Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.
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PMID:Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity. 135 9

Type A atrial natriuretic peptide (ANP) receptor was demonstrated to be present as a tetramer in the bovine adrenal cortex. Type A ANP receptor is composed of two functional domains, namely extracellular ANP-binding and cytoplasmic guanylate cyclase domains, and generally considered to be present as a single polypeptide chain of about 140 kDa based on its primary structure deduced from the cDNA sequence and its SDS/PAGE profile under reducing conditions. Characterization of the type A receptor or receptor/cyclase under non-reducing conditions led to the discovery stated in the title. The type A ANP receptor was partially purified from bovine adrenal cortex membranes by Blue-Sepharose and GTP-agarose chromatography. SDS-PAGE analysis of the receptor preparation revealed that although under reducing conditions it migrated as a 140-kDa band, the mobility of the receptor was greatly retarded in the absence of reducing agents, suggesting that the type A ANP receptor is present as a disulfide-linked oligomer in its native state. Further analysis using SDS-polyacrylamide-agarose gels suitable for determining the sizes of high-molecular-weight proteins revealed that the oligomer has an Mr of 500,000-550,000. This result clearly indicates that the native form of the type A receptor is a tetramer composed of four 140-kDa disulfide-linked receptor/cyclase molecules.
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PMID:Bifunctional atrial natriuretic peptide receptor (type A) exists as a disulfide-linked tetramer in plasma membranes of bovine adrenal cortex. 165

Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors.
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PMID:Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors. 167 87

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.
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PMID:Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase. 170 96

Some evidence suggests homologous and heterologous regulation of atrial natriuretic peptide (ANP) receptors. We have examined the effects of exposure to ANP, angiotensin II (ANG II), arginine vasopressin (AVP), and endothelin (ET), on binding of ANP to cultured rat vascular smooth muscle cells (VSMC) and on guanylate cyclase-coupled and -uncoupled ANP receptors. The latter were studied by examining production of guanosine 3',5'-cyclic monophosphate (cGMP) in response to ANP and binding of ANP to Triton X-100-solubilized VSMC membranes, irreversible cross-linking with disuccinimidyl suberate, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in presence of dithiothreitol, followed by radioautography. Exposure to 100 nmol/l ANP for 18 h reduced ANP sites to 43% of control. However, if acid wash of VSMC membranes at pH 5.0 was performed before binding, no decrease in density of ANP binding sites was detected. On SDS-PAGE, a 130-kDa band bound 42 vs. 46% of 125I-labeled ANP in acid-washed membranes from control vs. cells exposed to ANP; the remainder was bound to a 67-kDa band. ANG II (100 nmol/l), AVP (1 mumol/l), or ET-1 or ET-3 (100 nmol/l) did not produce changes in density of ANP sites or in binding to the 130- and 67-kDa bands. cGMP production in response to ANP showed exaggerated response in ANG II but not in AVP- or ET-treated VSMC. Effect of ANG II was abolished by the ANG II antagonist [Sar1-Ile8]ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of ANP, angiotensin, vasopressin, and endothelin on ANP receptors in cultured rat vascular smooth muscle cells. 184 18

Analysis of [125I]-ANP binding data in an isolated bovine ventricular sarcolemmal membrane fraction revealed a single high affinity binding site (Kd approximately 5 x 10(-11) M). The ring deleted ANP analogue des [QSGLG]-ANP (4-23)-NH2 bound with a 1000-fold lower affinity indicating the absence of C-type receptors in this preparation. ANP stimulated guanylate cyclase activity by up to 2-fold with half-maximal activation at approximately 10(-9) M. Crosslinking [125I]-ANP to its receptor with disuccinimidyl suberate (DSS) revealed two radiolabelled bands of 120 kDa and 65 kDa on non-denaturing SDS-PAGE. Radioactive signals from both bands were lost by reducing the sample with beta-mercaptoethanol prior to electrophoresis, in which case a radioactive fragment of less than 5 kDa migrated with the dye front. These results suggest that the binding of ANP to both high and low molecular weight "receptor" proteins may be associated with the hydrolysis of the peptide.
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PMID:Characterisation of atrial natriuretic peptide receptors in bovine ventricular sarcolemma. 196 33

