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

C Randak; A A Roscher; H B Hadorn; I Assfalg-Machleidt; E A Auerswald; W Machleidt

doi:10.1016/0014-5793(95)00314-Y

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

Journal article

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

C Randak, A A Roscher, H B Hadorn, I Assfalg-Machleidt, E A Auerswald and W Machleidt

FEBS letters, Vol.363(1-2), pp.189-194

04/17/1995

DOI: 10.1016/0014-5793(95)00314-Y

PMID: 7537226

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Abstract

CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a structured 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 occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s-1). 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.

Gene Expression

Sepharose

Adenosine Monophosphate - metabolism

Membrane Proteins - genetics

Glutathione Transferase - metabolism

Molecular Sequence Data

Trinitrobenzenes

Recombinant Fusion Proteins - chemistry

Glutathione Transferase - chemistry

Nucleotides - metabolism

Recombinant Fusion Proteins - metabolism

Cystic Fibrosis Transmembrane Conductance Regulator

Protein Folding

Hydrolysis

Glutathione Transferase - genetics

Membrane Proteins - chemistry

Escherichia coli - genetics

Adenosine Triphosphate - metabolism

Base Sequence

Membrane Proteins - metabolism

Adenosine Diphosphate - metabolism

Binding Sites

Details

Title: Subtitle: Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to glutathione-S-transferase
Creators: C Randak - Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität, München, Germany
A A Roscher
H B Hadorn
I Assfalg-Machleidt
E A Auerswald
W Machleidt
Resource Type: Journal article
Publication Details: FEBS letters, Vol.363(1-2), pp.189-194
DOI: 10.1016/0014-5793(95)00314-Y
PMID: 7537226
ISSN: 0014-5793
eISSN: 1873-3468
Language: English
Date published: 04/17/1995
Academic Unit: Pulmonary Medicine; Stead Family Department of Pediatrics
Record Identifier: 9984093486002771

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