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)

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.
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PMID:Purification and properties of the phosphorylated form of guanylate cyclase. 289 12

Atrial natriuretic peptide (ANP) contains a disulfide which is generally considered to be required for biological activity. A truncated linear ANP analog, des-Cys105,Cys121-ANP-(104-126) (referred to as analog I), that lacks the 2 cysteine residues of the parent peptide was synthesized. In competition binding studies using rabbit lung membranes, ANP-(103-126) and analog I displaced bound 125I-ANP-(103-126) from specific ANP binding sites 100 and 73%, respectively. The concentrations of ANP-(103-126) and analog I that produced 50% inhibition of radioligand binding to the membranes were 0.26 +/- 0.07 and 0.31 +/- 0.09 nM, respectively. Radioiodinated ANP-(103-126) and analog I were chemically cross-linked to binding sites on rabbit lung membranes, and the labeled membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. 125I-Analog I specifically labeled a 65,000-dalton protein and a 135,000-dalton protein which, under reducing conditions, dissociated into 65,000-dalton subunits. In contrast, 125I-ANP-(103-126) labeled specifically a nonreducible 135,000-dalton protein, in addition to the 65,000-dalton species and the reducible 135,000-dalton species. ANP-(103-126) (100 nM) stimulated rabbit lung particulate guanylate cyclase activity, whereas analog I, at the same concentration, had no effect on cyclic GMP production and did not antagonize the effect of ANP-(103-126). From these observations, we conclude that analog I is a selective ligand which binds to approximately 73% of the total ANP binding sites present in rabbit lung membranes. Unlike ANP-(103-126), analog I does not bind to the remaining 27% of the binding sites and does not activate guanylate cyclase. Binding to the cyclase-linked ANP receptor correlates with the specific labeling by 125I-ANP-(103-126) of the nonreducible 135,000-dalton membrane protein.
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PMID:A linear analog of atrial natriuretic peptide (ANP) discriminates guanylate cyclase-coupled ANP receptors from non-coupled receptors. 289 82

The receptor for the heat-stable enterotoxin (ST) from Escherichia coli was solubilized with Lubrol-PX from rat intestinal brush-border membranes and characterized. The binding kinetics and analog specificity of the solubilized receptor were virtually identical to those obtained with the membrane-bound receptor. Furthermore, the regulation of the receptor's affinity by cations was also maintained after solubilization, indicating a conservation of the toxin-binding site after removal of the receptor from its membrane environment. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 5.5 nm and a sedimentation coefficient of 7.0 S for the solubilized receptor. The isoelectric point of the receptor was determined as 5.5 using Sephadex isoelectric focusing electrophoresis. In all of these separation techniques, the ST receptor showed a single peak of activity that was clearly separated from that of guanylate cyclase. When 125I-ST was cross-linked to brush-border membranes with disuccinimidyl suberate, the affinity-labeled receptor solubilized with 0.1% Lubrol-PX eluted at a similar position as the native receptor on gel filtration chromatography. Analysis of the affinity-labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent and by autoradiography revealed the presence of three specifically labeled polypeptides with apparent molecular weights of 80,000, 68,000, and 60,000. These results suggest that the ST receptor is solubilized by Lubrol-PX in an active form with preservation of its regulation by cations. Also, the ST receptor is separable from particulate guanylate cyclase indicating that the receptor is coupled to the activation of guanylate cyclase by an as yet undefined mechanism. Three subunit peptides may constitute a binding region of the receptor.
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PMID:Characterization of the receptor for heat-stable enterotoxin from Escherichia coli in rat intestine. 394 95

Guanylate cyclase was purified from the soluble fraction of rat lung using a modification of procedures published previously. The purified enzyme exhibited specific activities, at pH 7.6, of 219-438 nmoles/mg protein/min and 34-60 nmoles/mg protein/min with Mn2+ and Mg2+ as cation cofactors, respectively. The specific activity changed as a function of the protein concentration due to a change in Vmax with no alteration of the Km for GTP. The enzyme migrated as a single band coincident wih guanylate cyclase activity on nondenaturing polyacrylamide and isoelectric focusing gels (isoelectric point = 5.9). Purified guanylate cyclase had an apparent molecular weight of 150,000 daltons as determined by gel filtration chromatography and polyacrylamide gel electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate revealed a single subunit of 72,000 daltons, suggesting that the enzyme is a dimer of an identical subunit. The purified enzyme could be activated by nitric oxide, indicating that this compound interacts directly with the enzyme.
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PMID:Purified guanylate cyclase: characterization, iodination and preparation of monoclonal antibodies. 611 Jun 82

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.
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PMID:Purification of a soluble, sodium-nitroprusside-stimulated guanylate cyclase from bovine lung. 611 59

The role of cyclic nucleotides was evaluated in the stimulation of cartilage metabolism by somatomedin-C (Sm-C). The effects of cAMP and cyclic guanosine monophosphate (cGMP) analogs on matrix synthesis were evaluated. The effects of Sm-C on tissue concentrations of these cyclic nucleotides were investigated. Likewise, the direct effects of Sm-C on the activities of cartilage adenylate cyclase, guanylate cyclase, and phosphodiesterase were determined. We found that tissue concentrations of cAMP in cartilage declined rapidly during organ culture, despite the presence of serum or Sm-C, cGMP concentrations in cartilage declined rapidly during control incubations but were augmented significantly at 30 and 60 min of incubation with the addition of serum or Sm-C. Thereafter, cGMP concentrations declined toward the levels of incubated control cartilages. Sm-C had no effect on phosphodiesterase activity. N6-Monobutyryl cAMP stimulated sulfate uptake, but dibutyryl cGMP did not. Sm-C did not stimulate adenylate cyclase in purified plasma membranes from chondrocytes, whereas it stimulated both plasma membrane and cytosol guanylate cyclase at concentrations of Sm-C as low as 10(-12) M. These data would indicate that cAMP is not the intracellular second messenger for Sm-C in cartilage. The data for cGMP are provocative and suggest it as a candidate for a second messenger mediating a portion of Sm's stimulation of cartilage metabolism.
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PMID:Cyclic nucleotides and somatomedin action in cartilage. 612 18

Guanylate cyclase activity was purified to apparent homogeneity from rat liver (7700-fold) and bovine lung (8600-fold) soluble fractions by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and isoelectric focussing. The purified enzymes did not contain heme and did not respond to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. By contrast, preformed NO-hemoglobin increased enzyme activity 10-12-fold or 60-80-fold when 4 mM MnCl2 or 4 mM MgCl2, respectively, were employed as the metal ion co-factor. Addition of hematin to the enzyme preparations restored responsiveness to NO, nitroprusside or NO-cysteine to levels seen with NO-hemoglobin. Partial purification of guanylate cyclase from the soluble fraction of bovine lung (2400-fold) by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and high pressure liquid chromatography (HPLC) resulted in a preparation which contained endogenous heme as indicated by absorbance at 436 nm and responded to NO, nitroprusside and NO-cysteine in the absence of added hematin. By contrast, guanylate cyclase purified from the hepatic supernatant by the identical procedure, did not contain detectable absorption due to heme and did not respond or responded poorly to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. Analogous to hepatic guanylate cyclase purified by isoelectric focussing, the HPLC purified hepatic enzyme was activated 14-fold by NO-hemoglobin in assays which contained 4 mM MnCl2 and 60-fold in assays with 4 mM MgCl2. Further, addition of hematin to the HPLC purified enzyme restored responsiveness to NO, nitroprusside and NO-cysteine to levels seen with NO-hemoglobin. These effects of hematin were specific for hematin and were not mimicked by albumin, sucrose or dithiothreitol. Moreover, the failure to observe stimulation of purified hepatic guanylate cyclase was not explained by a shift in the concentration response relationship between NO and guanylate cyclase activity. Several observations indicated that neither NO-thiol complexes nor [Fe(CN)5NO]-3 were the proximate moieties responsible for activation of guanylate cyclase by nitroprusside and related agents, as has been previously suggested. These results strongly support the proposal that activation of guanylate cyclase by NO and related agents specifically requires formation of an NO-heme complex.
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PMID:Requirement for heme in the activation of purified guanylate cyclase by nitric oxide. 613 53

