Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The target proteins for arrestin (48 kDa protein) action during the quench of cGMP phosphodiesterase (PDE) activation in retinal rod disk membranes were identified by the use of a cross-linking reagent. A heterobifunctional, cleavable, photo-activatable cross-linker (sulfo-SADP) was coupled to purified arrestin. Under precise weak visible light bleach and nucleotide conditions of quench, the cross-linker was UV flash-activated at a time when quench was well established. The target proteins covalently linked to arrestin by cross-linker activation were identified by immunoblotting. In the presence of ATP arrestin cross-linked to both PDE and rhodopsin during the quench phenomenon. Removal of ATP from the reaction mixture essentially abolished the cross-link with PDE, just as ATP omission abolishes quench, but significantly increased the cross-link to rhodopsin. The absence of a cross-link to the plentiful beta-subunit of transductin, as well as the results of competition studies employing arrestin without attached cross-linker, suggest that the observed cross-links are specific and reflect true binding interactions of arrestin during quench. The data are consistent with a model of quench in which photolyzed rhodopsin (R*) catalyzes the formation of an activated form of arrestin, which dissociates from R* in the presence of ATP, and binds to PDEs, thereby deactivating them.
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PMID:Sites of arrestin action during the quench phenomenon in retinal rods. 284 5

Cyclic GMP metabolism has been investigated in the retinas of mice that are heterozygous for a 'photoreceptor dystrophy' gene and have a lowered concentration of cGMP in their photoreceptor cells. The concentration of rhodopsin, retinal morphology and guanylate cyclase kinetics were normal. Cyclic GMP phosphodiesterase had a lowered affinity for cGMP. In accord with previous observations, chelation of exogenous calcium had no effect on cGMP levels in light-adapted retinas but increased them in dark-adapted tissue. The difference between cGMP concentrations in heterozygous and normal retinas in the dark was then eliminated. It was concluded that a modulator of cGMP phosphodiesterase activity is most likely to be causing the lowered steady-state level of cGMP in heterozygous retinas and that calcium is not involved.
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PMID:Cyclic GMP in the retinas of normal mice and those heterozygous for early-onset photoreceptor dystrophy. 286 61

In the membranous signal transduction process, hormone-binding to receptors causes receptor interaction with signal-transducing components; these components transfer the stimulus to effector systems, which generate intracellular signals. Several guanine nucleotide-binding proteins (N- or G-proteins) have been identified as membranous signal-transducing components. Two N-proteins are involved in the hormonal regulation of adenylate cyclase activity, one of which being stimulatory (Ns), the other one being inhibitory (Ni). Ns, Ni and a third N-protein, No, whose function is unknown, occur ubiquitously. On the other hand, transducin, an N-protein, which functionally couples light-activated rhodopsin to a cGMP phosphodiesterase, is specific for the retina. In addition to their established role as transducers regulating adenylate cyclase and retinal cGMP phosphodiesterase, N-proteins proteins may be involved in two mechanisms by which the cytoplasmic calcium concentration is elevated, i.e. hormonal stimulation of a phospholipase C catalyzing phosphatidyl-inositol 4,5-diphosphate hydrolysis (Pi response) and hormone-induced opening of receptor-operated calcium channels; the membrane-bound forms of cAMP phosphodiesterase and guanylate cyclase, stimulated by insulin and atrial natriuretic factor, respectively, are also likely to be regulated via N-proteins. Guanine nucleotide-binding proteins appear to play a universal role in transmembranous signalling processes, controlling effector systems (i.e. enzymes and ion channels) that regulate cytoplasmic concentrations of intracellular messengers such as cyclic AMP, cyclic GMP and calcium.
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PMID:[Principles of transmembranous signal transduction in the action of hormones and neurotransmitters]. 286 63

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.
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PMID:Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors. 287 93

We compare the retinal rod photocurrent before and after introduction of an hydrolysis-resistant analog of GTP into the outer segment by the whole-cell patch technique. Others have shown that GTP bound to transducin leads to the hydrolysis of cyclic GMP, causing the response to light--a decrease in dark current. The hydrolysis-resistant GTP analog prolongs the response to a bright flash, which leads us to suggest that prolonged transducin activation by bright light desensitizes the rod by a prolonged decrease in dark current. Recovery from the response to a bright flash does occur after introduction of the analog; that recovery requires acceleration of cyclase activity rather than inhibition of phosphodiesterase. The analog mimics light adaptation by desensitizing the rod and speeding the recovery from a dim flash. The analog plus light or light adaptation prolongs the activities of transducin and phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) to mediate desensitization by reducing the dark current. Hence, this faster recovery from a dim flash would be by increased activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] rather than by inhibited phosphodiesterase. Accelerated activity of guanylate cyclase may speed recovery by response truncation. We conclude that transducin, activated by photolyzed rhodopsin, may lead to increased activity of both phosphodiesterase and guanylate cyclase to mediate the desensitization and the faster recovery of the light-adapted response.
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PMID:Rod light adaptation may be mediated by acceleration of the phosphodiesterase-guanylate cyclase cycle. 289 82

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.
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PMID:Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits. 292 42

To test the hypothesis that cyclic GMP is the internal messenger coupling rhodopsin activation to membrane excitation in vertebrate rod photoreceptors, we used a novel technique combining measurement of membrane currents of isolated salamander rods with a suction electrode and the introduction of cyclic GMP through a whole-cell recording patch pipette. Rupture of an attached patch was followed by a rapid (approximately 10 s), approximately 10-fold increase in outer-segment membrane current, all of which was light-sensitive. There was little change in the rising phase of the response to a saturating flash, but the duration of the saturated phase of the response increased approximately 10-fold. The effects reversed completely within 3-4 min after withdrawal of the cyclic GMP-containing patch pipette. A formal kinetic analysis shows that the first two observations are inconsistent with the postulate that cyclic GMP opens the light-sensitive conductance by simple binding to channels, unless free cyclic GMP in the outer segment is assumed to be much lower than published estimates, and most of the outer-segment cyclic GMP is bound and inexchangeable on the timescale of 200 ms. Furthermore, our results suggest that rod cyclic GMP is not involved solely in keeping the light-sensitive conductance open, but may also affect the activity of the phosphodiesterase that mediates cyclic GMP hydrolysis.
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PMID:Cyclic GMP can increase rod outer-segment light-sensitive current 10-fold without delay of excitation. 298 8

ATP quenches light-dependent phosphodiesterase (PDE) activation in rod outer segments presumably due to rhodopsin phosphorylation. Here we compared the efficiency of phosphorylated and non-phosphorylated rhodopsins as PDE activators in a reconstituted cell-free system. It is shown that the ability of phosphorylated membranes to activate this enzyme is suppressed compared with non-phosphorylated ones.
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PMID:The effect of rhodopsin phosphorylation on the light-dependent activation of phosphodiesterase from bovine rod outer segments. 298 61

Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed.
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PMID:Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping. 298 63

Retinal S antigen chromatographically purified from whole retina, induces experimental autoimmune uveoretinitis in laboratory animals. The 48K protein, a soluble protein found in rod outer segments, is purified through its specific binding to photoexcited rhodopsin and is involved in the quenching of light-induced guanosine 3',5'-monophosphate-phosphodiesterase activity. Biochemical, immunological, functional, and pathological tests showed that retinal S antigen and the 48K protein are identical.
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PMID:Retinal S antigen identified as the 48K protein regulating light-dependent phosphodiesterase in rods. 298 24


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