Nitric oxide gas (NO) increased guanylate cyclase [GTP pyrophosphate-yase (cyclizing), EC 4.6.1.21 activity in soluble and particulate preparations from various tissues. The effect was dose-dependent and was observed with all tissue preparations examined. The extent of activation was variable among different tissue preparations and was greatest (19-to 33-fold) with supernatant fractions of homogenates from liver, lung, tracheal smooth muscle, heart, kidney, cerebral cortex, and cerebellum. Smaller effects (5-to 14-fold) were observed with supernatant fractions from skeletal muscle, spleen, intestinal muscle, adrenal, and epididymal fat. Activation was also observed with partially purified preparations of guanylate cyclase. Activation of rat liver supernatant preparations was augmented slightly with reducing agents, decreased with some oxidizing agents, and greater in a nitrogen than in an oxygen atmosphere. After activation with NO, guanylate cyclase activity decreased with a half-life of 3-4 hr at 4°but re-exposure to NO resulted in reactivation of preparations. Sodium azide, sodium nitrite, hydroxylamine, and sodium nitroprusside also increased guanylate cyclase activity as reported previously. NO alone and in combination with these agents produced approximately the same degree of maximal activation, suggesting that all of these agents act through a similar mechanism. NO also increased the accumulation of cyclic GMP but not cyclic AMP in incubations of minces from various rat tissues. We propose that various nitro compounds and those capable-of forming NO in incubations activate guanylate cyclase through a similar but undefined mechanism. These effects may explain the high activities of guanylate cyclase in certain tissues (e.g., lung and intestinal mucosa) that are exposed to environmental nitro compounds.

Partially purified soluble rat liver guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) METHODS AND MATERIALSMale Sprague-Dawley rats weighing 150-250 g were sacrificed by cervical dislocation. Livers were removed quickly and placed in cold 0.25 M sucrose containing 10 mM Tris-HCI buffer, pH 8.0/1 mM EDTA/1 mM dithiothreitol. Livers were homogenized in 8 volumes of this medium with a glass homogenizer and Teflon pestle at 4°. Homogenates were centrifuged at 105,000 X g for 60 min, and supernatant fractions were used for purification of guanylate cyclase as described (13,15,16). HC1 (1 M) was added to the supernatant fraction to yield pH 5.0. The precipitate was collected by centrifugation at 12,000 X g for 15 min, suspended in 50 mM Tris-HCl, pH 7.6/1 mM EDTA/1 mM dithiothreitol, and recentrifuged. Solid ammonium sulfate was added to the resulting supernatant fraction to obtain 20% saturation. The precipitate was removed by centrifugation at 12,000 X g for 15 min and discarded.Ammonium sulfate was added to the supernatant fraction to achieve 45% saturation. The resulting precipitate was dissolved in 10 mM Tris-HCl, pH 7.6/1 mM EDTA/1 mM dithiothreitol. The sample was desalted on a Sephadex G-25 column and chromatographed on DEAE-cellulose (15,16). Guanylate cyclase was eluted by using a NaCI gradient (0-0.5 M). Fractions containing guanylate cyclase were pooled and used as a source of partially purified enzyme. Fresh preparations or those stored at -70°for more than 2 years were qualitatively similar in these studies.Guanylate cyclase activity was determined in 100-g1 incu-4360

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Soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been purified from rat liver and exhibited a single protein band on polyacrylamide gels coincident with activity and indicative of a molecular weight of 150,000. The apparent specific activity of the purified enzyme was 276 nmol of cyclic GMP A number of agents increase guanylate cyclase activity in broken cell preparations and cyclic GMP levels in tissues. Azide, nitroprusside, nitroglycerin, and nitrosamines activate both particulate and soluble forms of the enzyme (5, 7-9). The common pathway for activation by these agents may be the formation of nitric oxide (10). Activation by hydroxyl radical (11) and unsaturated fatty acids (12, 13) has also been described. The precise mechanism(s) by which guanylate cyclase is activated by these different agents is unknown.The properties of guanylate cyclase activated by many of these agents are different from those of the native enzyme. For example, activated enzyme may use Mg2+ as cofactor as effectively as Mn2+ (14) and can catalyze the formation of cyclic AMP (15).Identification of the precise mechanism(s) by which agents activate guanylate cyclase as well as a study of the molecular changes occurring during activation will require highly purified enzyme. Purification of particulate guanylate cyclase from sea urchin sperm has been reported (16), and partial purifications of soluble guanylate cyclase from rat liver (15, 17), bovine lung (18), and human platelets (19) have been described. Bacterial guanylate cyclase has been partially purified from Caulobacter crescentus (20) and Escherichia coli (21).In this communication we report the purification of soluble guanylate cyclase from rat liver and describe some of the properties of the purified enzyme. Some of these observations have recently been presented in abstract form (22). MATERIALS AND METHODSLivers were removed from male Sprague-Dawley rats (150-200 g) and placed in cold 20 mM Tris-HCI buffer (pH 8.0) containing 0.25 M sucrose, 1 mM EDTA, and 1 mM dithiothreitol (buffer I). All subsequent procedures were at 4°C. Livers were homogenized in 4 vol of buffer I with a Waring blender for 30 sec followed by two strokes with a glass-Teflon homogenizer. Homogenates were centrifuged at 105,000 X g for 60 min. Supernatant fractions were adjusted to pH 5.0 with 1 M HCI and centrifuged at 12,000 X g for 20 min. The pH 5.0 precipitate was dissolved in 20 mM Tris-HCl buffer (pH 7.6)/1 mM EDTA/1 mM dithiothreitol (buffer II) and recentrifuged at 12,000 X g for 20 min. The supernatant fraction was brought to 20% saturation with ammonium sulfate and centrifuged at 12,000 X g for 20 min. The resulting supernatant fraction was brought to 50% saturation with ammonium sulfate and recentrifuged. The 20-50% ammonium sulfate precipitate was suspended in buffer II and dialyzed against 200 vol of the same buffer. The dialyzed preparation was applied to a DEAESephacel column (5 X 40 cm) equilibrated with buffer II. Enzyme was eluted with a linear NaC...

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