Interactions between fragments of trypsinized Na,K-ATPase detected by thermal inactivation of Rb+ occlusion and dissociation of the M5/M6 fragment.

This work provides evidence for interactions between fragments of "19-kDa membranes," a trypsinized preparation of Na,K-ATPase that retains cation occlusion and ouabain binding. Previously, we reported rapid thermal inactivation of Rb+ occlusion at 37 degreesC (Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). We describe here the detailed kinetics of thermal inactivation. In the range 25-35 degreesC, a two-step model (N left and right arrow U --> I, where N is the native species, U is the reversibly unfolded intermediate, and I is the irreversibly denatured form) fits the data. Reversibility of inactivation has been observed at 25 degreesC, consistent with the model. At 37 degreesC and higher temperatures, the data can be fitted to the simple mechanism N --> I, i.e. U is not significant as an intermediate. Occluded cations (Na+, Rb+, K+, Tl+, NH4+, and Cs+) and ouabain protect strongly against thermal inactivation. Ca2+, Ba2+, and La3+ ions do not protect. Proteolysis experiments provide independent evidence that disorganization can occur in stages, first in transmembrane segments and then in extra-membrane segments of the fragments. Analysis of selective dissociation of the M5/M6 fragment at 37 degreesC (Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940), using a specific antibody, showed that inactivation of Rb+ occlusion precedes dissociation of the fragment, and only approximately 50% of the fragment is released when occlusion is fully inactivated. In the presence of Ca2+ ions, occlusion is inactivated, but the M5/M6 fragment is not released. The experiments demonstrate that occlusion is inactivated by disruption of interactions between fragments of 19-kDa membranes, and only then does the M5/M6 fragment dissociate. Interactions between the M5/M6 and M7/M10 fragments seem to be essential for maintenance of Rb+ occlusion.