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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracellular nucleotides induce changes in cytosolic free Ca++ and also increase plasma membrane permeability to Ca++ ions in Chang human liver cells. Ca++ permeability induced by nucleotides is reversible and inactivated immediately upon removal of agonist. Stimulated cells transferred into the fresh medium and re-exposed to nucleotides demonstrate reopening of Ca++ channel without stimulation of Ca++ transient. Relative potencies of nucleotides to induce membrane permeability and Ca++ transient were: UTP > ATP > gamma-S-ATP > ADP (non-hydrolyzable ATP analogues and AMP had no effect). The permeability is not affected by specific inhibitors of voltage-operated calcium channels (verapamyl,cis-diltiazem and nifedipin). Nucleotides do not produce plasma membrane damages at concentrations up to 1 mM, as shown by exclusion of the propidium iodide. There are at least two types of nucleotides receptors in Chang cell membrane: P2y subtype receptors which is responsible for generation of the Ca++ transient, and P2x subtype receptors which lead to the opening of plasma membrane Ca++ channels upon activation.
Biochem Mol Biol Int 1995 Mar
PMID:Nucleotides-induced cytosolic calcium transient and plasma membrane permeability in Chang human liver cells. 777 94

The transcription of the 11 gene S10 operon of Escherichia coli is autogenously regulated by one of the operon's products, ribosomal protein L4. This protein stimulates termination of transcription in vivo at a specific site within the S10 leader. The in vivo effect can be reproduced in a purified transcription system but requires an additional factor, NusA. Our earlier in vitro studies showed that NusA is required for RNA polymerase pausing at the termination site; such paused complexes are further stabilized by L4, which presumably accounts for L4's stimulation of termination in vivo. Here we show that NusA is not absolutely required for RNA polymerase to recognize the attenuation site: at low (5 microM) UTP concentration, RNA polymerase pauses at the site, although the paused transcription complex formed in the absence of NusA can be further stabilized by subsequent addition of the protein. Furthermore, RNA polymerase pausing at the attenuation site is not sufficient for the L4 effect, since L4 cannot stabilize a transcription complex paused at the attenuation site in the absence of NusA. We have been able to isolate paused complexes formed without NusA and/or L4; such complexes are active upon re-addition of NTPs, and respond as expected to the addition of L4 or NusA. Our experiments are consistent with the notion that L4 is a stable component of a paused transcription complex.
J Mol Biol 1995 Feb 03
PMID:Role of NusA in L4-mediated attenuation control of the S10 r-protein operon of Escherichia coli. 784 21

Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP. Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites. The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP. These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain. Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding. These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model. This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.
J Mol Biol 1995 Feb 10
PMID:Intramolecular transmission of the ATP regulatory signal in Escherichia coli aspartate transcarbamylase: specific involvement of a clustered set of amino acid interactions at an interface between regulatory and catalytic subunits. 785 93

The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.
Mol Cell Biochem 1994 Nov 09
PMID:Physiological concentrations of purines and pyrimidines. 787 93

Our previous studies have shown several fold induction of neurotensin/neuromedin N (NT/N) primary transcripts and mature cytosolic mRNA in the dorsolateral region of the rat neostriatum (DLSt) following a single dose of the neuroleptic, haloperidol. The apparent enhancement of NT/N gene transcription by haloperidol is preceded by increases in the expression of c-fos mRNA in the same region. The present study used double-labeling in situ hybridization technique to study cellular localization of NT/N and c-fos mRNA following acute haloperidol treatment. Simultaneous detection of NT mRNA and c-fos mRNA was achieved using cRNA probes synthesized in vitro with digoxigenin- and 35S-labeled UTP, respectively. Interestingly, approximately 75% of DLSt neurons expressing NT/N mRNA also displayed c-fos mRNA in rats treated with haloperidol (1 mg/kg, i.p.) for 1 h. Colocalization of c-fos mRNA in haloperidol-responsive NT neurons in the DLSt suggests that haloperidol's induction of NT/N gene transcription may involve participation of the transcription factor Fos and the AP-1 consensus sequence in regulatory region of the NT/N gene.
Brain Res Mol Brain Res 1994 May
PMID:Coexpression of neurotensin and c-fos mRNAs in rat neostriatal neurons following acute haloperidol. 791 59

