Nucleotide binding to IAF-labelled Na+K+-ATPase measured by steady state fluorescence quenching by TNP-ADP

摘要: Abstract Nucleotide binding to 5-iodoacetamidofluorescein (IAF) labelled Na + K -ATPase was measured by steady state fluorescence quenching of the fluorescein label via energy transfer trinitrophenyl (TNP) nucleotide. TNP-nucleotides are valuable probes nucleotide ATPases. Interpretation these and other experiments in our laboratory using with rely on having a good model for interaction TNP-nucleotide enzyme. Sets curves obtained titrating enzyme TNP-ADP presence various concentrations ADP could not be adequately modelled simple single site. Therefore, we compare models which allow additional In two-site model, is second specific site unlabelled compete. two models, (in one case saturable, non-saturable) blocked or affected nucleotide, is, therefore, referred as non-specific TNP-nucleotide. The goal this work determine distinctly different physical pictures associated most accurately describes We find that IAF-labelled best described there classes binding: compete dissociation constants 0.13 μM 2.0 ADP: non-saturable TNP-ADP.