Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.     

Formation of pyrophosphate by soluble guanylate cyclase from rat lung.

The guanylate cyclase reaction was studied to determine the identity of the product(s) formed other than guanosine-3′,5′-monophosphate (cyclic GMP). Partially purified guanylate cyclase preparations from rat lung catalyzed the formation of nearly equal amounts of PP₁ and of cyclic GMP from GTP. Column chromatography of the enzyme preparation on DEAE-Sephadex or Bio-Gel A-5m failed to separate the enzyme(s) involved in formation of cyclic GMP and of PP₁. Nucleotide inhibitors of cyclic GMP formation also inhibited PP₁ formation, and Ca²⁺, a stimulant of cyclic GMP formation in the presence of Mn²⁺, also stimulated PP₁ formation. Detectable PP₁ formation was not observed when ATP was present instead of GTP.The results show that guanylate cyclase, in vitro, catalyzes the formation of pyrophosphate from GTP concomitant with the synthesis of cyclic GMP.     

Highly purified particulate guanylate cyclase from rat lung: characterization and comparison with soluble guanylate cyclase

Guanylate cyclase was purified 1000-fold from washed rat lung particulate fractions to a final specific activity of 500 nmoles cyclic GMP produced/min/mg protein by a combination of detergent extraction and chromatography on concanavalin A-Sepharose, GTP-agarose, and blue agarose. Particulate guanylate cyclase has a molecular weight of 200 000 daltons, a Stokes radius of 48 A and a sedimentation coefficient of 9.4 while the soluble form has a molecular weight of 150 000 daltons, a Stokes radius of 44 A, and a sedimentation coefficient of 7.0. Whereas the particulate enzyme is a glycoprotein with a specific affinity for concanavalin A and wheat germ agglutinin, the soluble form of guanylate cyclase did not bind to these lectins. Purified particulate guanylate cyclase did not cross-react with a number of monoclonal antibodies generated to the soluble enzyme. While both forms of the enzyme could be regulated by the formation of mixed disulfides, the particulate enzyme was relatively insensitive to inhibition by cystine. With GTP as substrate both forms of the enzyme demonstrated typical kinetics, and with GTP analogues negative cooperativity was observed with both enzyme forms. These data support the suggestion that the two forms of guanylate cyclase possess similar catalytic sites, although their remaining structure is divergent, resulting in differences in subcellular distribution, physical characteristics, and antigenicity.     

Inhibition of the soluble guanylate cyclase from rat lung by sulfated polyanions

Soluble guanylate cyclase was partially purified from rat lung homogenates, and shown to be inhibited by the following sulfated polyanions, with the I₅₀ in μg/ml in parentheses: Polyvinyl sulfate (0.33), 40,000-dalton dextran sulfate (0.45), polyanetholesulfonate (0.63) 500,000-dalton dextran sulfate (1.8), λ-carrageenan (2.9), τ-carrageenan (6.1), κ-carrageenan (48.0), heparin (68.0). There was a good correlation between inhibitory potency and sulfate content (as total sulfur). Inhibition by heparin and the carrageenans (but not the others) was potentiated by Mn²⁺, but not Ca²⁺ or Mg²⁺, when [Mn²⁺] exceeded [GTP]. Mn²⁺-potentiation could be blocked by high Na⁺. Heparin-agarose shows promise as an affinity matrix for guanylate cyclase.     

Immobilization of rat lung soluble guanylate cyclase on alkyl-agarose gels.

The soluble guanylate cyclase from rat lung was immobilized by absorption rather than covalent attachment on hexyl-, octyl-, or decyl-agarose. The enzyme retained activity after being bound to these matrices and could be compared to the soluble, mobile form of the enzyme. Compared to the soluble enzyme, the immobilized guanylate cyclase had a lower apparent maximal velocity and a higher apparent Km for MeGTP in the presence of Mg2+, Ca2+, or Mn2+. The apparent maximum velocity was reduced to the same extent by hexyl-, octyl-, or decyl-agarose, but the reduction in activity was greater with Mg2+ than with Ca2+ or Mn2+. Both the soluble and immobilized guanylate cyclase displayed concave downward patterns on double reciprocal polots as a function of Mn2+, and Ca2+ caused apparent activation of either form of the enzyme. MnATP appeared to be a linear competitive inhibitor with respect to MnGTP for both forms of the enzymes but the ki was 3 micron for the soluble form and 30 micron for the immobilized form. These results demonstrate that the soluble form of guanylate cyclase from rat lung retains many of its basic properties after being immobilized on a hydrophobic matrix; however, rather pronounced decreases in the maximum velocity and increases in the apparent Michaelis constant for MeGTP, particularly for MgGTP, are observed upon immobilization.     

Kinetic effects of the concentration-dependent stimulation of soluble guanylate cyclase from rat lung

Soluble guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from rat lung demonstrated concentration-dependent stimulation, that is, an increase in specific activity with increasing enzyme (protein) concentration. This phenomenon persisted through several steps of enzyme purification and was apparently due to the presence of a macromolecular activator, similar in size to the enzyme. Treatment of partially purified enzyme with N-ethylmaleimide destroyed catalytic activity, but did not effect the ability of the preparation to stimulate activity. Kinetic analysis demonstrated that the stimulation was due to an increased V value with no change in the apparent Km value for MnGTP. Stimulation occurred without a time lag, the activator apparently interacting reversibly with the enzyme to increase catalytic capability. Some nonionic detergents of the Triton series inhibited enzyme activity by decreasing the V value, with no change in the Km value, and also decreased concentration-dependent stimulation. However, the two phenomena were not directly related. While the physiological significance of the activator is unclear, its presence affects estimations of recovery during enzyme purification, V determinations, and determinations of the effect of hormone or drug treatment on the activity of tissue extracts.     

Superoxide dismutase catalyzes activation of synaptosomal soluble guanylate cyclase from rat brain

The hydrogen ion changes resulting from the photolysis of the rod visual pigment, rhodopsin, were investigated at acidic pH (5.2–6.5). After light-induced proton uptake, slow proton release occurred both in the dark and in the light. It was found that the amount of proton release in the dark was not equal to that in the light; about 0.9 proton remained bound to rhodopsin bleached in the dark, while all the bound protons were released in the light. Furthermore, the time course of proton release in the dark is not related to the decay of metarhodopsin II₃₈₀, but is closely related to the formation of metarhodopsin III₄₆₅.     

Characterization of ATP-stimulated guanylate cyclase activation in rat lung membranes

Many of the effects of ANP are mediated through the elevation of cellular cGMP levels by the activation of particulate guanylate cyclase. While the stimulation of this enzyme is receptor-mediated, the molecular mechanism of activation remains unknown. In this study we present evidence that ATP as well as its analogues adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) and adenylylimidophosphate (AMPPNP) activates guanylate cyclase from rat lung membranes and markedly potentiates the effect of ANP on the enzyme. The order of potency is ATP gamma S greater than ATP greater than AMPPNP. The enzyme activation by adenine nucleotide and ANP together is much more than the sum of the individual activations, suggesting that ATP may be the physiological component essential for the ANP-stimulated guanylate cyclase activation. The ATP gamma S-stimulated guanylate cyclase activity diminishes in the presence of various kinds of detergents, suggesting either that the conformation of an ATP binding site in guanylate cyclase is altered by detergents or that protein-protein interaction may be involved in the activation of guanylate cyclase by ATP. Guanylate cyclase from rat lung membranes is poorly activated by ANP and/or ATP gamma S after removing the cytosolic and weakly membrane-associated proteins or factors by centrifugation. Pre-incubation of the membranes with ATP gamma S retains enzyme activation after membrane washing. These results suggest either that ATP gamma S stabilizes the conformation of nucleotide binding site in guanylate cyclase from denaturation by membrane washing, or that the stimulatory effect of ATP on guanylate cyclase activity may be mediated by accessory proteins or non-protein cofactors which are lost during membrane washing, but remain bound to membranes by ATP gamma S pretreatment.     

Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung.

Complementary DNA clones corresponding to the 70- and 82-kDa subunits of soluble guanylate cyclase of rat lung have been isolated. Blot hybridization of total poly(A)+ RNA from rat tissues detected mRNA of about 3.4 kilobases for the 70-kDa subunit and about 5.5 kilobases for the 82-kDa subunit. Messenger RNA levels of both subunits were abundant in lung and cerebrum, moderate in cerebellum, heart, and kidney, and low in liver and muscle, consistent with previously described enzyme activities in these tissues. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated that the genes for the 70- and 82-kDa subunits are different. The carboxyl-terminal region of the 70- and 82-kDa subunits showed a high degree of homology and also had a partial homology with the putative catalytic domain of particulate guanylate cyclase and adenylate cyclase, indicating that both the 70- and 82-kDa subunits have catalytic domains. The cDNAs were subcloned to an expression vector and transfected to L cells. The cells transfected with cDNA of the 70-kDa subunit or the 82-kDa subunit showed no guanylate cyclase activity, whereas the cells transfected with both the 70- and 82-kDa subunit cDNAs showed significant guanylate cyclase activity that was activated markedly by sodium nitroprusside. These data suggest that both subunits are required for both the basal catalytic and regulatory activity of soluble guanylate cyclase. Presumably both catalytic subunits must be present and interactive to permit synthesis of cyclic GMP and nitrovasodilator activation.     

Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.     

可溶性鸟苷酸环化酶在低氧高二氧化碳大鼠肺组织中的表达

目的 :探讨慢性低氧高二氧化碳对大鼠肺动脉及支气管可溶性鸟苷酸环化酶 (sGC)蛋白表达的影响。方法 :雄性SD大鼠于低氧高二氧化碳饲养舱复制动物模型 ,免疫组织化学技术观察低氧高二氧化碳组及对照组肺组织sGCα1 、β1 亚基蛋白的表达。结果 :sGC在正常大鼠肺动脉、支气管平滑肌上阳性表达并呈梯度现象 ,低氧高二氧化碳组肺细小动脉及支气管平滑肌sGC蛋白与对照组相比逐渐减弱 (均P <0 .0 1)。结论 :低氧高二氧化碳抑制肺细小动脉及支气管平滑肌sGC蛋白的表达。     

Ultrastructural demonstration of guanylate cyclase in rat lung after activation by ANF.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.     

High yield purification of soluble guanylate cyclase from bovine lung

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3,5′-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg²⁺. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP–agarose. The purified enzyme migrated as a two-band protein on SDS–PAGE corresponding to sGC subunits α (M_r = 77,532) and β (M_r = 70,500) and had an A_280nm/A_430nm of 1 indicating one heme per heterodimer. The yield of enzyme was 8–10 mg from 4 to 5 kg bovine lungs. V_max and K_m of non-stimulated sGC were 22 nmol/mg/min and 180 μM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V_max increased to 1330 nmol/mg/min while the K_m dropped to 43 μM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.     

Adenine nucleotide regulation of particulate guanylate cyclase from rat lung.

Adenine nucleotides activate basal particulate guanylate cyclase in rat lung membranes. Activation is specific for adenine and not guanine, cytidine or uridine nucleotides. The concentration of adenine nucleotides yielding half-maximum activation of particulate guanylate cyclase is 0.1 mM and this nucleotide activates the enzyme by increasing maximum velocity 11-fold without altering affinity for substrate. Activation is specific for particulate guanylate cyclase, since soluble enzyme is inhibited by adenine nucleotides. Similarly, activation is specific for magnesium as the enzyme substrate cation cofactor, since adenine nucleotides inhibit particulate guanylate cyclase when manganese is used. Adenine nucleotide regulation of particulate guanylate cyclase may occur by a different molecular mechanism compared to other activators, since the effects of these nucleotides are synergistic with those of detergent, hemin and atrial natriuretic peptides. Cystamine inhibits adenine nucleotide activation of particulate guanylate cyclase at concentrations having minimal effects on basal enzyme activity suggesting a role for critical sulfhydryls in mechanisms underlying nucleotide regulation of particulate guanylate cyclase. Purification and quantitative recovery of particulate guanylate cyclase by substrate affinity chromatography results in the loss of adenine nucleotide regulation. These data suggest that adenine nucleotides may be important in the regulation of basal and activated particulate guanylate cyclase and may be mediated by an adenine nucleotide-binding protein which is separate from that enzyme.     

Purification of a soluble, sodium-nitroprusside-stimulated guanylate cyclase from bovine lung

A soluble, sodium-nitroprusside-stimulated guanylate cyclase as 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 mumol of 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 microM, 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 mumol min-1 mg-1.     

Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.