pivotal residue for cation-induced conformational changes
نویسندگان
چکیده
The cation binding characteristics of the mutant E327A formed in the sheep al isoform of the Na+,K+-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep al protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K+-ATPase (K, = 12.8 + 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 + 2.5 mM). Potassium inhibition of [3H]ouabain binding displayed a partially competitive nature with Hill co-
منابع مشابه
Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102.
The calcium-induced conformational changes of the 108-amino acid residue proteins, cod III parvalbumin and oncomodulin, were compared using tryptophan as a sensitive spectroscopic probe. As native oncomodulin is devoid of tryptophan, site-specific mutagenesis was performed to create a mutant protein in which tryptophan was placed in the identical position (residue 102) as the single tryptophan ...
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