Guanylate cyclase from 105,000 X g particulate fractions of rat liver homogenates (20 pmoles of cyclic GMP formed/min/mg protein) was solubilized in the absence of detergents by incubating fractions 12 min at 37 degrees with 5 ug/ml trypsin. Optimal solubilization was dependent upon trypsin and particulate preparation concentrations. Virtually no activation of particulate guanylate cyclase was observed at any time point or trypsin concentration tested. Guanylate cyclase solubilized with trypsin was purified about 500-fold (9.4 nmoles/min/mg protein) using ammonium sulfate precipitation, GTP-affinity chromatography, and preparative polyacrylamide gel electrophoresis. Activity eluted as a single peak on Sepadex G-200 (Stokes radius = 40 A) and migrated as a single peak on sucrose density gradients (S20,w = 4.6). Thus, the tryptic fragment was estimated to be about 80,000 daltons (Mr) with a frictional ratio (f/fo) of 1.4. These partially purified preparations exhibited linear double reciprocal plots with Mn-GTP and Hill coefficients of 1.0. This is in contrast to the crude membrane-associated enzyme which had a Hill co-efficient of 1.5. Membrane-bound and trypsin-solubilized guanylate cyclase were activated 3- and 4-fold with nitric oxide and were inhibited with 1mM cystamine. Cystamine inhibition could be partially reversed with 7.5 mM dithiothreitol. These studies indicate that particulate guanylate cyclase solubilized by limited proteolysis is amenable to purification by "classical" chromatographic techniques. The partially purified fragment contains the catalytic site, the site for nitric oxide activation, and at least one sulfhydryl group required for activity.
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PMID:Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin. 613 35

A guanylate cyclase of high specific activity was localized in the ciliary membrane from Tetrahymena pyriformis. Purity of cilia was checked by electron microscopy and purity of membrane fractions isolated by a sucrose density gradient by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Enzyme activity was due to the presence of endogenous calmodulin as evidenced by the inhibition of guanylate cyclase by addition of antiserum against calmodulin from Tetrahymena or soybean. Removal of endogenous calmodulin by La3+-treatment of ciliary membranes resulted in loss of guanylate cyclase activity. In addition to protozoan calmodulins, the original activity could also be restored by the nonhomologous calmodulins from soybean and pig brain but not by calcium-binding proteins like Dictyostelium calmodulin, parvalbumin, and troponin C, lacking the trimethyllysine characteristic for mammalian calmodulins. However, only calmodulins from the protozoans Tetrahymena and Paramecium stimulated guanylate cyclase activity in excess of the initial activity. This indicates that the guanylate cyclase either contains two binding sites for calmodulin with different specificities or that a single, but only partially occupied binding site is modified possibly by hydrolytic exo-proteases during membrane preparation. The ciliary membrane from Tetrahymena contains a discrete calcium-permeability as demonstrated by calcium-flux measurements using the calcium indicator dye arsenazo III. In analogy to the excitable ciliary membrane of the larger relative Paramecium, the ciliary membrane of Tetrahymena may thus carry the voltage-sensitive calcium-channels known from electrophysiological studies.
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PMID:Calcium/calmodulin-regulated guanylate cyclase and calcium-permeability in the ciliary membrane from Tetrahymena. 614 Jan 65

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.
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PMID:Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon. 614 53


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