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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