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)

In vertebrate photoreceptors, light induces hydrolysis of cGMP by activating cGMP phosphodiesterase (PDE), which results in closure of the cGMP-activated cation channel. During light adaptation, the cytoplasmic Ca2+ concentration decreases, and this decrease is one of the underlying mechanisms of light adaptation. Sensitivity-modulating protein (S-modulin) is a Ca(2+)-binding protein involved in light adaptation in frog rods; it regulates both the light sensitivity of PDE and the lifetime of activated PDE by controlling rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin has been reported as a Ca(2+)-dependent regulator of guanylate cyclase in bovine rods (Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A. Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Here, we show that recoverin has similar activity as S-modulin, and the amino acid sequences of both proteins are similar. The results strongly suggest that recoverin is bovine S-modulin and regulates PDE activation.
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PMID:Recoverin has S-modulin activity in frog rods. 839 55

A simple protocol was developed to isolate the integral membrane guanylate cyclase from bleached bovine photoreceptor outer segments. Hypotonic and hypertonic washes strip the photoreceptor outer segment membranes of peripheral proteins. The guanylate cyclase activity is solubilized by dodecyl-b-D-maltoside in a low salt concentration buffer. Phosphatidylcholine, glycerol, and dithiothreitol are used to stabilize the activity during chromatography. GTP-affinity chromatography achieves a 250-fold increase in specific activity over that of membranes stripped of peripheral proteins. From 100 retinas, the protocol yields 100-140 mg of purified guanylate cyclase composed of a 115-kDa subunit. The molar ratio of the guanylate cyclase to rhodopsin is estimated to be 1:440. A significant portion of the freshly solubilized enzyme behaves as a monomer with a Stokes radius of 48.7 A, whereas the purified protein forms homooligomers ranging from dimers to tetramers. These properties are similar to those of ANP and guanylin receptors, indicating that the photoreceptor protein shares characteristics of the membrane receptor guanylate cyclase family. For the physiological substrate MgGTP, the Km and Vmax are 1.07 +/- 0.20 mM and 3262 +/- 514 nmol cGMP min-1 mg-1, respectively, generating a turnover rate of approximately 3.9 nmol cGMP s-1 at physiological substrate concentrations. The relatively high Km suggests that in vivo changes in GTP concentration might modulate the rate of cGMP synthesis. These properties indicate that the photoreceptor membrane guanylate cyclase can sustain a rate of cGMP synthesis comparable to the dark-adapted (basal) rate of cGMP degradation by the cGMP phosphodiesterase.
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PMID:The bovine photoreceptor outer segment guanylate cyclase: purification, kinetic properties, and molecular size. 852 37

Neurocalcins belong to a family of neuronal specific EF hand Ca2+-binding proteins defined by recoverin. Previously, we reported the cloning and initial characterization of neurocalcin in Drosophila melanogaster (Teng, D. H.-F., Chen, C.-K., and Hurley, J. B. (1994) J. Biol. Chem. 269, 31900-31907). We showed that the Drosophila neurocalcin protein (DrosNCa) is expressed in neurons and that bacterially expressed recombinant DrosNCa (rDrosNCa) can be myristoylated. Here, we present two lines of evidence that DrosNCa is fatty acylated in vivo. First, the mobility of affinity-purified native DrosNCa on two-dimensional gel electrophoresis is identical to that of myristoylated rDrosNCa and distinct from that of nonacylated rDrosNCa. Second, the membrane binding properties of native DrosNCa are similar to those of myristoylated rDrosNCa; both of these proteins bind to membranes at 0.2 mM Ca2+, whereas nonacylated rDrosNCa always remains soluble. It has been shown that recoverin inhibits the phosphorylation of rhodopsin when Ca2+ is present (Kawamura et al., 1993) and that a dependent recoverin/rhodopsin kinase interaction underlies the inhibitory effect of recoverin (Chen et al., 1995). Given the similarities between recoverin and neurocalcin, we examined the effect of DrosNCa on rhodopsin phosphorylation. We find that rDrosNCa is capable of inhibiting bovine rhodopsin phosphorylation in vitro in a Ca2+-dependent manner. The inhibitory activity of rDrosNCa is enhanced by myristoylation, and the potency of its effect is similar to that of recoverin. Two other related EF hand proteins, guanylate cyclase-activating protein-2 and calmodulin, are only poor inhibitors in these phosphorylation assays. in vitro inhibition of rhodopsin phosphorylation therefore appears to be an assayable property of a subset of recoverin-like proteins.
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PMID:Drosophila neurocalcin, a fatty acylated, Ca2+-binding protein that associates with membranes and inhibits in vitro phosphorylation of bovine rhodopsin. 862 92

A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S-modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s.
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PMID:The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i. 874 28

