The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.
Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of ouabain play a crucial role. In the present study, a series of ouabain analogues were tested on baculovirus-expressed Na,K-ATPase and an ouabain-sensitive mutant of non-gastric H,K-ATPase (D312E/ S319G/ A778P/ I795L/ F802C). For each analogue, the results obtained by measuring ATPase inhibition and [(3)H]ouabain replacement agreed rather well. In Na,K-ATPase, strophanthidin had a 7-10 times higher and digoxin a 4-12 times lower affinity than ouabain. The results of the non-gastric H,K-ATPase mutant were rather similar to that of Na,K-ATPase with exception of dihydro-ouabain that showed a much lower affinity with the non-gastric H,K-ATPase mutant. Docking studies showed that all analogues bind to the same pocket in Na,K-ATPase. However, the amino acids to which hydrogen bonds were formed differed and depended on the availability of hydroxyl or keto groups in the ouabain analogues.
De Pont
Jan Joep H H M
JJ
Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. J.dePont@ncmls.ru.nl
Swarts
Herman G P
HG
Karawajczyk
Anna
A
Schaftenaar
Gijs
G
Willems
Peter H G M
PH
Koenderink
Jan B
JB
D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005768:Gastrointestinal Hormones / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010042:Ouabain / Q000031:analogs & derivatives / Q000378:metabolism*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D005768:Gastrointestinal Hormones; C052257:hydrogen potassium ATPase, gastric endogenous activator protein, mammal; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase