guanosine-triphosphate and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

Nitric oxide (NO) exerts physiological effects by activating specialized receptors that are coupled to guanylyl cyclase activity, resulting in cGMP synthesis from GTP. Despite its widespread importance as a signal transduction pathway, the way it operates is still understood only in descriptive terms. The present work aimed to elucidate a formal mechanism for NO receptor activation and its modulation by GTP, ATP, and allosteric agents, such as YC-1 and BAY 41-2272. The model comprised a module in which NO, the nucleotides, and allosteric agents bind and the protein undergoes a conformational change, dovetailing with a catalytic module where GTP is converted to cGMP and pyrophosphate. Experiments on NO-activated guanylyl cyclase purified from bovine lung allowed values for all of the binding and isomerization constants to be derived. The catalytic module was a modified version of one describing the kinetics of adenylyl cyclase. The resulting enzyme-linked receptor mechanism faithfully reproduces all of the main functional properties of NO-activated guanylyl cyclase reported to date and provides a thermodynamically sound interpretation of those properties. With appropriate modification, it also replicates activation by carbon monoxide and the remarkable enhancement of that activity brought about by the allosteric agents. In addition, the mechanism enhances understanding of the behavior of the receptor in a cellular setting.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Allosteric Regulation; Animals; Cattle; Cyclic GMP; Enzyme Activation; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Kinetics; Lung; Models, Chemical; Nitric Oxide; Protein Structure, Tertiary; Pyrazoles; Pyridines; Pyrophosphatases; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Thermodynamics

Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon binding of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved aqueous solubility as well as its effects on sGC.

Topics: Alcohols; Chemistry, Pharmaceutical; Drug Design; Electrons; Enzyme Activation; Enzyme Activators; Guanosine Triphosphate; Guanylate Cyclase; Heme; Indazoles; Kinetics; Models, Chemical; Nitric Oxide; Organophosphonates; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Temperature

Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (nuFe-CO)) appears at 492 cm(-1) in the presence of imidazole compared with 473 cm(-1) in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the nuFe-CO vs nuC-O plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O(2), sGC under the high concentration of imidazole (1.2 M) yielded nu4 at 1373 cm(-1) even after its removal by gel-filtration, but addition of dithionite gave the strong nu4 band at 1360 cm(-1). This indicated that imidazole caused autoxidation of sGC.

Topics: Animals; Cattle; Drug Synergism; Enzyme Activation; Guanosine Triphosphate; Guanylate Cyclase; Heme; Imidazoles; In Vitro Techniques; Indazoles; Solubility; Spectrum Analysis, Raman

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.

Topics: Benzoates; Chromatography, Ion Exchange; Cyclic GMP; Enzyme Activation; Enzyme Activators; Guanosine Triphosphate; Guanylate Cyclase; Heme; Hemeproteins; Histidine; Indazoles; Nitroprusside; Polysorbates; Proteins; Protoporphyrins; Spectrophotometry; Zinc

Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of approximately 4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.

Topics: Animals; Cattle; Enzyme Activation; Guanosine Triphosphate; Guanylate Cyclase; Humans; Indazoles; Kinetics; Mutagenesis, Site-Directed; Nitric Oxide

Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) and is the known mediator of the effects of NO on a variety of physiological processes. The rates at which sGC is activated and deactivated are therefore of wide interest since they determine the duration of a tissue's response to NO. The effect of NO on smooth muscle dissipates in 1-2 min, suggesting that both activation and deactivation are fast. In vitro measurements show that the activation of sGC occurs in less than a second, while the deactivation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deactivate the cyclase in several seconds to minutes, though the effectiveness of each of these agents is in dispute. We investigated the lifetime of NO-sGC in the cytosol of retina by monitoring its enzymatic activity at 20 degrees C. Our results show that Mg-GTP, the substrate of NO-sGC, has no influence on the deactivation. Similarly, reducing agents glutathione and dithiothreitol shortened the half-life of NO-sGC only by about 30%. The greatest effect on the deactivation was caused by scavengers of NO: oxyhemoglobin reduced the half-life of NO-sGC from 106 min to 18 s; another NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), reduced it to 42 s (20 degrees C). Similarly rapid deactivation was observed with the enzyme from bovine lung, immunoprecipitated enzyme from bovine retina, and heme-deficient enzyme from bovine retina reconstituted with heme. On the other hand, YC-1, an activator of sGC, stabilized the activated enzyme, preventing NO dissociation, as was evident from the inability of oxyhemoglobin or CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, also inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deactivation of the enzyme. Lithium, which is also known to inhibit NO-sGC, had no effect on the deactivation rate of the enzyme. These results, taken together, suggest that two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cell.

Topics: Animals; Calcium; Cattle; Cytosol; Dithiothreitol; Enzyme Activation; Enzyme Activators; Enzyme Stability; Free Radical Scavengers; Glutathione; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Nitric Oxide; Oxyhemoglobins; Reducing Agents; Solubility; Substrate Specificity

Previous work has proved that the enzyme-soluble guanylate cyclase, GC, is activated several 100-fold by the combination of carbon monoxide plus a benzylindazole derivative called YC-1. That is about the same as activation by nitric oxide, which has a well-established role both in vivo and in vitro. This report addresses several spectroscopic, equilibrium, and kinetic effects wrought by YC-1 on carboxyl guanylate cyclase, including the following: a shift in the Soret absorption band by 4 nm to shorter wavelength; an increase in CO affinity by an order of magnitude; a dramatic change in the kinetics of CO association. After photolytic dissociation of CO, the majority, but not all, of bimolecular ligand recombination occurs with a time constant about 1000-fold faster than in the absence of YC-1, while a smaller fraction recombines almost, but not quite, the same as usual. This is reminiscent of the kinetics of NO association with GC, which also shows two prominent phases. The results just listed pertain in the presence of GTP/cGMP, which would be present during enzyme catalysis. Qualitatively similar, but smaller, effects occur in the absence of GTP/cGMP. Measurements are reported to characterize other changes in buffer conditions. The results are consistent with a mechanistic model that attributes a crucial role to the proximal bond that connects the heme iron to a histidine side chain in GC but also requires protein control of the distal environment.

Topics: Animals; Carbon Monoxide; Cattle; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Kinetics; Ligands; Models, Chemical; Nitric Oxide; Photolysis; Platelet Aggregation Inhibitors; Solubility; Spectrophotometry, Ultraviolet

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in various signaling cascades and are structurally closely related. The crystal structure of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the heterodimeric soluble GC (sGC), has been identified which acts like forskolin on AC. Here, we investigated the respective substrate and potential activator domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost complete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and higher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain which corresponds to the forskolin site of the ACs may comprise the binding site for YC-1.

Topics: Animals; Binding Sites; Catalytic Domain; Cattle; Conserved Sequence; Cyclic GMP; Dimerization; Enzyme Activators; Guanosine Triphosphate; Guanylate Cyclase; Indazoles; Magnesium; Manganese; Mutagenesis, Site-Directed; Nitric Oxide; Point Mutation; Solubility

Soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to the second messenger cGMP, functions as the receptor for nitric oxide (NO) and nitrovasodilator drugs. Three distinct cDNA species of each subunit (alpha1-alpha3, beta1-beta3) have been reported from various species. From human sources, none of these have been expressed as functionally active enzyme. Here we describe the expression of human alpha/beta heterodimeric sGC in Sf9 cells yielding active recombinant enzyme that was stimulated by the nitrovasodilator sodium nitroprusside or the NO-independent activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1). At the protein level, both alpha and beta subunits were detected in human tissues, suggesting co-expression also in vivo. Moreover, resequencing of the human cDNA clones [originally termed alpha3 and beta3; Giuili, Scholl, Bulle and Guellaen (1992) FEBS Lett. 304, 83-88] revealed several sequencing errors in human alpha3; correction of these eliminated major regions of divergence from rat and bovine alpha1. As human beta3 also displays more than 98% similarity to rat and bovine beta1 at the amino acid level, alpha3 and beta3 represent the human homologues of rat and bovine alpha1 and beta1, and the isoenzyme family is decreased to two isoforms for each subunit (alpha1, alpha2; beta1, beta2). Having access to the human key enzyme of NO signalling will now permit the study of novel sGC-modulating compounds with therapeutic potential.

Topics: Amino Acid Sequence; Animals; Cattle; Cyclic GMP; Guanosine Triphosphate; Guanylate Cyclase; Humans; Indazoles; Isoenzymes; Molecular Sequence Data; Nitric Oxide; Nitroprusside; Platelet Aggregation Inhibitors; Protein Conformation; Rats; Recombinant Proteins; Solubility; Spodoptera; Vasodilator Agents