Studies of the ATPase activity of the ABC protein SUR1.
The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits. Binding and/or hydrolysis of Mg-nucleotides at the nucleotide-binding domains of sulfonylurea receptor 1 stimulates channel opening and leads to membrane hyperpolarization and inhibition of insulin secretion. We report here the first purification and functional characterization of sulfonylurea receptor 1. We also compared the ATPase activity of sulfonylurea receptor 1 with that of the isolated nucleotide-binding domains (fused to maltose-binding protein to improve solubility). Electron microscopy showed that nucleotide-binding domains purified as ring-like complexes corresponding to approximately 8 momomers. The ATPase activities expressed as maximal turnover rate [in nmol P(i).s(-1).(nmol protein)(-1)] were 0.03, 0.03, 0.13 and 0.08 for sulfonylurea receptor 1, nucleotide-binding domain 1, nucleotide-binding domain 2 and a mixture of nucleotide-binding domain 1 and nucleotide-binding domain 2, respectively. Corresponding K(m) values (in mm) were 0.1, 0.6, 0.65 and 0.56, respectively. Thus sulfonylurea receptor 1 has a lower K(m) than either of the isolated nucleotide-binding domains, and a lower maximal turnover rate than nucleotide-binding domain 2. Similar results were found with GTP, but the K(m) values were lower. Mutation of the Walker A lysine in nucleotide-binding domain 1 (K719A) or nucleotide-binding domain 2 (K1385M) inhibited the ATPase activity of sulfonylurea receptor 1 by 60% and 80%, respectively. Beryllium fluoride (K(i) 16 microm), but not MgADP, inhibited the ATPase activity of sulfonylurea receptor 1. In contrast, both MgADP and beryllium fluoride inhibited the ATPase activity of the nucleotide-binding domains. These data demonstrate that the ATPase activity of sulfonylurea receptor 1 differs from that of the isolated nucleotide-binding domains, suggesting that the transmembrane domains may influence the activity of the protein.
de Wet
Heidi
H
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Mikhailov
Michael V
MV
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Fotinou
Constantina
C
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Dreger
Mathias
M
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Craig
Tim J
TJ
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Vénien-Bryan
Catherine
C
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
Ashcroft
Frances M
FM
Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy and Genetics, University of Oxford, UKLaboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, UK.
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D058619:Maltose-Binding Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D011485:Protein Binding; D051381:Rats; D011955:Receptors, Drug; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011994:Recombinant Proteins; D064233:Sulfonylurea Receptors; C577509:Abcc8 protein, rat