Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to

FEBS Letters

Machleidt, Werner¹  Assfalg-Machleidt, Irmgard¹  Auerswald, Ennes A.²  Hadorn, Hans-Beat³  Randak, Christoph³  Roscher, Adelbert A.³ 

[1] Institut für Physiologische Chemie, Physikalische Biochemie and Zellbiologie der Ludwig-Maximilians-Universitdt, München, Germany;Abteilung fur Klinische Chemie and Klinische Biochemie in der Chirurgischen Klinik and Poliklinik, Klinikum Innenstadt der Ludwig-Maximilians-Universität, München, Germany;Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität, München, Lindwurmstraße 4, D-80337 München, Germany

关键词: Cystic fibrosis;    Cystic fibrosis transmembrane conductance regulator (CFTR);    Nucleotide binding;    Fluorescence enhancement;    Inner filter effect;    ATPase activity;    CFTR;    cystic fibrosis transmembrane conductance regulator;    CF;    cystic fibrosis;    G Cl;    Cl− conductance;    GST;    glutathione-S-transferase;    GST-NBF-2;    glutathione-S-transferase-CFTR-NBF-2 fusion protein;    IPTG;    isopropyl-β-d-thiogalactoside;    NBF;    nucleotide binding fold;    PBS;    phosphate-buffered saline;    PMSF;    phenylmethylsulfonylfluoride;    TNP-ATP;    2′(3′)-O-(2;    4;    6-trinitrophenyl) adenosine 5′-triphosphate;    TNP-ADP;    2′(3′)-O-(2;    4;    6-trinitrophenyl) adenosine 5′-diphosphate;    TNP-AMP;    2′(3′)-O-(2;    4;    6-trinitrophenyl) adenosine 5′-monophosphate;    AK;    adenylate kinase;   

DOI  :  10.1016/0014-5793(95)00314-Y

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown