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)

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.
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PMID:Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells. 131 16

Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA. The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP. About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides. Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A'. Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer. Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer. The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand. The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA. This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases. Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA. Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP. About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA.
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PMID:Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs. 824 Nov 39

The equilibrium nucleotide binding and oligomerization of bacteriophage T7 gene 4 helicases have been investigated using thymidine 5'-triphosphate (dTTP), deoxythymidine 5'-(beta, gamma-methylenetriphosphate)(dTMP-PCP), thymidine 5'-diphosphate (dTDP), adenosine 5'-triphosphate (ATP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). In the presence of nucleotide ligands, T7 helicases self-assemble into hexamers with six potential nucleotide binding sites that are nonequivalent both in the absence and in the presence of single-stranded DNA. All nucleotides tested bind with high affinity to three sites (K(d) = 5 x 10(-6) M, dTTP; 6 x 10(-7) M, dTMP-PCP; 4 x 10(-6) M, dTDP; 3 x 10(-5) M, ATP; 2 x 10(-6) M, ATP gamma S), while binding to the remaining sites is undetectable. Interestingly, nucleotide binding to the high-affinity sites exhibits positive cooperativity which is sensitive to protein concentration. This effect is a result of ligand binding-linked oligomerization wherein helicase oligomer equilibrium changes as a function of both nucleotide and protein concentration. A study of DNA binding shows that 1-2 NTPs bound per hexamer are sufficient for stoichiometric interaction between the helicase and DNA. Thus, the ring-shaped helicase hexamers assemble around DNA with one, two, or three NTPs bound to each hexamer. This study also examines the preferred use of dTTP for T7 helicase-catalyzed DNA unwinding by comparison with ATP, the more commonly used nucleotide ligand. ATP binds to the helicase with 6-fold weaker affinity than dTTP and promotes hexamerization as well as DNA binding. Nevertheless, DNA unwinding with ATP is at least 100-fold slower than with dTTP. Thus, the difference in ATP and dTTP utilization probably lies in a highly specific step in the coupling of NTP hydrolysis to DNA unwinding.
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PMID:Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases. 865 63

Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.
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PMID:DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required. 1082 54