EVIDENCE THAT THE CATION OCCLUSION DOMAIN OF NA K-ATPASE CONSISTS OF A COMPLEX OF MEMBRANE-SPANNING SEGMENTS - ANALYSIS OF LIMIT MEMBRANE-EMBEDDED TRYPTIC FRAGMENTS/

Digestion of renal Na/K-ATPase with trypsin, in the presence of rubidi um and absence of calcium ions, produces so called ''19-kDa membranes, '' containing a C-terminal 19-kDa and smaller fragments (8-12 kDa) of the alpha chain, and a beta chain either intact or split into two frag ments (Karlish, S. J. D., Goldshleger, R., and Stein, W. D. (1990) Pro c. Natl. Acad. Sci. U. S. A. 87, 4566-4570). Cation occlusion is intac t. The cation sites are thought to be located within trans-membrane se gments, but the identity and number of segments involved is unknown. A nalysis of Ca2+-induced sensitization of 19-kDa membranes to proteolys is, and characterization of the limit membrane-embedded fragments, has provided some insight into this question. Calcium ions have been show n to compete with two rubidium ions for occlusion sites on 19-kDa memb ranes, with a high affinity (K-D approximate to 2.8 mu M, pH 7.5, 20 d egrees C). The kinetics of displacement of rubidium by calcium ions in dicate that competition is direct and is not an allosteric antagonism. At 37 degrees C, reversible displacement of rubidium ions by calcium ions is followed by an irreversible thermal inactivation of rubidium o cclusion. Calcium ions partially protect rubidium occlusion sites agai nst modification by the carboxyl reagent, N,N'-dicyclohexylcarbodiimid e. We propose that calcium ions, like rubidium ions, recognize carboxy l groups at the entrance to the cation sites, but the calcium ions do not become occluded and thus fail to protect 19-kDa membranes against further proteolysis or thermal inactivation. Upon displacement of occl uded rubidium, trypsin digests the Ca2+-bound and thermally inactivate d 19-kDa membranes, and all of the membrane-embedded fragments are tru ncated or are split in these conditions. A related finding is that the C-terminal sequence of the 19-kDa fragment (and alpha chain), E-T-Y-Y , is digested by carboxypeptidase Y only when the rubidium occlusion i s inactivated. Identification of the limit tryptic fragments indicates that polypeptide loops and the C-terminal tail of the 19-kDa fragment , N and C termini of the smaller fragments of the alpha chain, and bot h N and C termini of a 16-kDa fragment of the beta chain are split by proteolytic enzymes upon displacement of occluded rubidium. We conclud e that all fragments of 19-kDa membranes form a complex, which is stab ilized and protected against proteolytic enzymes upon occlusion of rub idium ions, and which relaxes upon displacement of occluded rubidium. The cation occlusion ''cage'' presumably consists of ligating groups f rom several trans-membrane segments. The interactions between the rubi dium occlusion sites and protected segments of the protein are necessa rily reciprocal, and so they must also affect properties of rubidium o cclusion. These interactions may be either direct or indirect such as those between the 16-kDa fragment of the beta chain and occlusion site s within transmembrane segments of the alpha chain. The arrangement of the limit tryptic fragments in the membrane is discussed within the f ramework of possible topological models. The results are suggestive of a 10-segment model of the alpha chain but do not exclude certain 8-se gment models.