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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Photoreceptor membranes derived from isolated bovine rod outer segments, are subjected to treatment with phospholipase C (Bacillus cereus). This results in varying degrees of hydrolysis of the membrane phospholipids into diglycerides and water soluble phosphate esters without loss of rhodopsin. Electron microscopic observations of thin sections and freeze-fractured preparations indicate extrusion of diglycerides from the membranes and their coalescence to lipid droplets, beginning at 20% hydrolysis of phospholipids. After 90% hydrolysis of phospholipids membranous structures are still present. The rhodopsin is located in these structures, presumably in the form of two-dimensional lateral aggregates. This explains the cross-fracturing of the membranous structures, regularly observed upon freeze-fracturing of the phospholipase-treated photoreceptor membranes.
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PMID:Biochemical aspects of the visual process. XXXVII. Evidence for lateral aggregation of rhodopsin molecules in phospholipase C-treated bovine photoreceptor membranes. 64 3

Treatment of bovine rod outer segments with phospholipase C leads to largely lipid-depleted membranous structures. Under these conditions rhodopsin remains spectrally intact, but its thermal stability and regeneration capacity are decreased, whereas upon illumination the metarhodopsin I to II transition is blocked. These observations can be explained on the basis of the previously demonstrated lateral aggregation of rhodopsin molecules which, on the one hand leads to a (partial) shielding of these molecules and, on the other hand, might impose constraints on the flexibility of the molecule to undergo light-induced conformational changes. Upon reconstitution of these lipid-depleted preparations with amphipathic lipids by means of a detergent dialysis procedure, the aggregates are apparently rearranged to lipid bilayer structures with complete recovery of the original rhodopsin properties. Under our conditions the nature of the polar head groups and the fatty acids is not critical in this respect. Simple addition of amphipathic lipids, without the use of detergent, restores the rhodopsin properties only in the case of rod outer segment lipids and of didecanoylphosphatidylcholine, and even then only occasionally. These results are discussed in the light of the strong analogy in properties between phospholipase C-treated rod outer segment membranes and lipid- and detergent-free rhodopsin obtained by affinity chromatography. It is concluded that rhodopsin must be in a freely dispersed state in order to function properly. Apparently, a non-specific lipid bilayer fulfills this condition for the regeneration capacity, whereas normal photolytic behaviour requires, in addition, a minimal membrane fluidity according to the observations of other investigators. Presumably, the uniquely high phospholipid unsaturation of rod outer segment membranes is important for another, as yet unassessed, function of rhodopsin or the photoreceptor membrane.
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PMID:Biochemical aspects of the visual process. XXXVIII. Effects of lateral aggregation on rhodopsin in phospholipase C-treated photoreceptor membranes. 64 4

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.
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PMID:Oxidative damage of retinal rod outer segment membranes and the role of vitamin E. 96 69

Rhodopsin kinase activity of Musca domestica was characterized in a reconstitution assay, using urea-treated eye membranes as substrate and a purified fraction of eye cytosol as the enzyme. Analysis of kinase activity in fly eye, brain and abdomen extracts by reconstitution assays revealed that fly rhodopsin kinase is an eye-specific enzyme. It preferentially phosphorylates the light-activated form of rhodopsin (metarhodopsin) and has little activity with other protein substrates. Rhodopsin kinase binds to metarhodopsin and is released from rhodopsin-containing membranes. Metarhodopsin is a poor substrate for kinases from tissues other than the eye, making it a unique substrate for rhodopsin kinase. Rhodopsin kinase is inhibited by heparin, but not by the protein inhibitor of cAMP-dependent protein kinase. Its Km for ATP is 9 microM. Since fly rhodopsin is coupled to phospholipase C, studies of the interaction of rhodopsin with rhodopsin kinase can be useful in analysis of the reactions that lead to termination of the inositol-phospholipid-signaling pathway.
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PMID:Characterization of fly rhodopsin kinase. 142 85

We have previously demonstrated that human bronchial smooth muscle cells possess a single class of high-affinity binding sites for endothelin 1. In this study, we further characterized the receptor for endothelin 1 and evaluated the signal transduction mechanisms of this peptide. Stimulation of cultured human bronchial smooth muscle cells with endothelin 1 induced mobilization of Ca2+ from both intracellular and extracellular pools with a biphasic increase in cytoplasmic free Ca2+ concentration. Endothelin 1 increased cellular levels of inositol phosphates and diacylglycerol, indicating activation of phospholipase C, but induced production of inositol phosphates in smooth muscle cell membranes only in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Treatment of smooth muscle cells with pertussis toxin failed to block the endothelin 1-induced increase in inositol phosphate production and Ca2+ mobilization. These results suggest that the receptor for endothelin 1 in bronchial smooth muscle is coupled to phospholipase C through a pertussis toxin-insensitive G protein. Affinity crosslinking experiments identified the endothelin 1 receptor as a single band with an apparent molecular weight of approximately 70,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, further supporting the functional evidence that endothelin 1 receptor belongs to the G protein-linked rhodopsin type of receptor superfamily.
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PMID:Mechanisms of calcium mobilization and phosphoinositide hydrolysis in human bronchial smooth muscle cells by endothelin 1. 165 61

A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively.
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PMID:Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods. 190 Dec 31

