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)

Isolated pancreatic islets from rats and humans express a plasmalogen-preferring ATP-stimulatable, Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme which participates in the glucose-stimulated hydrolysis of arachidonate from membrane phospholipids and in insulin secretion. Here we report that clonal insulin-secreting HIT beta-cells contain substantial amounts of endogenous plasmalogens and express a similar ASCI-PLA2 activity with the following properties: (1) Enzymatic activity as well as glucose-induced eicosanoid release and insulin secretion are inhibited by a mechanism-based suicide substrate directed towards ASCI-PLA2. (2) HIT cell ASCI-PLA2 is selectively activated and protected against thermal denaturation by ATP. (3) The magnitude of ASCI-PLA2 activation by the nonhydrolyzable ATP analog AMP-PCP is similar to that by ATP. (4) The ATP concentrations required to activate ASCI-PLA2 fall within physiologic ranges in the presence of Mg2+. (5) ADP induces a concentration-dependent attenuation of the activation of ASCI-PLA2 by ATP. HIT cell ASCI-PLA2 exhibited an apparent isoelectric point of 7.5 on chromatofocusing analysis and was quantitatively adsorbed to an ATP-agarose matrix and selectively desorbed from this column by ATP. Mono-Q anion-exchange analysis of the active ATP-agarose eluant yielded a peak of ASCI-PLA2 activity associated with a single protein band with an apparent molecular mass of 40 kDa. Similar chromatographic behavior of the rat pancreatic islet ASCI-PLA2 activity was observed during sequential ATP-agarose and Mono-Q anion-exchange steps. These results indicate that HIT cells express an ASCI-PLA2 similar to the analogous islet enzyme and suggest that expression of this enzyme and of its preferred plasmalogen substrates may be a general property of insulin-secreting beta-cells.
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PMID:Characterization of an ATP-stimulatable Ca(2+)-independent phospholipase A2 from clonal insulin-secreting HIT cells and rat pancreatic islets: a possible molecular component of the beta-cell fuel sensor. 800 9

3H-labeled 9-methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, binds to the caffeine binding site of terminal cisternae (TC) of skeletal muscle sarcoplasmic reticulum (SR) (Fang, Y-I., Adachi, M., Kobayashi, J., and Ohizumi, Y. (1993). J. Biol. Chem. 268, 18622-18625.) and activates Ca(2+)-induced Ca2+ release (CICR). [3H]MBED, however, bound to rabbit hepatic microsomes with a comparable affinity (Kd = 50 nM) and with a more than 30-fold greater receptor density (Bmax = 350 pmol/mg of protein), compared with those in SR. Caffeine (0.1-10 mM) caused a concentration dependent inhibition of [3H]MBED binding to hepatic microsomes with the IC50 value of 0.3 mM. The mode of inhibition by caffeine was allosteric, indicating that the binding site of the ligand is distinct from but related to that of caffeine. Procaine (1-10 mM), a representative inhibitor of CICR, which suppresses [3H]MBED binding to TC-SR, inhibited ligand binding to hepatic microsomes only slightly. Moreover, ligand binding to the hepatic binding site was not affected by adenosine 5'-(beta, gamma-methylene) triphosphate (AMP-PCP) (10-100 microM), which is an activator of CICR and potentiates [3H]MBED binding to TC-SR. Inhibitors of [3H]MBED binding to liver microsomes other than caffeine were nucleotides such as ADP, ATP, GTP, UTP (1 mM), while CTP, cAMP, AMP, adenosine (1 mM), ryanodine (0.1-100 mM) and inositol 1,4,5-trisphosphate (1 microM) were not effective. These features of the hepatic microsomal [3H]MBED binding site distinguish it from that of skeletal muscle SR. [3H]MBED, which binds to the different sites which are both sensitive to caffeine, is useful as a probe to investigate the actions of caffeine at the molecular level.
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PMID:The specific binding site of 9-[3H]methyl-7-bromoeudistomin D, a caffeine-like Ca2+ releaser, in liver microsomes in distinct from that in skeletal sarcoplasmic reticulum. 801 Nov 74

Activation of rat basophilic leukaemia cells (RBL-2H3) leads to the secretion of allergic and inflammatory mediators. These cells can be permeabilized, yet still retain their ability to secrete in response to antigen. Secretion can also be induced in permeabilized cells by the addition of the ATP analogue, ATP gamma S [adenosine-5'-O-(3-thiotriphosphate)], which is relatively resistant to phosphatase activity. ATP gamma S-induced secretion (35-50% of total amine) is temperature and concentration-dependent. Calcium enhances secretion, but unlike antigen-induced secretion, it does occur in the absence of calcium and without the requirement for inositol phospholipid hydrolysis. Other ATP analogues induced secretion in the rank order AMP-PNP > or = ATP gamma S >>> AMP-PCP > ATP alpha S = ATP [AMP-PNP, adenylyl-imidodiphosphate; AMP-PCP, adenylyl (beta,gamma-methylene)-diphosphonate; ATP alpha S, adenosine-5'-O-(1-thiotriphosphate)]. At equimolar concentrations, ATP inhibits ATP gamma S-induced secretion by 50%, but prolonged incubation in the presence of ATP gamma S surmounts the ATP inhibition. ADP is nearly as effective an inhibitor, but GTP and ITP are ineffective. It is likely that secretion occurs through attachment at an ATP-binding site, effectively blocking the action of a phosphatase, active later in the normal secretory pathway.
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PMID:Calcium-independent secretion by ATP gamma S from a permeabilized rat basophilic leukaemia cell line (RBL-2H3). 808 86

The relaxant action of adenine nucleotides was studied in isolated rabbit trachealis to assess the presence of P2-purinoceptors in the airways, their cellular location, and pharmacologic properties. Strips of tracheal smooth muscle with intact epithelium were incubated in tissue baths and contracted with 1 microM acetylcholine. Over a dose range of 0.1 microM to 1 mM, ATP and ADP were significantly more potent than adenosine in relaxing tracheal smooth muscle. Significant relaxations were also elicited by AMP-PCP, AMP-CPP, and AMP-PNP, three ATP analogs stable to enzymatic hydrolysis to adenosine. In the absence of acetylcholine, neither ATP nor AMP-CPP exerted any contractile effect on the tracheal strips. In tissues selectively denuded of epithelium, ATP-, ADP-, and AMP-PCP-induced relaxations were markedly reduced. ATP-induced relaxation was also inhibited by the P2y-purinoceptor antagonist Reactive Blue 2 (RB2) (50 to 300 microM) and partially reduced by the cyclooxygenase inhibitor indomethacin (10 microM), whereas adenosine-induced relaxation was not significantly affected by these agents. These results suggest that ATP can induce smooth muscle relaxation in acetylcholine-contracted tracheal strips through a distinct P2-purinoceptor. This receptor appears to be located on the epithelium where its relaxant effect is mediated in part by release of one or more cyclooxygenase products. Additional relaxation at high ATP concentrations may occur through enzymatic hydrolysis of ATP to adenosine and interaction at P1-purinoceptors.
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PMID:Relaxation of rabbit tracheal smooth muscle by adenine nucleotides: mediation by P2-purinoceptors. 811 Apr 78

The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2_, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.
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PMID:Conformation of Ca(2+)-ATPase in two crystal forms. Effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate), and chromium(III)-ATP on crystallization. 815 94

The hepatocyte has an organic anion transport system that recognizes compounds such as bilirubin and sulfobromophthalein. These anions circulate bound tightly to albumin from which they are extracted rapidly by hepatocytes by an electroneutral process that requires extracellular inorganic anions such as Cl- for activity. Transport activity is reduced by depletion of intracellular ATP, but whether ATP interacts directly with this transporter is not known. In this study, the influence of extracellular ATP on the hepatocyte organic anion transport mechanism has been characterized. In the presence of 2.5 mM Ca2+ and 2 mM Mg2+, initial uptake of [35S]sulfobromophthalein was reduced by 50% at 1 mM ATP. In the absence of divalent cations sensitivity to ATP was 10-fold greater. Other nucleotides including UTP, CTP, GTP, ADP, AMP, and AMP-PCP (adenosine 5'-(beta,gamma-methylene)triphosphate) were inactive. Decreased transport activity was rapidly reversible, was non-competitive with respect to ATP, did not require ATP hydrolysis, and did not correlate with P2y purinergic receptor activity. Differential activity of ATP on sulfobromophthalein transport in the presence and absence of divalent cations was not due to ecto-ATPase activity but rather to alteration in [ATP4-]. Although an ATP4- receptor in macrophages mediates increased cellular permeability, reduced organic anion permeability is seen in hepatocytes. This effect is not seen in the hepatoma cell line HepG2. Modulation of activity of the organic anion transporter by extracellular ATP may have important pathophysiological consequences in conditions resulting in liver cell injury.
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PMID:Extracellular ATP4- modulates organic anion transport by rat hepatocytes. 834 Mar 70

In the epithelial cell line FRT, derived from rat thyroid, extracellular ATP, at a concentration as low as 1 x 10(-7) M, specifically increases cytosolic Ca++ two fold over the basal level of 255 +/- 45 nM. A maximum increase of 5 fold over basal is seen at 1 x 10(-5) M ATP. The effect occurs in the absence of any measurable phosphatidyl inositol metabolism and requires the presence of extracellular Ca++, but is independent of extracellular Na+; it is duplicated by ATP gamma S but not by adenosine, AMP, ADP, AMP-PNP, AMP-CPP, or AMP-PCP. In the presence of the P2-receptor antagonist suramin, the ATP induced Ca++ influx is completely inhibited, whereas Mg++, La , and verapamil are ineffective. It appears that the most likely (and unique) mechanism of ATP induced increase of cytosolic Ca++ in FRT cells in an increased influx through the activation of a P2 receptor operated Ca++ channel.
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PMID:Purinergic (P2) receptor-operated calcium entry into rat thyroid cells. 836 91

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.
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PMID:Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: characterization using [32P]3'-O-(4-benzoyl)benzoyl ATP, a photoaffinity label. 853 Dec 2

1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.
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PMID:Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. 854 25

Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.
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PMID:Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product. 862 34


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