Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in α- and β-subunits of Na+,K+-ATPase by Raman spectroscopy

AbstractRaman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including α-subunit hydrophobic regions as well as the intact β-subunit and hydrophobic regions of α- and β-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the α-helical conformation; (ii) essential contents of the α-helix as well as β-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase α-subunit; (iii) the exoplasmic hydrophilic domain of the β-subunit is shown to include several antiparallel β-pleated sheets and a small amount of the α-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations