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:3.4.16.2 (PCP)
3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The possible role of protein phosphorylation in modulation of [3H]glibenclamide binding to the sulphonylurea receptor, a putative ATP-sensitive K-channel, was investigated in the cloned pancreatic beta-cell line, HIT T15. Diazoxide, an opener of ATP-sensitive K-channels, increased HIT cell 86Rb-efflux, inhibited insulin secretion and decreased non-competitively [3H]glibenclamide binding to intact HIT cells. ATP-depletion reduced the [3H]glibenclamide binding activity of intact cells but did not change diazoxide-insensitive binding. Although diazoxide alone did not change the binding of [3H]glibenclamide to HIT cell membranes, the simultaneous presence of MgATP revealed an inhibition of [3H]glibenclamide binding by diazoxide. This effect of MgATP was reproduced by MgATP gamma S, but not by MgADP, MgAMP-PNP or MgAMP-PCP. These findings suggest that protein phosphorylation may be involved in the response of ATP-sensitive K-channels to diazoxide.
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PMID:Possible involvement of protein phosphorylation in the regulation of the sulphonylurea receptor of a pancreatic beta-cell line, HIT T15. 183 60

ATP-induced increases of intracellular calcium concentration ([Ca2+]i) were measured as a function of flow rate in single cell recordings within a confluent endothelial cell monolayer. Although flow and its associated shear stress did not per se significantly alter basal [Ca2+]i, ATP-induced [Ca2+]i was exquisitely sensitive to flow. Step increases of flow in the presence of ATP triggered large [Ca2+]i transients that slowly (60-150 s) returned to basal values. ATP-releasable [Ca2+]i was mobilized from intracellular stores, as well as obtained from the extracellular medium. Since potent ectonucleotidases on the cell surface are expected to influence local ATP concentrations, experiments were repeated using the poorly hydrolyzable ATP analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). Comparison between ATP and AMP-PCP responses suggested that flow regulates the mass transport of agonist to the endothelial cell surface by overcoming the local effects of degradative enzymes. An additional, quite different phenomenon of flow-mediated [Ca2+]i regulation in endothelial cells was observed when [Ca2+]i oscillations induced by AMP-PCP in the absence of flow were shown to be reversibly inhibited by step increases in flow. These results imply that the effectiveness of local or systemic agonists in stimulating endothelial transduction will vary with flow rates. Regional variations in hemodynamic shear stresses associated with altered flow patterns throughout the arterial system are predicted to result in large variations of vessel wall responsiveness to physiological and pathological agonists.
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PMID:Flow modulation of agonist (ATP)-response (Ca2+) coupling in vascular endothelial cells. 185 15

The motility of bile canaliculi was examined in hepatocyte couplets permeabilized with palmitoyl lysophosphatidyl choline in a dosage regimen that drastically affected secretory function, yet maintained relative integrity of the cellular cytoskeleton. The permeabilized cells showed no exclusion of trypan blue, notable cytoplasmic organelle and membrane damage, and no uptake or secretion of either fluorescein diacetate or sodium fluorescein. However, bile canalicular structure remained relatively intact and actin and myosin were localized immunocytochemically in the pericanalicular region. Coincident with the administration of 1 mM ATP, 2 mM Mg2+, and 1 microM Ca2+, the canaliculi contracted with partial or complete luminal closure. ADP, AMP, or AMP-PCP could not be substituted for ATP. A dose-dependent relationship was shown between ATP concentration and canalicular contraction rate. The permeabilization procedure also provided enhanced visualization of pericanalicular microfilaments, believed to be actin filaments, and their organization into two layers: an inner membrane-associated network, and an outer filament bundle that inserted into belt junctions (zonulae adherentes). The organization of the microfilament belt of contiguous hepatocytes was such that it formed a circumferential band of microfilaments around the canaliculus. It is analogous to contractile filament belts found in the apical terminal web region of other epithelia. It was also observed that with canalicular luminal closure, there was a change in the organization of the pericanalicular microfilaments. It is concluded that in hepatocyte couplets, differential sensitivity of cell components to permeabilization can be achieved with palmitoyl lysophosphatidyl choline. In addition, the results provide evidence that the bile canaliculus has the capacity to be a contractile structure even in the absence of secretion, that canalicular contraction is ATP-dependent, and hence is a dynamic process.
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PMID:Permeabilized hepatocyte couplets. Adenosine triphosphate-dependent bile canalicular contractions and a circumferential pericanalicular microfilament belt demonstrated. 188 Nov 22

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.
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PMID:Inhibition of Escherichia coli glutamine synthetase by alpha- and gamma-substituted phosphinothricins. 196 48

A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.
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PMID:Characterization of the mechanism of endocytic vesicle fusion in vitro. 212 Feb 6

Uptake of the catecholamines (CA), dopamine (DA) and norepinephrine (NE) into synaptosomes prepared from rat and bovine brains was potentiated by ATP (from 0.1 to 5.0 mM) in a dose-dependent manner. Other nucleotides, particularly the nonhydrolyzable ATP analogs beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP) and beta,gamma-methyladenosine-5'-triphosphate (AMP-PCP) also potentiated [3H]DA and [3H]NE uptake. Several endogenous 5'-nucleotide triphosphates (e.g. GTP, UTP and CTP) potentiated [3H]CA uptake, but were less effective than ATP. Among the ATP metabolites, only ADP potentiated uptake whereas AMP and adenosine did not. [3H]Dopamine uptake measured in Krebs bicarbonate buffer had a Km of 2.1 microM and a Vmax of 163.9 pmol/mg prot./min. In presence of ATP, [3H]DA uptake had much higher affinity (Km = 0.56 microM) and larger capacity (Vmax = 333 pmol/mg prot./min) than uptake in absence of added ATP. Furthermore, [3H]DA uptake in presence of ATP had faster rate of uptake, and was independent of temperature while in absence of added ATP it was temperature-dependent. This ATP-dependent [3H]DA uptake was retained by synaptosomal ghosts that were obtained after lysing the striatal synaptosomes and removing their contents of synaptic vesicles and mitochondria. It is proposed that, in addition to the carrier-mediated (neuronal) uptake of CA, there is neuronal uptake that is regulated by ATP and inhibited by cocaine, which may be more relevant for terminating the synaptic action of CA because of its faster rate of uptake and larger capacity.
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PMID:ATP-regulated neuronal catecholamine uptake: a new mechanism. 240 89

Ionic gradients imposed by choline Cl replacement of K methanesulfonate (Mes) at constant [K][Cl] product stimulate 45Ca efflux from skinned muscle fibers; a small, sustained Ca2+-insensitive efflux component, observed in EGTA, appears to grade a much larger Ca2+-dependent component responsible for contractile activation and is likely to reflect intermediate steps in excitation-contraction coupling. The present studies examined ATP-related effects on the Ca2+-insensitive stimulation. 45Ca efflux was measured on segments of frog semitendinosus muscle skinned by microdissection, with isometric force monitored continuously. The Ca2+-insensitive component was potentiated by quercetin, a flavonoid thought to inhibit the sarcoplasmic reticulum (SR) Ca pump by stabilizing a phosphorylated intermediate. Quercetin increased the stimulated net 45Ca release in the absence of EGTA, as expected from inhibition of reaccumulation, but its effectiveness in EGTA indicated potentiation of unidirectional efflux as such. Quercetin also increased unstimulated (control) 45Ca efflux in EGTA, to a smaller extent; potentiation appeared to be a function of efflux, with stimulation above control loss increased approximately 2.6-fold. ATP removal before stimulation, which led to rigor force and increased stiffness, prevented all quercetin effects in EGTA. ATP removal by itself inhibited ionic stimulation of the Ca2+-insensitive component, with little residual increase above the parallel control loss. Addition of the nonhydrolyzable ATP analogue AMP-PCP ([adenylyl-beta,gamma-methylene]diphosphate) (0.8 mM) after ATP removal gave similar results to ATP-free solution, which suggests that adenine nucleotide binding alone does not support stimulation by choline Cl. These results imply a fundamental role for ATP in the excitation of skinned fibers by imposed diffusion potentials; they also suggest that ATP regulates the SR Ca efflux channel, in a manner that could provide the positive feedback in Ca2+-dependent Ca release.
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PMID:Excitation of skinned muscle fibers by imposed ion gradients. II. Influence of quercetin and ATP removal on the Ca2+-insensitive component of stimulated 45Ca efflux. 241 70

