INTERACTIONS BETWEEN FRAGMENTS OF TRYPSINIZED NA,K-ATPASE DETECTED BYTHERMAL 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 retain s cation occlusion and ouabain binding. Previously, we reported rapid thermal inactivation of Rb+ occlusion at 37 degrees C (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 the rmal inactivation. In the range 25-35 degrees C, a two-step model (N < -> U --> I, where N is the native species, U is the reversibly unfolde d intermediate, and I is the irreversibly denatured form) fits the dat a. Reversibility of inactivation has been observed at 25 degrees C, co nsistent with the model. At 37 degrees C and higher temperatures, the data can be fitted to the simple mechanism N --> I, i.e. U is not sign ificant as an intermediate. Occluded cations (Na+, Rb+, K+, Tl+, NH4+, and Cs+) and ouabain protect strongly against thermal inactivation. C a2+, Ba2+, and La3+ ions do not protect. Proteolysis experiments provi de independent evidence that disorganization can occur in stages, firs t in transmembrane segments and then in extra-membrane segments of the fragments. Analysis of selective dissociation of the M5/M6 fragment a t 37 degrees C (Lutsenko, S., and Kaplan, J. H. (1995) Proc. Natl. Aca d Sci. U.S.A. 92, 7936-7940), using a specific antibody, showed that i nactivation of Rb+ occlusion precedes dissociation of the fragment, an d only similar to 50% of the fragment is released when occlusion is fu lly inactivated. In the presence of Ca2+ ions, occlusion is inactivate d, but the M5/M6 fragment is not released. The experiments demonstrate that occlusion is inactivated by disruption of interactions between f ragments of 19-kDa membranes, and only then does the M5/M6 fragment di ssociate. Interactions between the M5/M6 and M7/M10 fragments seem to be essential for maintenance of Rb+ occlusion.