The interactions of Mn2+, Pi and nucleotide substrate and substrate analogs with highly purified membrane-bound (Na+ + K+)-ATPase from sheep kidney medulla were examined using EPR techniques. EPR studies of both native and partially delipidated ATPase preparations indicated that the enzyme binds Mn2+ at 1 tight site with a KD of 0.21 .times. 10-6 M. A 2nd class of 24 .+-. 3 weaker binding sites for Mn2+ was also observed in the native enzyme, but these were removed upon removal of 50% of the essential phospholipids in the enzyme preparation, confirming that these are lipid binding sites for Mn2+ as was previously suggested. The X-band EPR spectrum of the binary Mn2+-ATPase complex exhibited a powder or quasi-solid state line shape consisting of a braod transition with partial resolution of the 55Mn nuclear hyperfine structure and a broad component to the low-field side of the main pattern. The spectrum of the delipidated enzyme was significantly broadened compared with the native enzyme, with a loss in resolution of the hyperfine lines. Low concentrations of phosphate produced a shift toward the center of the spectrum for the broad low-field component, with additional smaller effects on the hyperfine structure of the spectrum. ATP, ADP, AMP-PNP and high concentrations of Pi all broadened the hyperfine lines of the Mn2+ spectrum, consistent with a change in coordination geometry of the bound Mn2+, a change in accessibility of the Mn2+ site to solvent, or both. Probably the true substrate for the enzyme is ATP and not Mn2+-ATP. In contrast to these effects, AMP caused a substantial narrowing of the Mn2+ spectrum, including the narrowing and eventual disappearance of the broad, low-field signal. These changes were consistent with a greatly reduced axial distortion of the Mn2+ geometry in the ternary ATPase-Mn2+-AMP complex. Addition of phosphate to this complex provided a very slight broadening, while the further addition of Na+ restored the broad, asymmetric spectrum observed in the binary ATPase-Mn2+ complex. By contrast, the addition of AMP to the delipidated enzyme-Mn2+ complex produced much different spectra, indicating unusual Mn2+ distortions and suggesting that lipid removal greatly alters the ability of the enzyme to interact with nucleotides. Apparently while the divalent metal and substrate sites are preserved in the delipidated enzyme, their conformations are substantially altered.
