Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na,K-ATPase.
The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per ATPase alpha beta dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 +/- 11 microseconds at 0 degree C and 44 +/- 4 microseconds at 20 degrees C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.
Mahaney
J E
JE
Department of Chemistry, University of Virginia, Charlottesville 22901.
Girard
J P
JP
Grisham
C M
CM
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D007668:Kidney / Q000201:enzymology; D010042:Ouabain; D012399:Rotation; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013113:Spin Labels; D013696:Temperature
D003497:Cyclic N-Oxides; D013113:Spin Labels; D010042:Ouabain; C042847:4-amino-2,2,6,6-tetramethylpiperidin-1-oxy-ouabain; D000254:Sodium-Potassium-Exchanging ATPase