A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase

Christoph Randak; Peter Neth; Ennes A Auerswald; Christoph Eckerskorn; Irmgard Assfalg-Machleidt; Werner Machleidt

doi:10.1016/S0014-5793(97)00574-7

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A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase

Journal article

Peer reviewed

A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase

Christoph Randak, Peter Neth, Ennes A Auerswald, Christoph Eckerskorn, Irmgard Assfalg-Machleidt and Werner Machleidt

FEBS letters, Vol.410(2), pp.180-186

1997

DOI: 10.1016/S0014-5793(97)00574-7

PMID: 9237625

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Abstract

CFTR–NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP→ADP+P i by three different assays, monitoring ATP turnover, formation of ADP and release of P i ( K m 86 μM, rate constant 0.37 min −1). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and P i was demonstrated ( K m 40 μM, rate constant 0.29 min −1). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR–NBF-2 by the inability of TNP–ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap 5A. As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.

Cystic fibrosis

ATPase activity

Adenylate kinase

Cystic fibrosis transmembrane conductance regulator

GTPase activity

Details

Title: Subtitle: A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase
Creators: Christoph Randak - Kinderklinik, Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität München, Lindwurmstraße 4, D-80337 München, Germany
Peter Neth - Kinderklinik, Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität München, Lindwurmstraße 4, D-80337 München, Germany
Ennes A Auerswald - Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgischen Klinik und Poliklinik, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, München, Germany
Christoph Eckerskorn - Max-Planck-Institut für Biochemie, Martinsried, Germany
Irmgard Assfalg-Machleidt - Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universität München, München, Germany
Werner Machleidt - Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universität München, München, Germany
Resource Type: Journal article
Publication Details: FEBS letters, Vol.410(2), pp.180-186
Publisher: Elsevier B.V
DOI: 10.1016/S0014-5793(97)00574-7
PMID: 9237625
ISSN: 0014-5793
eISSN: 1873-3468
Language: English
Date published: 1997
Academic Unit: Pulmonary Medicine; Stead Family Department of Pediatrics
Record Identifier: 9984093595802771

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