The cDNA encoding bovine opsin was transfected into Chinese hamster ovary (CHO) cells to generate stable clones expressing the rod cell photoreceptor protein. Cells expressing opsin, when incubated in 11-cis retinal and exposed to light, inhibited forskolin-stimulated adenylyl cyclase activity. Rhodopsin-mediated inhibition of adenylyl cyclase was prevented by treatment of cells with pertussis toxin. In the same cells, thrombin stimulated phosphatidylinositol hydrolysis through G protein-mediated pathways, but rhodopsin neither significantly influenced the action of thrombin nor stimulated phosphatidylinositol hydrolysis. Our findings indicate that rhodopsin selectively regulates a Gi protein in intact CHO cells that is coupled to adenylyl cyclase but not to phospholipase C.
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PMID:Rhodopsin expressed in Chinese hamster ovary cells regulates adenylyl cyclase activity. 210 93

We examined a white-eyed strain of the norpA mutant (norpA;cn bw) and white (w)norpA+ controls using microspectrophotometry (MSP), electron microscopy (EM), and electroretinography (ERG). These studies revealed that light mediates receptor demise in norpA even though norpA lacks phototransduction. Rhodopsin and the rhabdomere which houses it decrease with increasing age in norpA but not in w with rearing on a 12 h light/12-h dark cycle or in constant light. At higher temperature in norpA;cn bw and w reared in constant light, visual pigment decreases, rhabdomeres diminish, and cells die. Importantly, dark rearing blocked visual pigment loss in norpA;cn bw; the M-potential, an ERG reflection of visual pigment level, corroborated this finding. MSP showed that norpA's visual pigment loss was not due to acute loss of metarhodopsin, rhodopsin's photoproduct. NorpA blocks certain processes expected to be light elicited. The alteration of visual pigment as a function of time of day, present in w controls, is absent in white-eyed norpA, suggesting that light-induced depolarization may be necessary to entrain the rhythm. Microspectrofluorometry using the fluorescent dye, Lucifer yellow, suggested that norpA lacks a light-induced uptake mechanism; using control flies, we determined the stimulus parameters required for uptake in vivo. An attempt to "cure" norpA;cn bw by replacement "therapy" using phospholipase C, missing in norpA's phototransduction cascade, was largely unsuccessful.
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PMID:Microphotometric, ultrastructural, and electrophysiological analyses of light-dependent processes on visual receptors in white-eyed wild-type and norpA (no receptor potential) mutant Drosophila. 212 52

The photoreceptor cells of invertebrate animals differ from those of vertebrates in morphology and physiology. Our present knowledge of the different structures and transduction mechanisms of the two animal groups is described. In invertebrates, rhodopsin is converted by light into a meta-rhodopsin which is thermally stable and is usually re-isomerized by light. In contrast, photoisomerization in vertebrates leads to dissociation of the chromophore from opsin, and a metabolic process is necessary to regenerate rhodopsin. The electrical signals of visual excitation have opposite character in vertebrates and invertebrates: the vertebrate photoreceptor cell is hyperpolarized because of a decrease in conductance and invertebrate photoreceptors are depolarized owing to an increase in conductance. Single-photon-evoked excitatory events, which are believed to be a result of concerted action (the opening in invertebrates and the closing in vertebrates) of many light-modulated cation channels, are very different in terms of size and time course of photoreceptors for invertebrates and vertebrates. In invertebrates, the single-photon events (bumps) produced under identical conditions vary greatly in delay (latency), time course and size. The multiphoton response to brighter stimuli is several times as long as a response evoked by a single photon. The single-photon response of vertebrates has a standard size, a standard latency and a standard time course, all three parameters showing relatively small variations. Responses to flashes containing several photons have a shape and time scale that are similar to the single-photon-evoked events, varying only by an amplitude scaling factor, but not in latency and time course. In both vertebrate and invertebrate photoreceptors the single-photon-evoked events become smaller (in size) and faster owing to light adaptation. Calcium is mainly involved in these adaptation phenomena. All light adaptation in vertebrates is primarily, or perhaps exclusively, attributable to calcium feedback. In invertebrates, cyclic AMP (cAMP) is apparently another controller of sensitivity in dark adaptation. The interaction of photoexcited rhodopsin with a G-protein is similar in both vertebrate and invertebrate photoreceptors. However, these G-proteins activate different photoreceptor enzymes (phosphodiesterases): phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates. In the photoreceptors of vertebrates light leads to a rapid hydrolysis of cGMP which results in closing of cation channels. At present, the identity of the internal terminal messenger in invertebrate photoreceptors is still unsolved.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Phototransduction: different mechanisms in vertebrates and invertebrates. 215 Aug 59

The gene encoding the 49-kilodalton protein that undergoes light-induced phosphorylation in the Drosophila photoreceptor has been isolated and characterized. The encoded protein has 401 amino acid residues and a molecular mass of 44,972 daltons, and it shares approximately 42 percent amino acid sequence identity with arrestin (S-antigen), which has been proposed to quench the light-induced cascade of guanosine 3',5'-monophosphate hydrolysis in vertebrate photoreceptors. Unlike the 49-kilodalton protein, however, arrestin, which appears to bind to phosphorylated rhodopsin, has not itself been reported to undergo phosphorylation. In vitro, Ca2+ was the only agent found that would stimulate the phosphorylation of the 49-kilodalton protein. The phosphorylation of this arrestin-like protein in vivo may therefore be triggered by a Ca2+ signal that is likely to be regulated by light-activated phosphoinositide-specific phospholipase C.
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PMID:A 49-kilodalton phosphoprotein in the Drosophila photoreceptor is an arrestin homolog. 215 71


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