Abstract
Information on cation occlusion sites of renal NA,K-ATPase has been obtained by comparing the ability of competitive Na-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149) with that of transported alkali metal cations to protect against covalent modification and structural perturbations of the protein. Sodium antagonists include p- or m-xylylenebisguanidium, guanidinium ions, and ethylenediamine. Experiments with proteoliposomes reconstituted with Na,K-ATPase demonstrate that p-xylylenebisguanidium has pronounced selectivity for the cytoplasmic surface. Tryptic digestion of Na,K-ATPase leading to "19-kDa membranes," a specifically truncated enzyme with intact cation occlusion sites, requires the presence of alkali metal cations. Sodium antagonists do not protect 19-kDa membranes against further digestion, and occlusion is destroyed. Incubation of 19-kDa membranes at 37 degrees C, in the absence of occluded ions, leads rapidly to loss of ability to occlude rubidium ions. Rubidium, sodium, or other alkali metal cations protect fully, whereas sodium antagonists do not protect against this thermal inactivation. Like the alkali metal cations, sodium antagonists protect Na,K-ATPase and, somewhat less effectively, 19-kDa membranes against inactivation by the carboxyl reagent N,N'-dicyclohexylcarbodiimide. Cation occlusion from the cytoplasmic surface is suggested to occur in two steps. In an initial recognition, either transported cations or sodium antagonists interact with carboxyl groups. The second step is selective for transported cations and involves occlusion of cations (either potassium or sodium ions) and a conformational change to a compact structure, which is resistant to proteolysis and thermal inactivation. Sodium antagonists are sterically hindered from becoming occluded and block Na,K-ATPase activity. Implications for the structural basis of cation specificity of the Na/K pump are discussed.
Highlights
ATPase has been obtained by comparing the abiliotyf refers to the fact that ions are trapped within the protein and competitive Na-like antagonists
Chem. 267,1141-1149) with thatof transported characterized thoroughlyat the kinetilcevel (Glynn and Richalkali metal cations to protect against covalentmodi- ards, 1982; Glynn et al, 1985; Forbush, 1987), little is known fication and structural perturbations of the protein. about the protein structureisnvolved in cation occlusion
Like the alkali metal cations, These compounds block Na,K-ATPase activity and stabilize sodium antagonists protect Na,K-ATPase and, some- the E, conformation of the enzyme(David et al, 1992), what less effectively, 19-kDa membranes against in- suggesting that mXBG and pXBG cabne classed as competactivation by the carboxyl reagent N,N”dicyclohex- itive sodium antagonists
Summary
ATPase has been obtained by comparing the abiliotyf refers to the fact that ions are trapped within the protein and competitive Na-like antagonists The resultsof Na/K exchange experiments are compatible V,,, of Rb/Rb exchange sustained by right side-out pumps with the notion that pXBG is a competitive antagonist of by about 20%, andcaused a small but insignificant decrease sodium ions at the cytoplasmic surface (see Table II), in apparent rubidium affinity from 158 k 35 to 211 k 30 FM
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