Characterization of ATP binding sites of sheep kidney medulla (Na+ + K+)--ATPase using CrATP.
The nucleotide substrate sites of sheep kidney medulla (NA+ + K+)-ATPase are characterized using CrATP, a paramagnetic, substitution-inert substrate analogue probe. The paramagnetic effect of CrATP on 1/T1 of water protons of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP in the presence of Na+ and K+ yielded characteristic enhancements for the binary enzyme-CrATP and ternary enzyme-Mg2+-CrATP complexes of 3.3 and 3.6 and dissociation constants for CrATP of 5 and 12 microM, respectively. Substitution of Li+ for K+ in these titrations did not substantially alter the titration behavior. From the frequency dependence of 1/T1, the correlation time, tau c, for the dipolar water proton-CrATP interaction is 2.7 x 10(-10) sec, indicating that tau c is dominated by tau s, the electron spin relaxation time of Cr3+. The paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn2+ complex with CrATP decreases the characteristic enhancement due to Mn2+ from 6.6-8.0 to 1.5. The failure to observe free Mn2+ epr signals in solutions of the ATPase, Mn2+, and CrATP demonstrate that this decrease in epsilon Mn is due to cross-relaxation between Mn2+ and Cr3+ bound simultaneously to the enzyme, and not to displacement of Mn2+ from the enzyme by CrATP. The relaxation rate, 1/T1, of 7Li+ is increased upon addition of CrATP to solutions of the ATPase, indicating that the sites for Li+ and CrATP are close on the enzyme. A Cr3+-Li+ distance of 4.8 +/- 0.5 angstrom is calculated from that data.
D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002857:Chromium / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D011485:Protein Binding; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D002857:Chromium; D008345:Manganese; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase