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6-Aminopurines

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M. Kohlhardt – One of the best experts on this subject based on the ideXlab platform.

  • Na+ channel blockade by cyclic AMP and other 6-Aminopurines in neonatal rat heart
    The Journal of Membrane Biology, 1991
    Co-Authors: J. W. Herzig, M. Kohlhardt

    Abstract:

    Elementary Na+ currents were recorded at 19°C in cell attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the effect of cAMP and other 6-Aminopurines.

  • Na^+ channel blockade by cyclic AMP and other 6-Aminopurines in neonatal rat heart
    The Journal of Membrane Biology, 1991
    Co-Authors: J. W. Herzig, M. Kohlhardt

    Abstract:

    Elementary Na^+ currents were recorded at 19°C in cell attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the effect of cAMP and other 6-Aminopurines. The treatment of the cardiocytes with db-cAMP (1×10^−3 mol/liter) led to a decline of reconstructed macroscopic peak I _Na to 62±7.6% of the initial control value. This reduction in NP_0 was mostly accompanied by a decrease in burst activity. Openstate kinetics were preserved even in DPI-modified, noninactivating Na^+ channels. Since the stimulator of the adenylate cyclase, forskolin (1×10^−6 mol/liter), evoked a similar pattern of response, the NP_0 decrease can be considered as the functional correlate of Na^+ channel phosphorylation brought about by cAMP-dependent protein kinase. As found in inside-out patches, cAMP (1×10^−3 mol/liter) remained effective under cell-free conditions and reduced reconstructed macroscopic peak I _NA to about 50% of the initial control value when the absence of Mg-ATP at the cytoplasmic membrane surface prevents phosphorylation reactions. A very similar response developed in the cytoplasmic presence of other 6-Aminopurines including ATP (1×10^3 mol/liter), adenosine (1×10^−4 mol/liter), adenine (1×10^−5 mol/liter) and hypoxanthine (1×10^−5 mol/liter). This susceptibility to adenine suggests that cardiac Na^+ channels in situ could sense intracellular fluctuations of adenine nucleotides, most likely of ATP.

David T F Dryden – One of the best experts on this subject based on the ideXlab platform.

  • 2 aminopurine flipped into the active site of the adenine specific dna methyltransferase m taqi crystal structures and time resolved fluorescence
    Journal of the American Chemical Society, 2007
    Co-Authors: Thomas Lenz, David T F Dryden, Robert K Neely, Anita C Jones, Eleanor Y M Bonnist, Goran Pljevaljcic, Axel J Scheidig, Elmar G. Weinhold

    Abstract:

    We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state:  base flipping is accompanied by the loss of the very short (∼50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by π-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the propo…

  • evidence of tautomerism in 2 aminopurine from fluorescence lifetime measurements
    Journal of Physical Chemistry B, 2004
    Co-Authors: Robert K Neely, David T F Dryden, Steven W Magennis, Anita C Jones

    Abstract:

    The fluorescence decay characteristics of 2-aminopurine (2AP) and 2-aminopurine riboside (2APr) have been investigated as a function of excitation and emission wavelength in aqueous and ethanolic solutions. Global analysis of the decay data shows that 2AP exists as two emitting species, whereas 2APr exists as a single species. This is attributed to 9H/7H tautomerism of 2AP. The proportion of 7H tautomer is estimated to be 20% in ethanol and 40% in water.

  • unusual 2 aminopurine fluorescence from a complex of dna and the ecoki methyltransferase
    Nucleic Acids Research, 2004
    Co-Authors: Tsueuju Su, Bernard A Connolly, C Darlington, R Mallin, David T F Dryden

    Abstract:

    The methyltransferase, M.EcoKI, recognizes the DNA sequence 5¢-AACNNNNNNGTGC-3¢ and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosylmethionine to the M.EcoKI:DNA complex, the 2aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.

Elmar G. Weinhold – One of the best experts on this subject based on the ideXlab platform.

  • 2 aminopurine flipped into the active site of the adenine specific dna methyltransferase m taqi crystal structures and time resolved fluorescence
    Journal of the American Chemical Society, 2007
    Co-Authors: Thomas Lenz, David T F Dryden, Robert K Neely, Anita C Jones, Eleanor Y M Bonnist, Goran Pljevaljcic, Axel J Scheidig, Elmar G. Weinhold

    Abstract:

    We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state:  base flipping is accompanied by the loss of the very short (∼50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by π-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the propo…

  • 2 aminopurine as a fluorescent probe for dna base flipping by methyltransferases
    Nucleic Acids Research, 1998
    Co-Authors: Birgit Holz, Saulius Klimasauskas, Saulius Serva, Elmar G. Weinhold

    Abstract:

    DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3′- or 5′-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.