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1.
BMC Vet Res ; 20(1): 196, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38741109

ABSTRACT

BACKGROUND: Hypoxia is a detrimental factor in solid tumors, leading to aggressiveness and therapy resistance. OMX, a tunable oxygen carrier from the heme nitric oxide/oxygen-binding (H-NOX) protein family, has the potential to reduce tumor hypoxia. [18F]Fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) is the most widely used and investigated method for non-invasive imaging of tumor hypoxia. In this study, we used [18F]FMISO PET/CT (computed tomography) to assess the effect of OMX on tumor hypoxia in spontaneous canine tumors. RESULTS: Thirteen canine patients with various tumors (n = 14) were randomly divided into blocks of two, with the treatment groups alternating between receiving intratumoral (IT) OMX injection (OMX IT group) and intravenous (IV) OMX injection (OMX IV group). Tumors were regarded as hypoxic if maximum tumor-to-muscle ratio (TMRmax) was greater than 1.4. In addition, hypoxic volume (HV) was defined as the region with tumor-to-muscle ratio greater than 1.4 on [18F]FMISO PET images. Hypoxia was detected in 6/7 tumors in the OMX IT group and 5/7 tumors in the OMX IV injection group. Although there was no significant difference in baseline hypoxia between the OMX IT and IV groups, the two groups showed different responses to OMX. In the OMX IV group, hypoxic tumors (n = 5) exhibited significant reductions in tumor hypoxia, as indicated by decreased TMRmax and HV in [18F]FMISO PET imaging after treatment. In contrast, hypoxic tumors in the OMX IT group (n = 6) displayed a significant increase in [18F]FMISO uptake and variable changes in TMRmax and HV. CONCLUSIONS: [18F]FMISO PET/CT imaging presents a promising non-invasive procedure for monitoring tumor hypoxia and assessing the efficacy of hypoxia-modulating therapies in canine patients. OMX has shown promising outcomes in reducing tumor hypoxia, especially when administered intravenously, as evident from reductions in both TMRmax and HV in [18F]FMISO PET imaging.


Subject(s)
Dog Diseases , Misonidazole , Neoplasms , Positron Emission Tomography Computed Tomography , Tumor Hypoxia , Animals , Dogs , Misonidazole/analogs & derivatives , Positron Emission Tomography Computed Tomography/veterinary , Positron Emission Tomography Computed Tomography/methods , Dog Diseases/diagnostic imaging , Dog Diseases/drug therapy , Female , Tumor Hypoxia/drug effects , Male , Neoplasms/veterinary , Neoplasms/drug therapy , Neoplasms/diagnostic imaging , Thiosemicarbazones/therapeutic use , Thiosemicarbazones/pharmacology , Coordination Complexes
2.
Shock ; 62(1): 103-110, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38662597

ABSTRACT

ABSTRACT: Hemorrhagic shock is a major source of morbidity and mortality worldwide. While whole blood or blood product transfusion is a first-line treatment, maintaining robust supplies presents significant logistical challenges, particularly in austere environments. OMX is a novel nonhemoglobin (Hb)-based oxygen carrier derived from the H-NOX (heme-nitric oxide/oxygen binding) protein family. Because of their engineered oxygen (O 2 ) affinities, OMX proteins only deliver O 2 to severely hypoxic tissues. Additionally, unlike Hb-based oxygen carriers, OMX proteins do not scavenge nitric oxide in the vasculature. To determine the safety and efficacy of OMX in supporting tissue oxygen delivery and cardiovascular function in a large animal model of controlled hemorrhage, 2-3-week-old lambs were anesthetized, intubated, and mechanically ventilated. Hypovolemic shock was induced by acute hemorrhage to obtain a 50% reduction over 30 min. Vehicle (n = 16) or 400 mg/kg OMX (n = 13) treatment was administered over 15 min. Hemodynamics, arterial blood gases, and laboratory values were monitored throughout the 6-h study. Comparisons between groups were made using t tests, Wilcoxon rank sum test, and Fisher's exact test. Survival was assessed using Kaplan-Meier curves and the log-rank test. We found that OMX was well-tolerated and significantly improved lactate and base deficit trends, and hemodynamic indices ( P < 0.05). Median survival time was greater in the OMX-treated group (4.7 vs. 6.0 h, P < 0.003), and overall survival was significantly increased in the OMX-treated group (25% vs. 85%, P = 0.004). We conclude that OMX is well-tolerated and improves metabolic, hemodynamic, and survival outcomes in an ovine model of controlled hemorrhagic shock.


