A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein

Christoph Randak; Peter Neth; Ennes A Auerswald; Irmgard Assfalg-Machleidt; Adelbert A Roscher; Hans-Beat Hadorn; Werner Machleidt

doi:10.1016/S0014-5793(96)01217-3

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A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein

Journal article

Peer reviewed

A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein

Christoph Randak, Peter Neth, Ennes A Auerswald, Irmgard Assfalg-Machleidt, Adelbert A Roscher, Hans-Beat Hadorn and Werner Machleidt

FEBS letters, Vol.398(1), pp.97-100

1996

DOI: 10.1016/S0014-5793(96)01217-3

PMID: 8946960

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Abstract

Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a K d value of 3.9 μM. The corrected K d values for unlabelled guanine nucleotides were determined to be 33 μM for GTP, 92 μM for GDP and 217 μM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min −1. Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.

Nucleotide binding

Cystic fibrosis

G-protein

Guanosine triphosphate

Cystic fibrosis transmembrane conductance regulator

GTPase activity

Details

Title: Subtitle: A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein
Creators: Christoph Randak - Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Lindwurmstraβe 4, D-80337 Munich, Germany
Peter Neth - Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Lindwurmstraβe 4, D-80337 Munich, Germany
Ennes A Auerswald - Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Klinikum Innendtadt der Ludwig-Maximilians-Universität, Munich, Germany
Irmgard Assfalg-Machleidt - Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-Universität, Munich, Germany
Adelbert A Roscher - Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Lindwurmstraβe 4, D-80337 Munich, Germany
Hans-Beat Hadorn - Kinderklinik im Dr. von Haunerschen Kinderspital der Ludwig-Maximilians-Universität München, Lindwurmstraβe 4, D-80337 Munich, Germany
Werner Machleidt - Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-Universität, Munich, Germany
Resource Type: Journal article
Publication Details: FEBS letters, Vol.398(1), pp.97-100
DOI: 10.1016/S0014-5793(96)01217-3
PMID: 8946960
NLM abbreviation: FEBS Lett
ISSN: 0014-5793
eISSN: 1873-3468
Publisher: Elsevier B.V
Language: English
Date published: 1996
Academic Unit: Pulmonary Medicine; Stead Family Department of Pediatrics
Record Identifier: 9984093481102771

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