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

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide, and on various other cells as a receptor for atrial natriuretic peptides. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphorylation. Unexpectedly, the enzyme is homologous to the protein kinase family.
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PMID:Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases. 290 Oct 39

The particulate form of guanylate cyclase from sea urchin spermatozoa was purified to apparent homogeneity by chromatography on GTP-Sepharose and DEAE-Sepharose and by preparative gel electrophoresis. The sedimentation coefficient (S20,w) was 6.8 and the Stokes radius was 5.1 nm, from which a native molecular weight of 157,000 was calculated. A single protein or periodic acid-Schiff staining band of 135,000 Da was observed after Na dodecyl SO4 gel electrophoresis. Antibody was produced to guanylate cyclase and was shown by electrophoretic transfer experiments (Western blot) to interact with only the Mr = 135,000 band in cases where all of the detergent-extracted protein from spermatozoa was added to the Na dodecyl SO4 gels. Although guanylate cyclase was normally bound to concanavalin A-Sepharose, after endoglycosidase H treatment it failed to bind. Treatment of the enzyme with endoglycosidase H did not alter guanylate cyclase activity, but the apparent size of the enzyme decreased to 72,000 Da on Na dodecyl SO4 gels. An analysis of carbohydrate composition indicated that the oligosaccharides contained N-acetylglucosamine, mannose, galactose, and 2-aminoerythritol in molar ratios (1:3:0.75:2); after endoglycosidase H treatment the enzyme contained essentially no carbohydrate. Major amino acids in the enzyme were aspartic (Asn) and glutamic (Gln) which accounted for approximately 25 mol % of the enzyme amino acid composition. The purified enzyme displayed linear kinetics on double reciprocal plots and had a KMnGTP = 133 microM, KM2+ = 138 microM, KiMnGTP = 122 microM, KiMn2+ = 127 microM, and a V max in excess of 15 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C. Sodium nitroprusside did not stimulate the enzyme in either the presence or absence of added hemeproteins. These results indicate that the particulate form of guanylate cyclase from sea urchin spermatozoa is a glycoprotein which is distinctly different than the soluble form of the enzyme found in mammalian tissues.
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PMID:Purification and characterization of particulate guanylate cyclase from sea urchin spermatozoa. 613 28

A peptide (resact) associated with the eggs of the sea urchin, Arbacia punctulata, which stimulates sperm respiration rates by 5-10-fold, was purified and its amino acid sequence was determined. The sequence was found to be Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2. The peptide was subsequently synthesized by solid phase methods, amidated at the carboxyl-terminal Leu, and shown to be identical to the isolated, native material. The peptide half-maximally stimulated A. punctulata spermatozoan respiration at 0.5 nM and half-maximally elevated cyclic GMP concentrations at 25 nM at an extracellular pH of 6.6. The increase in oxygen consumption was coupled with a stimulation of motility. However, at elevated extracellular pH (pH 8.0), resact failed to appreciably stimulate respiration while the elevations of cyclic GMP continued to occur. Resact did not cross-react with sperm cells obtained from Lytechinus pictus or Strongylocentrotus purpuratus; a peptide (speract) obtained from S. purpuratus eggs (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) which activates S. purpuratus sperm respiration did not stimulate A. punctulata spermatozoa. Resact caused a shift in the apparent molecular weight (160,000-150,000) of a major sperm plasma membrane protein; as with cyclic GMP elevations, this response was evident at extracellular pH values of both 6.6 and 8.0. The protein exists in the cell as a phosphoprotein and 32P is released coincident with the molecular weight change. Approximately 115 nM resact caused one-half-maximal conversion of the 160,000-dalton protein after 1 min of incubation. Resact caused the apparent molecular weight conversion of the protein within 5 s and appeared to do so in an irreversible manner. The molecular weight change of the protein was also observed after the addition of monensin A (25 microM) and NH4Cl (40 mM), two agents known to elevate intracellular pH and to increase sperm respiration rates. The membrane protein appears to be the enzyme guanylate cyclase, but since concentrations of resact causing one-half-maximal conversion of the Mr = 160,000 form of the enzyme are about 250 times higher than those causing one-half-maximal stimulation of respiration, the relationship of the apparent molecular weight conversion to a subsequent physiological event remains unclear.
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PMID:A peptide associated with eggs causes a mobility shift in a major plasma membrane protein of spermatozoa. 615 45

Cycl AMP concentrations were elevated and acrosome reactions were induced in intact sea urchin spermatozoa by Nigericin, A23187, and pH 9.0 seawater. To determine whether or not the metabolism of cyclic AMP was being altered in sperm heads, the heads were mechanically separated from the flagella, and the flagella-less heads were then isolated by differential centrifugation. The isolated heads contained 1 to 2 nmol of ATP and 1 to 2 pmol of cyclic AMP/mg wet weight and retained these concentrations for several hours if stored at 0 degrees C. The flagella-less heads also retained the mitochondria of the midpiece area. The heads retained their functional status and could be stimulated to undergo acrosome reactions (filament extension) in response to Nigericin, A23187, or pH 9.0 seawater. Furthermore, the isolated heads could activate sea urchin eggs after induction of an acrosome reaction by Nigericin or pH 9.0 seawater. The isolated heads contained appreciable adenylate cyclase, cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase, guanylate cyclase, cyclic AMP-dependent protein kinase, and calmodulin. Nigericin, pH 9.0 seawater, and A23187 caused not only the induction of an acrosome reaction but also elevations of cyclic AMP in the isolated heads, and extracellular Ca2+ was an absolute requirement for both responses. At 16 degrees C, Nigericin caused elevations of cyclic AMP within 5 s, but maximal elevations were not observed until 1 min; it induced a maximal percentage of acrosome reactions by 40 s. Incubation of cells at 0 degrees C resulted in a delay of maximal acrosome reactions until between 10 and 20 min after addition of Nigericin. Under these conditions, maximal elevations of cyclic AMP were observed by 5 min, demonstrating that cyclic AMP elevations precede the complete morphological change associated with an acrosome reaction. ATP concentrations within the sperm heads declined in response to Nigericin, pH 9.0 seawater, or A23187, and its decrease also required the presence of extracellular Ca2+. The decline in ATP concentrations was slightly more rapid in the presence of rotenone, suggestive of some ATP synthetic capabilities of the isolated head preparation. 45Ca2+ uptake was increased by Nigericin elevated pH, and A23187 but was not appreciably altered by monensin. Monensin also did not cause appreciable elevations of cyclic AMP concentrations, induction of an acrosome reaction, or decreases of ATP concentrations. Here, we describe for the first time that cyclic AMP concentrations can be increased in flagella-less heads of spermatozoa and show that these changes are associated with an acrosome reaction.
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PMID:The elevation of cyclic AMP concentrations in flagella-less sea urchin sperm heads. 625 63

