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)

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.
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PMID:Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor. 249 80

Turkey erythrocyte membranes possess a phospholipase C that is markedly activated by P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of [3H]inositol-labeled turkey erythrocyte membranes with guanine nucleotide regulatory protein (G-protein) beta gamma subunits resulted in inhibition of both AlF-4-stimulated adenylate cyclase and AlF-4-stimulated phospholipase C activities. The apparent potency (K0.5 approximately 1 microgram or 20 pmol of beta gamma/mg of membrane protein) of beta gamma subunits for inhibition of each enzyme activity was similar and occurred with beta gamma purified by different methodologies from turkey erythrocyte, bovine brain, or human placenta membranes. In contrast to the effect on AlF-4-stimulated activity, the stimulatory effect on phospholipase C of the P2Y-purinergic receptor agonist 2-methylthioadenosine 5'-triphosphate in the presence of guanine nucleotides was potentiated by 50-100% in a concentration-dependent manner by reconstitution of beta gamma subunits. beta gamma subunits did not affect the K0.5 value of 2-methylthioadenosine 5'-triphosphate for the stimulation of phospholipase C activity. These results indicate that beta gamma subunits influence phospholipase C activity in a concentration range similar to that necessary for regulation of adenylate cyclase activity and suggest the involvement of a G-protein possessing an alpha beta gamma heterotrimeric structure in coupling hormone receptors to phospholipase C.
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PMID:Modification of AlF-4- and receptor-stimulated phospholipase C activity by G-protein beta gamma subunits. 250 7

Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an "inhibitory action" and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as "stimulatory actions" in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the "inhibitory actions" of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.
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PMID:A permissive role of pertussis toxin substrate G-protein in P2-purinergic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells. Cooperative mechanism of signal transduction systems. 254 44

Various adenine nucleotides activated phospholipase C of FRTL-5 cell membranes in the following order of activity, ATP gamma S greater than ATP greater than AppNp greater than AppCp = ADP greater than MeSATP. This order was well consistent with that observed in intact cells. Such activation occurred only in the presence of appropriate concentrations of GTP gamma S and Ca2+, in a way similar to the norepinephrine-induced activation. NaF, a non-specific GTP-binding protein (G-protein) activator, also stimulated the enzyme. These adenine nucleotides, norepinephrine and NaF-induced activations were inhibited by GDP beta S. We conclude that a G-protein is involved in the adenine nucleotides-induced activation of phospholipase C via P2-purinergic receptor in FRTL-5 cells.
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PMID:P2-purinergic agonists activate phospholipase C in a guanine nucleotide- and Ca2+-dependent manner in FRTL-5 thyroid cell membranes. 254 54

A guanine nucleotide-dependent P2Y-purinergic receptor-regulated phospholipase C activity of turkey erythrocyte membranes has been characterized in detail previously (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890). The occurrence of agonist-induced desensitization of this receptor-regulated phospholipase C is now described. Preincubation of turkey erythrocytes with the P2Y-purinergic receptor agonist ADP beta S resulted in a marked loss of capacity of ADP beta S plus GTP to stimulate phospholipase C in membranes derived from these cells. The half-time of occurrence of desensitization was 0.5-2.0 min, and within 10 min responsiveness had reached a new quasi-steady state level representing 40-55% of control. Transfer of agonist-preincubated erythrocytes to agonist-free medium resulted in recovery of agonist plus GTP responsiveness of the membrane phospholipase C activity to control levels with a half-time of 10-20 min. The change in ADP beta S plus GTP responsiveness occurred as a loss of maximal effect with little or no change in the apparent affinity of agonist for stimulation of inositol phosphate production. Induction of desensitization occurred with an agonist-specificity that followed that expected of a P2Y-purinergic receptor. Neither the rate of activation nor the final phospholipase C activity attained in the presence of GTP gamma S alone was altered in membranes from cells preincubated with ADP beta S for 15 min. AlF-4-stimulated inositol phosphate production was also not modified in membranes from agonist-preincubated erythrocytes. In contrast, the capacity of ADP beta S to increase the rate of activation of phospholipase C by GTP gamma S was markedly reduced in membranes from agonist-preincubated cells. The amount of 3H-radioactivity in phosphoinositides, as well as the ratio of labeling among the phosphoinositides, was not altered by incubation of erythrocytes with a P2Y-purinergic receptor agonist. Taken together these data suggest that P2Y-purinergic receptor agonist-induced desensitization occurs as a consequence of a modification at the level of the receptor or at the level of receptor-guanine nucleotide regulatory protein (G-protein) coupling with no change occurring in the capacity of the G-protein to activate phospholipase C.
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PMID:Agonist-induced desensitization of a P2Y-purinergic receptor-regulated phospholipase C. 255 22

3'-O-(4-Benzoyl)benzoyladenosine 5'-triphosphate (BzATP), a photoaffinity analog of ATP, was used as a ligand for the phospholipase C-linked P2Y-purinergic receptor in turkey erythrocytes. In membranes from [3H]inositol-labeled turkey erythrocytes, BzATP stimulated inositol phosphate formation in a concentration-dependent manner (K0.5 = 172 +/- 4 nM). The effect of BzATP was strictly dependent on the presence of guanine nucleotides and was not additive with the effect of other full P2Y-purinergic receptor agonists. Photolysis of [3H]-inositol-labeled membranes in the presence of BzATP had no effect on the magnitude of the maximally attainable response of phospholipase C to the P2Y-purinergic receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) plus guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, the effects of submaximally effective concentrations of GTP gamma S were markedly increased in membranes previously photolyzed in the presence of BzATP. In addition, the rate of activation of phospholipase C by GTP gamma S in membranes photolyzed in the presence of BzATP was increased 3-fold, as compared with control membranes. BzATP effects on phospholipase C were prevented by photolysis in the presence of ATP or ADP, but not by the presence of the weak P2Y-purinergic receptor agonist beta,gamma-methyleneadenosine 5'-triphosphate. These results suggest that BzATP is a full P2Y-purinergic receptor agonist, which after photolysis becomes irreversibly associated with turkey erythrocyte membranes and promotes P2Y-purinergic receptor-mediated guanine nucleotide-dependent activation of phospholipase C.
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PMID:Irreversible activation of phospholipase C-coupled P2Y-purinergic receptors by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate. 268 59

Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with the idea that P2Y receptor activation increases the rate of exchange of GTP and GTP analogs for GDP on the relevant guanine nucleotide regulatory protein. Once the active enzymic species is formed, hydrolysis of guanine nucleotide reverts the enzyme to the inactive state.
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PMID:Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides. 291 Aug 69

The effects of GTP gamma S, a stable GTP analogue that can activate guanine nucleotide-binding proteins, on phospholipase C activation/calcium mobilization were studied in intact cultured bovine aortic endothelial cells (BAEC). Phosphoinositide metabolism and cytosolic free Ca2+ concentration [( Ca2+]i; fura-2 fluorescence) were studied after the cells were transiently permeabilized, loaded with different guanine nucleotides, and then allowed to reseal and recover. Intracellular GTP gamma S stimulated a dose-dependent [median effective concentration (EC50) 2.5 microM] decrease in basal [3H]phosphoinositide content. Phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-bisphosphate, and phosphatidylinositol levels decreased to 57 +/- 9, 63 +/- 8, and 74 +/- 8% control levels, respectively, in BAEC loaded with approximately 85 microM GTP gamma S. Basal inositol trisphosphate (IP3) and [Ca2+]i were increased in GTP gamma S-loaded BAEC when compared with sham-loaded BAEC. In control BAEC, the purinergic receptor agonist ATP (100 microM) induced rapid increases in [Ca2+]i and IP3. However, BAEC that had been intracellularly loaded with GTP gamma S [median inhibitory constant (IC50) 1 microM] or 5'-guanylyl-imidodiphosphate exhibited decreased calcium mobilization in response to ATP. Ionomycin (calcium ionophore)-releasable pools of calcium were similar in sham- and GTP gamma S-loaded cells, suggesting that total intracellular calcium had not been depleted by the permeabilization protocol. The diminished calcium mobilization in response to ATP was associated with decreases in ATP-stimulated PIP2 hydrolysis and IP3 formation. In addition, GTP gamma S loading did not increase basal levels of cyclic AMP. Intracellular GDP beta S, GDP, or GTP did not inhibit ATP-stimulated increases in [Ca2+]i or IP3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:GTP gamma S loading of endothelial cells stimulates phospholipase C and uncouples ATP receptors. 305 27

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.
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PMID:The guanine-nucleotide-binding protein subunit G alpha i2 is involved in calcium activation of phospholipase A2. Effects of the dominant negative G alpha i2 mutant, [G203T]G alpha i2, on activation of phospholipase A2 in Chinese hamster ovary cells. 760 Oct 96

The regulation of phospholipase A2 by G protein-coupled receptors is examined in CHO cells which normally express the purinergic receptor and have been transfected with bovine rhodopsin. The purinergic receptor has been reported to activate both phospholipase C and phospholipase A2 in this cell line. In contrast, bovine rhodopsin by itself is not able to activate phospholipase A2. However, the photoreceptor does potentiate purinergic receptor-mediated phospholipase A2 activation in a light-dependent manner. Both the purinergic receptor stimulation of phospholipase A2 and the enhanced activity mediated by rhodopsin are completely pertussis toxin-sensitive, suggesting the regulation of phospholipase A2 by a member of the Gi family of G proteins. Both of these receptors also inhibit adenylyl cyclase activity. Rhodopsin-mediated inhibition of adenylyl cyclase is pertussis toxin-sensitive, whereas inhibition by the purinergic receptor is calcium-sensitive but not pertussis toxin-sensitive. These results suggest (1) that rhodopsin is similar to other receptors that normally couple to Gi when expressed in cultured cells and (2) that regulation of adenylyl cyclase and PLA2 in CHO cells by rhodopsin and the purinergic receptor occur via distinct pathways.
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PMID:The coupling of pertussis toxin-sensitive G proteins to phospholipase A2 and adenylyl cyclase in CHO cells expressing bovine rhodopsin. 781 32


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