Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1

András Kern, Ferenc Felföldi, B. Sarkadi, A. Váradi

Research output: Article

11 Citations (Scopus)

Abstract

The His6-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F4 inhibited the ATPase activity of both nucleotide binding domains. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)913-919
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume273
Issue number3
DOIs
Publication statusPublished - júl. 14 2000

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Adenosine Triphosphate
Nucleotides
Adenosine Triphosphatases
Maltose-Binding Proteins
Multidrug Resistance-Associated Proteins
Bacterial Proteins
Adenosine Monophosphate
Purification
Hydrolysis
Bacteria
Fusion reactions
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1",
abstract = "The His6-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F4 inhibited the ATPase activity of both nucleotide binding domains. (C) 2000 Academic Press.",
keywords = "ABC-transporters, ATP-binding, ATPase-activity, Multidrug resistance, Nucleotide binding domains",
author = "Andr{\'a}s Kern and Ferenc Felf{\"o}ldi and B. Sarkadi and A. V{\'a}radi",
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TY - JOUR

T1 - Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1

AU - Kern, András

AU - Felföldi, Ferenc

AU - Sarkadi, B.

AU - Váradi, A.

PY - 2000/7/14

Y1 - 2000/7/14

N2 - The His6-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F4 inhibited the ATPase activity of both nucleotide binding domains. (C) 2000 Academic Press.

AB - The His6-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F4 inhibited the ATPase activity of both nucleotide binding domains. (C) 2000 Academic Press.

KW - ABC-transporters

KW - ATP-binding

KW - ATPase-activity

KW - Multidrug resistance

KW - Nucleotide binding domains

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