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 rubidium 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 fragments (Karlish, S. J. D., Goldshleger, R., and Stein, W.D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4566-4570). Cation occlusion is intact. The cation sites are thought to be located within trans-membrane segments, but the identity and number of segments involved is unknown. Analysis of Ca(2+)-induced sensitization of 19-kDa membranes to proteolysis, and characterization of the limit membrane-embedded fragments, has provided some insight into this question. Calcium ions have been shown to compete with two rubidium ions for occlusion sites on 19-kDa membranes, with a high affinity (KD approximately 2.8 microM, pH 7.5, 20 degrees C). The kinetics of displacement of rubidium by calcium ions indicate 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 occlusion. Calcium ions partially protect rubidium occlusion sites against modification by the carboxyl reagent, N,N'-dicyclohexylcarbodiimide. We propose that calcium ions, like rubidium ions, recognize carboxyl 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 occluded rubidium, trypsin digests the Ca(2+)-bound and thermally inactivated 19-kDa membranes, and all of the membrane-embedded fragments are truncated 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 is 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 both N and C termini of a 16-kDa fragment of the beta chain are split by proteolytic enzymes upon displacement of occluded rubidium.4+ We conclude that all fragments of 19-kDa membranes form a complex, which is stabilized and protected against proteolytic enzymes upon occlusion of rubidium ions, and which relaxes upon displacement of occluded rubidium. The cation occlusion "cage" presumably consists of litigating groups from several trans-membrane segments.(ABSTRACT TRUNCATED AT 400 WORDS)
Shainskaya
A
A
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
Karlish
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002462:Cell Membrane / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
D010446:Peptide Fragments; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D002118:Calcium