Subject(s)
Disease Models, Animal , Oxygen , Shock, Hemorrhagic , Animals , Shock, Hemorrhagic/therapy , Sheep , Hemodynamics , Blood Substitutes/therapeutic use , Blood Substitutes/pharmacology
3.
Stroke ; 53(4): 1363-1372, 2022 04.
Article in English | MEDLINE | ID: mdl-35306836

ABSTRACT

BACKGROUND: Tissue hypoxia plays a critical role in the events leading to cell death in ischemic stroke. Despite promising results in preclinical and small clinical pilot studies, inhaled oxygen supplementation has not translated to improved outcomes in large clinical trials. Moreover, clinical observations suggest that indiscriminate oxygen supplementation can adversely affect outcome, highlighting the need to develop novel approaches to selectively deliver oxygen to affected regions. This study tested the hypothesis that intravenous delivery of a novel oxygen carrier (Omniox-Ischemic Stroke [OMX-IS]), which selectively releases oxygen into severely ischemic tissue, could delay infarct progression in an established canine thromboembolic large vessel occlusion stroke model that replicates key dynamics of human infarct evolution. METHODS: After endovascular placement of an autologous clot into the middle cerebral artery, animals received OMX-IS treatment or placebo 45 to 60 minutes after stroke onset. Perfusion-weighted magnetic resonance imaging was performed to define infarct progression dynamics to stratify animals into fast versus slow stroke evolvers. Serial diffusion-weighted magnetic resonance imaging was performed for up to 5 hours to quantify infarct evolution. Histology was performed postmortem to confirm final infarct size. RESULTS: In fast evolvers, OMX-IS therapy substantially slowed infarct progression (by ≈1 hour, P<0.0001) and reduced the final normalized infarct volume as compared to controls (0.99 versus 0.88, control versus OMX-IS drug, P<0.0001). Among slow evolvers, OMX-IS treatment delayed infarct progression by approximately 45 minutes; however, this did not reach statistical significance (P=0.09). The final normalized infarct volume also did not show a significant difference (0.93 versus 0.95, OMX-IS drug versus control, P=0.34). Postmortem histologically determined infarct volumes showed excellent concordance with the magnetic resonance imaging defined ischemic lesion volume (bias: 1.33% [95% CI, -15% to 18%). CONCLUSIONS: Intravenous delivery of a novel oxygen carrier is a promising approach to delay infarct progression after ischemic stroke, especially in treating patients with large vessel occlusion stroke who cannot undergo definitive reperfusion therapy within a timely fashion.


Subject(s)
Brain Ischemia , Stroke , Animals , Brain Ischemia/diagnostic imaging , Brain Ischemia/drug therapy , Brain Ischemia/pathology , Dogs , Humans , Infarction , Magnetic Resonance Imaging/methods , Oxygen , Stroke/diagnostic imaging , Stroke/drug therapy
5.
PLoS Biol ; 16(10): e2005924, 2018 10.
Article in English | MEDLINE | ID: mdl-30335746

ABSTRACT

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.


Subject(s)
Heme/therapeutic use , Hypoxia/therapy , Oxygen/therapeutic use , Animals , Biological Therapy/methods , Heart/physiology , Heart Rate/drug effects , Heart Ventricles/drug effects , Lung , Muscle Contraction/drug effects , Myocardial Contraction/drug effects , Myocardium/metabolism , Nitric Oxide/metabolism , Nitric Oxide/therapeutic use , Oxygen/metabolism , Oxygen Consumption/physiology , Protein Engineering/methods , Sheep , Vascular Resistance/drug effects
6.
SLAS Discov ; 22(3): 324-331, 2017 03.
Article in English | MEDLINE | ID: mdl-27932698

ABSTRACT

ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.


Subject(s)
High-Throughput Screening Assays , Immunologic Factors/pharmacology , Protein Kinase Inhibitors/pharmacology , Receptors, Antigen, T-Cell/genetics , Small Molecule Libraries/pharmacology , ZAP-70 Protein-Tyrosine Kinase/genetics , Cell-Free System/chemistry , Fluorescence Polarization/methods , Fluorescence Resonance Energy Transfer/methods , Gene Expression , Humans , Immunologic Factors/chemistry , Peptides/antagonists & inhibitors , Peptides/genetics , Peptides/immunology , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/chemistry , Receptors, Antigen, T-Cell/antagonists & inhibitors , Receptors, Antigen, T-Cell/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction , Small Molecule Libraries/chemistry , ZAP-70 Protein-Tyrosine Kinase/antagonists & inhibitors , ZAP-70 Protein-Tyrosine Kinase/immunology
7.
Biochem J ; 465(1): 149-61, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25287889

ABSTRACT

Zeta-chain associated protein of 70 kDa (ZAP-70) and spleen tyrosine kinase (Syk) are non-receptor tyrosine kinases that are essential for T-cell and B-cell antigen receptor signalling respectively. They are recruited, via their tandem-SH2 (Src-homology domain 2) domains, to doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) on invariant chains of immune antigen receptors. Because of their critical roles in immune signalling, ZAP-70 and Syk are targets for the development of drugs for autoimmune diseases. We show that three thiol-reactive small molecules can prevent the tandem-SH2 domains of ZAP-70 and Syk from binding to phosphorylated ITAMs. We identify a specific cysteine residue in the phosphotyrosine-binding pocket of each protein (Cys39 in ZAP-70, Cys206 in Syk) that is necessary for inhibition by two of these compounds. We also find that ITAM binding to ZAP-70 and Syk is sensitive to the presence of H2O2 and these two cysteine residues are also necessary for inhibition by H2O2. Our findings suggest a mechanism by which the reactive oxygen species generated during responses to antigen could attenuate signalling through these kinases and may also inform the development of ZAP-70 and Syk inhibitors that bind covalently to their SH2 domains.


Subject(s)
Cysteine/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Phosphopeptides/metabolism , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , ZAP-70 Protein-Tyrosine Kinase/chemistry , ZAP-70 Protein-Tyrosine Kinase/metabolism , src Homology Domains , Amino Acid Motifs , Binding Sites , Humans , Hydrogen Peroxide/pharmacology , Models, Molecular , Oxidation-Reduction/drug effects , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Protein Binding/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Sulfhydryl Compounds/metabolism , Syk Kinase , ZAP-70 Protein-Tyrosine Kinase/antagonists & inhibitors
8.
BMC Struct Biol ; 9: 7, 2009 Feb 24.
Article in English | MEDLINE | ID: mdl-19236722

ABSTRACT

BACKGROUND: Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line. RESULTS: We examined the inhibition of NQO2 activity by the Abl kinase inhibitors imatinib, nilotinib, and dasatinib, and obtained IC50 values of 80 nM, 380 nM, and >100 microM, respectively. Using electronic absorption spectroscopy, we show that imatinib binding results in a perturbation of the protein environment around the flavin prosthetic group in NQO2. We have determined the crystal structure of the complex of imatinib with human NQO2 at 1.75 A resolution, which reveals that imatinib binds in the enzyme active site, adjacent to the flavin isoalloxazine ring. We find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function. CONCLUSION: The structure of the imatinib-NQO2 complex demonstrates that imatinib inhibits NQO2 activity by competing with substrate for the active site. The overall conformation of imatinib when bound to NQO2 resembles the folded conformation observed in some kinase complexes. Interactions made by imatinib with residues at the rim of the active site provide an explanation for the binding selectivity of NQO2 for imatinib, nilotinib, and dasatinib. These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds. Taken together, these studies provide insight into the mechanism of NQO2 inhibition by imatinib, with potential implications for drug design and treatment of chronic myelogenous leukemia in patients.


Subject(s)
Antineoplastic Agents/metabolism , Piperazines/metabolism , Protein Kinase Inhibitors/metabolism , Pyrimidines/metabolism , Quinone Reductases/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Benzamides , Crystallography, X-Ray , Dasatinib , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Molecular Structure , NAD(P)H Dehydrogenase (Quinone)/chemistry , NAD(P)H Dehydrogenase (Quinone)/metabolism , Phosphorylation , Piperazines/chemistry , Piperazines/therapeutic use , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Quinone Reductases/antagonists & inhibitors , Quinone Reductases/chemistry , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/metabolism , Thiazoles/therapeutic use
9.
BMC Struct Biol ; 8: 42, 2008 Oct 07.
Article in English | MEDLINE | ID: mdl-18842118

ABSTRACT

BACKGROUND: Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases. RESULTS: We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 A resolution, and show that it is a dimeric molecule. CONCLUSION: Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of approximately 1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.


Subject(s)
Catalytic Domain , Guanylate Cyclase/chemistry , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Animals , Base Sequence , Chlamydomonas reinhardtii/chemistry , Crystallography, X-Ray , Dimerization , Enzyme Activation , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment
10.
J Biol Chem ; 282(2): 897-907, 2007 Jan 12.
Article in English | MEDLINE | ID: mdl-17098738

ABSTRACT

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.


Subject(s)
Guanylate Cyclase/chemistry , Guanylate Cyclase/metabolism , Heme/metabolism , Nitric Oxide/metabolism , Oxygen/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Binding Sites/physiology , Cells, Cultured , Enzyme Activation/physiology , Kinetics , Models, Chemical , Protein Structure, Tertiary , Rats , Soluble Guanylyl Cyclase , Spodoptera , Temperature
11.
Trends Biochem Sci ; 31(4): 231-9, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16530415

ABSTRACT

Nitric oxide (NO) triggers various physiological responses in numerous tissues by binding and activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. In vivo, basal NO/cGMP signaling maintains a resting state in target cells (for example, resting tone in smooth muscle), but an acute burst of NO/cGMP signaling triggers rapid responses (such as smooth muscle relaxation). Recent studies have shown that the sGC heterodimer comprises at least four modular domains per subunit. The N-terminal heme domain is a member of the H-NOX family of domains that bind O(2) and/or NO and are conserved in prokaryotes and higher eukaryotes. Studies of these domains have uncovered the molecular basis for ligand discrimination by sGC. Other work has identified two temporally distinct states of sGC activation by NO: formation of a stable NO-heme complex results in a low-activity species, and additional NO produces a transient fully active enzyme. Nucleotides also allosterically modulate the duration and intensity of enzyme activity. Together, these studies suggest a biochemical basis for the two distinct types of NO/cGMP signal observed in vivo.


Subject(s)
Guanylate Cyclase/metabolism , Nitric Oxide/metabolism , Signal Transduction , Animals , Cyclic GMP/metabolism , Heme/metabolism , Humans , Models, Chemical , Nitric Oxide Synthase/metabolism
12.
Proc Natl Acad Sci U S A ; 102(37): 13064-9, 2005 Sep 13.
Article in English | MEDLINE | ID: mdl-16131543

ABSTRACT

Nitric oxide (NO) affects many physiological systems by activating cGMP signaling cascades through soluble guanylate cyclase (sGC). In the accepted model, NO binds to the sGC heme, activating the enzyme. Here, we report that in the presence of physiological concentrations of ATP and GTP, NO dissociation from the sGC heme is approximately 160 times slower than the rate of enzyme deactivation in vitro. Deactivated sGC still has NO bound to the heme, and full activation requires additional NO. We propose an activation model where, in the presence of both ATP and GTP, tonic NO forms a stable heme complex with low sGC activity; acute production of NO transiently and fully activates this NO-bound sGC.


Subject(s)
Guanylate Cyclase/metabolism , Nitric Oxide/metabolism , Signal Transduction , Adenosine Triphosphate , Animals , Enzyme Activation , Guanosine Triphosphate , Heme/metabolism , Kinetics , Models, Chemical , Nitric Oxide/physiology , Rats , Solubility
13.
Biochemistry ; 44(10): 4083-90, 2005 Mar 15.
Article in English | MEDLINE | ID: mdl-15751985

ABSTRACT

The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.


Subject(s)
Catalytic Domain , Guanylate Cyclase/chemistry , Heme/metabolism , Protein Subunits/chemistry , Animals , Catalytic Domain/genetics , Dimerization , Enzyme Activation/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Guanosine Triphosphate/metabolism , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/genetics , Guanylate Cyclase/isolation & purification , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Protein Subunits/isolation & purification , Rats , Solubility , Substrate Specificity/genetics
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