg2+, respectively. Formation of cGMP was proportional to the incubation time and to the amount of enzyme added. The stimulatory effect of sodium nitroprusside was half‐maximal at about 2 μM. was observed immediately after addition and could be reversed either by dilution or by removal of sodium nitroprusside on a Sephadex G‐25 column. The purified enzyme in the absence of catalase was stimulated by sodium nitroprusside, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and 3‐morpholino‐sydnonimine and in the presence of catalase by sodium nitrite and sodium azide. In the presence of Mn2+ and sodium nitroprusside, the purified enzyme catalyzed the formation of cAMP from ATP at a rate of 0.6 μmol min−1 mg−1.

Soluble guanylate cyclase purified from bovine lung contains heme and copper

“…Soluble guanylate cyclase was purified from bovine lung as in [10]. Visible absorption spectroscopy was performed using a Beckman ACTA M VI spectrophotometer in cuvettes of 1 cm pathlength.…”

“…Values obtained in purified enzyme preparations were corrected for these blanks. Guanylate cyclase activity was determined with 3 mM Mg 2+ as in [ 10], with omissions of cyclic GMP and EGTA in the assay. All values represent means of duplicate determinations from representative experiments.…”

“…Purification of soluble guanylate cyclase from bovine lung can be achieved without major loss in stimulation by sodium nitropmsside and related agents [10]. In addition, it was observed that in the last purification step a chromophore with an absorption maxhnum at ~430 nm copurified with the enzyme activity [ 11 ].…”

Heme oxygenase-1 induction depletes heme and attenuates pulmonary artery relaxation and guanylate cyclase activation by nitric oxide

“…Organ culturing BPA with heme (hemin) reversed the attenuation of relaxation to NO under the conditions used to increase HO-1 expression. Early studies on NO regulation of sGC demonstrated that heme was easily lost as sGC was purified (8)(9)(10)(11)(18)(19)(20), suggesting that heme depletion could be a mechanism of regulating sGC stimulation by NO. Thus, the control of heme availability could be an important factor regulating the responsiveness of sGC and vascular tissue to NO.…”

“…Early studies on how nitric oxide (NO) regulates the soluble form of guanylate cyclase identified heme as an essential cofactor in mediating the stimulation of cGMP formation (8)(9)(10)(11)(18)(19)(20). These studies detected evidence for the presence of heme-containing and hemedeficient forms of sGC, where heme was easily lost from sGC as the enzyme was purified.…”