A subpopulation of canine cardiac sarcoplasmic reticulum vesicles has been found to contain a "Ca2+ release channel" which mediates the release of intravesicular Ca2+ stores with rates sufficiently rapid to contribute to excitation-contraction coupling in cardiac muscle. 45Ca2+ release behavior of passively and actively loaded vesicles was determined by Millipore filtration and with the use of a rapid quench apparatus using the two Ca2+ channel inhibitors, Mg2+ and ruthenium red. At pH 7.0 and 5-20 microM external Ca2+, cardiac vesicles released half of their 45Ca2+ stores within 20 ms. Ca2+-induced Ca2+ release was inhibited by raising and lowering external Ca2+ concentration, by the addition of Mg2+, and by decreasing the pH. Calmodulin reduced the Ca2+-induced Ca2+ release rate 3-6-fold in a reaction that did not appear to involve a calmodulin-dependent protein kinase. Under various experimental conditions, ATP or the nonhydrolyzable ATP analog, adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and caffeine stimulated 45Ca2+ release 2-500-fold. Maximal release rates (t1/2 = 10 ms) were observed in media containing 10 microM Ca2+ and 5 mM AMP-PCP or 10 mM caffeine. An increased external Ca2+ concentration (greater than or equal to 1 mM) was required to optimize the 45Ca2+ efflux rate in the presence of 8 mM Mg2+ and 5 mM AMP-PCP. These results suggest that cardiac sarcoplasmic reticulum contains a ligand-gated Ca2+ channel which is activated by Ca2+, adenine nucleotide, and caffeine, and inhibited by Mg2+, H+, and calmodulin.
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PMID:Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. 243 95

K+ currents through ATP-dependent channels were recorded from inside-out patches of beta-cell membrane as previously described (Rorsman and Trube 1985). Channels were opened by removing ATP from the intracellular side of the membrane. The open probability and/or the number of active channels declined spontaneously ("run-down") when ATP was absent for periods longer than about 30 s. Channels subject to the run-down could be activated again after applying a blocking concentration (greater than 0.1 mM) of ATP in presence of 1 mM MgCl2 for at least 2 min. ATP in absence of Mg and the ATP-analogues AMP-PNP, AMP-PCP and ATP gamma S were ineffective in reactivating the channels. This suggests that phosphorylation of the channels or associated proteins or hydrolysis of ATP may be necessary for keeping the channels available. In contrast to the differential effects on the run-down, ATP in presence and absence of Mg and the ATP analogues were similarly effective in blocking the channels at concentrations above 0.1 mM. Using an experimental protocol avoiding the run-down the dose-inhibition curve for ATP was found to reach 50% at 18 microM.
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PMID:Dual effects of ATP on K+ currents of mouse pancreatic beta-cells. 243 38

Micromolar concentrations of copper (Cu2+) and cysteine induce rapid efflux of calcium from sarcoplasmic reticulum (SR) vesicles. This effect appears to be due to a Cu2+-catalyzed oxidation of the added cysteine to a critical sulfhydryl group on the release protein from sarcoplasmic reticulum (J. L. Trimm, G. Salama, and J. J. Abramson (1986) J. Biol. Chem. 261, 16092-16098). The data presented here indicate that adenine nucleotides synergistically stimulate copper/cysteine (oxidation)-induced calcium efflux from SR vesicles. The order of effectiveness in stimulating calcium efflux is ATP greater than AMP-PCP greater than cAMP greater than AMP greater than adenine approximately NAD approximately NADH. Non-adenine-containing nucleotides such as GTP, CTP, UTP, and ITP and the high energy phosphate compound, acetyl phosphate, were ineffective in stimulating oxidation-induced calcium efflux. The relative effectiveness of various adenine nucleotides in stimulating calcium-induced calcium efflux and oxidation-induced calcium efflux are identical, suggesting that a common mode of action is involved when calcium release is triggered by either method. The stimulatory effect of the adenine nucleotides on oxidation-induced efflux is independent of external magnesium concentration and independent of the magnesium gradient across the SR membrane.
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PMID:Adenine nucleotides stimulate oxidation-induced calcium efflux from sarcoplasmic reticulum vesicles. 245 34


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