The induction of acrosomal exocytosis in capacitated bull spermatozoa by atrial natriuretic peptide (ANP) was studied in vitro. ANP markedly stimulated acrosomal exocytosis in a calcium-dependent manner. Typically, ANP exerts its action via activation of the ANP receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and subsequent formation of guanosine 3',5'-cyclic monophosphate (cGMP). We found that the ANP-induced acrosome reaction was inhibited by the competitive ANPR-A receptor antagonist-anantin, indicating a receptor-mediated effect. We could mimic the effect of ANP on the acrosome reaction by using 8-bromo-cGMP, suggesting that cGMP may serve as a signal transducer mediating the acrosome reaction. Indeed, the ANP-induced acrosome reaction was associated with elevation of cGMP levels. cGMP can also be formed by activation of the soluble form of guanylyl cyclase. Sodium nitroprusside (SNP) stimulated cGMP accumulation and acrosome reaction of capacitated spermatozoa. Thus ANP and the nitric oxide-releasing compound SNP, via activation of guanylyl cyclase (the former activating the particulate and the latter activating the soluble form of the enzyme), may play a significant role in the induction of the acrosome reaction.
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PMID:Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa. 765 38

Nitric oxide (NO) has recently been found to function as an intra and extra cellular messenger by activating guanylate cyclase. Its role in sperm hyperactivation was examined by adding to the capacitating medium a classical donor of NO (Sodium nitroprusside, NP) in two different concentrations (150 microM and 300 microM). In both treatments, the percentage of motile cells was evaluated, showing a significant decrease on motility and viability at 90 and 120 minutes when sodium nitroprusside was used in a concentration of 300 microM; no modifications were observed with 150 microM. The effect obtained with 300 microM of sodium nitroprusside was avoided by hemoglobin (20 micrograms/ml), a scavenger of the NO. The percentage of hyperactivated spermatozoa in the presence of 300 microM sodium nitroprusside increased significantly more than the control during the first 30 and 60 minutes of capacitation; but with 150 microM sodium nitroprusside a significant increase was observed at 60 and 90 minutes of incubation. Thus, the data strongly suggests that nitric oxide plays an important role in sperm hyperactivation "in vitro".
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PMID:Effect of nitric oxide on mouse sperm hyperactivation. 766 15

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.
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PMID:Purification and partial characterization of acrosome reaction inhibiting glycoprotein from human seminal plasma. 766 49

Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as egg-derived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half-maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5-27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Atrial natriuretic peptide: a chemoattractant of human spermatozoa by a guanylate cyclase-dependent pathway. 777 48

A decapeptide (GFDLNGGGVG) isolated from the solubilized jelly layer of the sea urchin Hemicentrotus pulcherrimus stimulates the respiration and motility of H. pulcherrimus spermatozoa and, in addition, produces a number of biological effects on H. pulcherrimus spermatozoa including increases in cAMP and cGMP levels, activation of a Na+/H+ exchange system, and increases in intracellular pH (pHi) and [Ca2+] ([Ca2+]i). The peptide activates the metabolism of endogenous phosphatidylcholine and promotes the acrosome reaction as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate. The peptide also induces an electrophoretic mobility change in the guanylate cyclase of the sperm plasma membrane with concomitant dephosphorylation and inactivation of the enzyme. Seventy-four peptides producing similar biological effects, named sperm-activating peptide (SAP), have since been purified from the solubilized jelly layer of seventeen species of sea urchins distributed over five taxonomic orders. These peptides show essentially the same biological effects on sea urchin spermatozoa although their activity and structures are specific at the ordinal level. Equilibrium binding experiments using a radioiodinated SAP-I analogue [GGGY(125I)GFDLNGGGVG] to H. pulcherrimus spermatozoa suggests the presence of two classes of receptors (high affinity and low affinity) specific for SAP-I binding. Based on the Kd values and EC50's for SAP-I's biological activity, we presume that the high affinity receptor is associated with respiration-stimulating activity and elevations in pHi, while the low affinity receptor is coupled to elevations in cGMP and [Ca2+]i. The radioiodinated SAP-I analogue crosslinks to a 71 kDa protein which contains a single membrane-spanning domain at almost near C-terminus. A SAP-I precursor which is synthesized in the accessory cells contains five SAP-I and seven SAP-I-like decapeptides, each separated by a single lysine residue.
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PMID:Structure, function and biosynthesis of sperm-activating peptides and fucose sulfate glycoconjugate in the extracellular coat of sea urchin eggs. 779 87


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