1. Using Fura-2, we measured cytosolic free calcium concentrations in rod outer segments of the bullfrog (Rana catesbeiana) under a wide range of steady-state, adapting light intensities. We also measured rod circulating currents under the same adapting conditions. 2. Both the steady-state cytosolic free calcium concentration and the steady-state circulating current were halved by background lights that isomerized approximately 100 rhodopsin molecules per rod per second. A just-measurable reduction in calcium was evoked by a background 1/10 as bright. The steady-state calcium concentration and the steady-state circulating current were proportional to each other over the full range of intensities tested, the brightest of which suppresses the circulating current by 75% and reduces flash sensitivity by about two orders of magnitude relative to the rods' sensitivity in darkness. 3. Additional experiments, in which the Na+:Ca2+,K+ exchanger in the rod outer segment was inactivated while maintaining intracellular calcium at physiological levels, demonstrated that steady-state calcium concentrations in the outer segment are set only by the influx of calcium through the light-sensitive channels and its efflux via the Na+:Ca2+,K+ exchanger. We found no measurable light-induced release of calcium from internal stores and no evidence of any other calcium flux between the cytosol and intracellular compartments. 4. Taken together, these findings lead us to conclude that over the normal operating range of the rod, the selectivity of the cGMP-gated channels for calcium does not change as a result of background illumination. 5. Our data also suggest that a major role of calcium-dependent cGMP synthesis by guanylyl cyclase is to stabilize both the circulating current and the cytosolic free calcium concentration in darkness. This would minimize the dark noise of the rod and thereby increase the reliability with which dim stimuli can be detected in the dark-adapted state.
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PMID:Light-dependent control of calcium in intact rods of the bullfrog Rana catesbeiana. 882 63

Guanylate cyclase is sensitive to changes of light and dark periods in incubated extracts obtained from soluble fractions of the retina, optic nerve and optic chiasm. The changes in guanylate cyclase activity found, about 100-fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibits strongly the retinal guanylate cyclase activity, while it increases the activity of this enzyme in the optic nerve. A generalized photo-inhibited response of guanylate cyclase was observed in all studied tissues in animals adapted to the dark. This suggests that light could act as a double stimulus gating the central circuit which promotes the hydrolysis of cGMP via cGMP phosphodiesterase-rhodopsin-transducin cascade, and by direct inhibition of the retinal guanylate cyclase activity. Finally, different responses have been observed in the guanylate cyclase activity in relation with the ion exposure depending on the studied tissue. In summary, all indicate an important role for the soluble guanylate cyclase activity in retina, and other tissues involved in the visual process such as optic nerve and optic chiasm, which have not been examined until now.
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PMID:Effect of different illumination conditions and ionic environment on the guanylate cyclase activity in retina, optic nerve and optic chiasm of the rat. 932 37

Biochemical experiments by others have indicated that protein kinase C activity is present in the rod outer segment, with potential or demonstrated targets including rhodopsin, transducin, cGMP-phosphodiesterase (PDE), guanylate cyclase, and arrestin, all of which are components of the phototransduction cascade. In particular, PKC phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE ) have been studied in some detail, and suggested to have roles in downregulating the sensitivity of rod photoreceptors to light during illumination. We have examined this question under physiological conditions by recording from a single, dissociated salamander rod with a suction pipette while exposing its outer segment to the PKC activators phorbol-12-myristate,13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu), or to the PKC-inhibitor GF109203X. No significant effect of any of these agents on rod sensitivity was detected, whether in the absence or presence of a background light, or after a low bleach. These results suggest that PKC probably does not produce any acute downregulation of rod sensitivity as a mechanism of light adaptation, at least for isolated amphibian rods.
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PMID:Protein kinase C activity and light sensitivity of single amphibian rods. 937 74

Leber's congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies responsible for congenital blindness. Genetic heterogeneity of LCA has been suspected since the report by Waardenburg of normal children born to affected parents. In 1995, we localized the first disease causing gene, LCA1, to chromosome 17p13 and confirmed the genetic heterogeneity. In 1996, we ascribed LCA1 to mutations in the photoreceptor-specific guanylate cyclase gene (retGC1). RetGC1 is an essential protein implicated in the phototransduction cascade, especially in the recovery of the dark state after the excitation process of photoreceptor cells by light stimulation. In 1997, mutations in a second gene were reported in LCA, the RPE65 gene, which is the first specific retinal pigment epithelium gene. The protein RPE65 is implicated in the metabolism of vitamin A, the precursor of the photoexcitable retinal pigment (rhodopsin). Finally, a third gene, CRX, implicated in photoreceptor development, has been suspected of causing a few cases of LCA. Taken together, these three genes account for only 27% of LCA cases in our series. The three genes encode proteins that are involved in completely different physiopathologic pathways. Based on these striking differences of physiopathologic processes, we reexamined all clinical physiopathological discrepancies and the results strongly suggested that retGC1 gene mutations are responsible for congenital stationary severe cone-rod dystrophy, while RPE65 gene mutations are responsible for congenital severe but progressive rod-cone dystrophy. It is of tremendous importance to confirm and to refine these genotype-phenotype correlations on a large scale in order to anticipate the final outcome in a blind infant, on the one hand, and to further guide genetic studies in older patients on the other hand.
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PMID:Leber congenital amaurosis. 1052 70

When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.
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PMID:Adaptation in vertebrate photoreceptors. 1115 56

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.
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PMID:Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors. 1188 95


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