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Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to
Author(s) -
Randak Christoph,
Roscher Adelbert A.,
Hadorn Hans-Beat,
Assfalg-Machleidt Irmgard,
Auerswald Ennes A.,
Machleidt Werner
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00314-y
Subject(s) - chemistry , allosteric regulation , nucleotide , adenine nucleotide , recombinant dna , cystic fibrosis transmembrane conductance regulator , atp hydrolysis , biochemistry , biophysics , stereochemistry , microbiology and biotechnology , enzyme , atpase , biology , gene
CFTR‐NBF‐2 was expressed in Escherichia coli in fusion with glutathione‐ S ‐transferase, the soluble portion was purified and identified as a stuctured protein by its CD spectrum. Association reactions of the recombinant NBF‐2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP‐, ADP‐ and AMP‐affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)‐labelled adenine nucleotides occuring as a result of binding to NBF‐2. Best‐fit monophasic binding curves to the fluorescence data indicated K d values of 22 μM for TNP‐ATP, 39 μM for TNP‐ ADP and 2.1 μM for TNP‐AMP. The corrected K values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 μM for ATP, 92 μM for ADP and 12 μM for AMP. The recombinant NBF‐2 did not show any hydrolytic activity on ATP (detection limit 0.001 s'). Our findings support the concept of a central role of NBF‐2 in CFTR activity regulation acting as an allosteric switch between channel opening and closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF‐2.