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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)

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 microM. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S), adenylyl (beta,gamma-methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 micrograms/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.
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PMID:Evidence for a nucleotide-dependent topoisomerase activity from yeast mitochondria. 775 Jan 44

The effect of a keto-derivative of cholesterol, namely, 6-ketocholestanol (5 alpha-cholestan-3 beta-ol-6-one; kCh) on the uncoupling of oxidation and phosphorylation by various uncouplers was studied in rat heart mitochondria. kCh was found to completely abolish the uncoupling effect (the increase in the respiration rate under the respiratory control conditions and the decrease in the membrane potential) caused of FCCP, CCCP and SF6847 and partially by TTFB at low concentrations of uncouplers. It was without effect on the uncoupling by PCP, DNP and palmitate. Carboxyatractylate, a specific inhibitor of the ADP/ATP-antiporter, was shown to almost completely abolish the uncoupling induced by palmitate and partially by low concentration of TTFB, PCP and DNP. Effects of high concentrations of all these uncouplers as well as of any concentrations of gramicidin proved to be kCh- and carboxyatractilate-insensitive. The data are discussed in terms of the hypothesis on the protein-mediated mechanism of the protonophorous uncoupling.
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PMID:6-ketocholestanol abolishes the effect of the most potent uncouplers of oxidative phosphorylation in mitochondria. 798 94

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 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

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 microM extracellular GTP or 300 microM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 microM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40-65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics. GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP >> ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms. The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1,3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the A1 receptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the P1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.
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PMID:GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells. 877 5


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