Characterization of Disulfide Cross-links between Fragments of Proteolyzed Na,K-ATPase

This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10 in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864). Upon solubilization, cross-links form spontaneously between the beta subunit, 19- and 11.7-kDa fragments of the alpha subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of beta-19 kDa dimers ( approximately 65%) and beta-19 kDa-11.7 kDa trimers ( approximately 35%). The alpha-beta cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that betaCys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced by o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197-8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the alpha and beta subunits.