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.     

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.     

Effect of GTP analogues on purified soluble guanylate cyclase

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.     

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.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Inhibition of soluble guanylate cyclase by bovine serum albumin

Bovine serum albumin (BSA) and to a lesser extent beta-lactoglobulin produced concentration-dependent inhibition of the guanylate cyclase activity in supernatant fraction and partially purified enzyme (PPE) prepared from rat lung homogenates. Ovalbumin had little effect. Some activity was lost when PPE was applied to a BSA-agarose column, however the loss disappeared when the enzyme reaction mixture contained Lubrol PX. Also, BSA no longer inhibited PPE after BSA-agarose treatment. BSA inhibition of PPE was not apparent when activity was maximally stimulated by arachidonate. These data were interpreted as indicating that the enzyme had bound to it an amphiphilic activator, possibly a fatty acid, the removal of which by BSA decreased activity.     

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.     

Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside

Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.     

Preparation of heme-free soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.     

NO-independent stimulators of soluble guanylate cyclase

SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure lowering effect in rats after oral application.     

Activation of purified guanylate cyclase by nitric oxide requires heme comparison of heme-deficient,heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg²⁺ or Mn²⁺. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO nitroso compounds.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

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.     

Purification of soluble guanylate cyclase enzyme from human platelets

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.     

Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme

The mechanism by which arachidonic acid activates soluble guanylate cyclase purified from bovine lung is partially elucidated. Unlike enzyme activation by nitric oxide (NO), which required the presence of enzyme-bound heme, enzyme activation by arachidonic acid was inhibited by heme. Human but not bovine serum albumin in the presence of NaF abolished activation of heme-containing guanylate cyclase by NO and nitroso compounds, whereas enzyme activation by arachidonic acid was markedly enhanced. Addition of heme to enzyme reaction mixtures restored enzyme activation by NO but inhibited enzyme activation by arachidonic acid. Whereas heme-containing guanylate cyclase was activated only 4- to 5-fold by arachidonic or linoleic acid, both heme-deficient and albumin-treated heme-containing enzymes were activated over 20-fold. Spectrophotometric analysis showed that human serum albumin promoted the reversible dissociation of heme from guanylate cyclase. Arachidonic acid appeared to bind to the hydrophobic heme-binding site on guanylate cyclase but the mechanism of enzyme activation was dissimilar to that for NO or protoporphyrin IX. Enzyme activation by arachidonic acid was insensitive to Methylene blue or KCN, was inhibited competitively by metalloporphyrins, and was abolished by lipoxygenase. Whereas NO and protoporphyrin IX lowered the apparent Km and Ki for MgGTP and uncomplexed Mg2+, arachidonic and linoleic acids failed to alter these kinetic parameters. Thus, human serum albumin can promote the reversible dissociation of heme from soluble guanylate cyclase and thereby abolish enzyme activation by NO but markedly enhance activation by polyunsaturated fatty acids. Arachidonic acid activates soluble guanylate cyclase by heme-independent mechanisms that are dissimilar to the mechanism of enzyme activation caused by protoporphyrin IX.     

The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase

The primary structure of the 70 kDa subunit of soluble bovine guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been determined. The alignment of six different clones out of two bovine libraries yielded a total of 3.1 kb with a coding region of 1857 bases. The open reading frame encodes a protein of 619 amino acids and a molecular mass of 70.5 kDa. Antibodies raised against a synthetic peptide, which corresponded to the C-terminus of the deduced sequence precipitated guanylate cyclase activity from guanylate cyclase-enriched preparations.     

Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.     

The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.