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.
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PMID:Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method. 197 95

Cultured human skin fibroblasts possessed the high-affinity and low-capacity binding sites for [125I]alpha-human atrial natriuretic peptide (hANP), in which the dissociation constant and maximal binding capacity were computed to 68.7 +/- 11.3 pM and 7.3 +/- 1.2 fmols/mg protein, respectively, from Scatchard plot analysis. The specific [125I] alpha-hANP binding sites of cultured human fibroblast were displaced by unlabeled atriopeptin I, a truncated analogue, to the same extent as the case of alpha-hANP. In human adrenal membrane fractions, [125I] alpha-hANP binding sites were suppressed only by unlabeled alpha-hANP, while the high concentrations of atriopeptin I could slightly inhibit the binding sites for alpha-hANP. As it was reported that atriopeptin I had more significant affinity to the low-molecular weight ANP receptor (60-70 KD) than that to the high-molecular weight form (130-140 KD), the specific bindings may be attributed by the low-molecular weight ANP receptor in cultured human fibroblasts. Furthermore, alpha-hANP up to 10(-8)M failed to induce the significant cGMP formation in cultured human skin fibroblasts. The molecular weight of [125I]alpha-hANP binding sites of human fibroblasts was identified only at the region of 67 KD and no radioactive band was visualized around the region of large molecular weight ANP receptor in the SDS gel electrophoresis of a crosslinked [125I]alpha-hANP-receptor complex. In contrast, the affinity labeling of [125I]alpha-hANP to the human adrenal membrane fractions showed that 135 KD binding sites were responsible to the human adrenal ANP receptor. In conclusion, cultured human skin fibroblasts have a high-affinity low-capacity receptor for ANP. The molecular weight of ANP receptor is approximately 67 KD, and ANP-specific guanylate cyclase may not be linked to the receptor, suggestive that so-called C receptor may be localized in cultured human fibroblasts.
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PMID:[Structure and function of the receptor for human atrial natriuretic peptide in cultured human skin fibroblasts]. 217 31

1. Receptors for atrial natriuretic peptide (ANP) have been identified on mouse astrocytes in primary culture, and have similar characteristics to those found on previously recognized ANP-target tissues. 2. Scatchard analysis revealed one class of high affinity receptors with a Kd of 0.32 nmol/L. The IC50 for specific binding was 0.5 nmol/L. 3. Ligand binding resulted in stimulation of guanylate cyclase. 4. Under reducing conditions, the covalently cross-linked receptor-ANP complex migrated on SDS-polyacrylamide gels as a single band with an apparent molecular weight of 66 kDa. 5. Although the physiological relevance of our observations remains to be determined, these data document that cultured mouse astrocytes contain specific high-affinity ANP receptors which are linked to the production of cGMP.
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PMID:Mouse astrocytes possess specific ANP receptors which are linked to cGMP production. 254 96

Atrial natriuretic factor (ANF) specifically stimulated the endogenous phosphorylation of a protein band in an isolated membrane fraction of human placenta. The apparent molecular weight of the substrate protein as determined by SDS-polyacrylamide gel electrophoresis is 160-170,000. In the same membrane fraction, ANF also stimulated guanylate cyclase activity in a dose-dependent manner. Guanosine 3':5'-cyclic monophosphate (cyclic GMP), added to the membrane fraction in lieu of ANF, also stimulated the phosphorylation of several protein bands, one of which have the same apparent molecular weight as the one stimulated by ANF. In contrast, adenosine 3':5'-cyclic monophosphate (cyclic AMP) at a similar concentration and hormones such as angiotensin II, insulin and vasopressin had no effect on the phosphorylation state of this protein band. The finding that ANF alters the phosphorylation state of a certain membrane protein and that this effect is mimicked by cyclic-GMP suggests that at least some of the biological action of ANF may be mediated by the phosphorylation of membrane protein involving a cyclic GMP-dependent protein kinase.
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PMID:Atrial natriuretic factor induced phosphorylation of human placental membrane protein: an effect mimicked by guanosine 3':5'-cyclic monophosphate. 287 3


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