1. In pheochromocytoma PC12 cells ATP and, to a lesser extent, 2-methylthioATP stimulate phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, leading to stimulation of norepinephrine release. In contrast, although UTP also stimulates phosphoinositide breakdown, release of intracellular calcium, and influx of external calcium, there is no stimulation of norepinephrine release. 2. 2-MethylthioATP, presumably acting at P2y receptors, and UTP, presumably acting at P2u receptors, in combination elicit a phosphoinositide breakdown greater than that elicited by either alone. Intracellular levels of calcium measured with Fura-2 increase to greater levels with ATP than with UTP and are sustained, while the UTP intracellular levels of calcium rapidly return to basal values. Both ATP and UTP cause a similar influx of 45 Ca2+ presumably by stimulation of a P2 receptor directly linked to a cation channel. 3. It is proposed that PC12 cells contain two distinct G protein-coupled P2 receptors that activate phospholipase C and a P2 receptor linked to a cation channel. The P2y receptor sensitive to ATP (and to 2-methylthioATP) causes the depletion of a pool of intracellular calcium, sufficient to activate so-called "receptor-operated calcium entry". The sustained elevation of intracellular calcium after ATP treatment is proposed to result in stimulation of norepinephrine release and activation of calcium-dependent potassium channels and sodium-calcium exchange pathways. 4. The P2u receptor sensitive to UTP (and to ATP) causes only a transient elevation in levels of intracellular calcium, perhaps from a different pool, insufficient to activate so-called receptor-operated calcium entry. Further sequelae do not ensue, and the functional role of the UTP-sensitive P2u receptor is unknown.
Cell Mol Neurobiol 1994 Feb
PMID:Effects of ATP and UTP in pheochromocytoma PC12 cells: evidence for the presence of three P2 receptors, only one of which subserves stimulation of norepinephrine release. 795 59

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [3H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274-279, 1984) to near homogeneity. A 23 degrees C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [gamma-32P]ATP,Mg2+ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [gamma-32P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn2+ or Ca2+, could not be substituted for Mg2+ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.
Mol Cell Biochem 1994 Mar 30
PMID:Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase. 796 99

P2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxin-sensitive members of the G1 family of G proteins. These and other pharmacological data strongly suggest that multiple P2Y purinergic receptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cDNA that, when expressed in frog oocytes, displayed the general pharmacological characteristics of a P2Y purinergic receptor but whose second messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P2Y purinergic receptor and have stably expressed it in a heterologous human cell line (1321N1 astrocytoma cells) to establish its signaling properties. The purinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a marked activation of phospholipase C in 1321N1 cells stably expressing the meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expressed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'-O-(2-thio)diphosphate > ADP > 2-chloro-ATP = adenosine 5'-O-(3-thio)triphosphate > or = ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P2Y purinergic receptors in turkey erythrocytes and many other tissues and was markedly different from those of the P2U and P2X purinergic receptor subtypes. Stimulation of inositol lipid hydrolysis by P2Y purinergic agonists was not affected by preincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and variable inhibition of cAMP accumulation. Ribonuclease protection analysis of turkey tissues showed that this P2Y purinergic receptor is most highly expressed in blood and brain. Taken together, these results indicate that a phospholipase-C-activating P2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.
Mol Pharmacol 1994 Jul
PMID:Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. 805 61

The artificial UTP-analogue 5-bromouridine 5'-triphosphate (BrUTP) has been used to label pre-mRNA in vitro and in vivo [1,2]. We have investigated the effect of bromouridine (BrU) in pre-mRNA on the efficiency of splicing. An adenovirus major late II construct was used to prepare four different transcripts, each containing a different amount of BrU. These four transcripts were tested in an in vitro splicing assay. We found that splicing is strongly inhibited if all uridines (U) in the transcript were substituted for BrU. Splicing was restored to some extent if 50% of the Us were replaced by BrU. The splicing efficiency returned to an almost normal level if only 1 our of every 10 Us was substituted for BrU. This demonstrates that only a pre-mRNA containing a small amount of BrU can be spliced normally in vitro. Furthermore, these results strongly suggest that some Us in the adenoviral transcript, probably those at the splice sites, cannot be replaced by BrU and are therefore critical in the splicing reaction.
Mol Biol Rep 1994 Mar
PMID:In vitro splicing of pre-mRNA containing bromouridine. 807 91

The regulatory chain of E. coli aspartate transcarbamylase (E.C. 2.1.3.2) is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.
J Mol Biol 1994 Sep 16
PMID:The activation of Escherichia coli aspartate transcarbamylase by ATP. Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains. 808 37


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