Abstract
Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.
Highlights
The lanthanides appear to biantda singlesite and Martonosi, 1986;Hanel and Jencks, 1990;Squieroetal., and actas competitive antagonists, without themselves1990)
Characterization of Lanthanum as a Competitor of Rb+ and Nu' Occlusion-Because lanthanidesaretrivalent cations they might affect (Na+,K')-ATPase activity in a variety of ways, including binding a t monovalent and/or divalent metal sites orbinding to ATP
La3+appears to behave competi- andJencks, 1990; Fujimori and Jencks, 1990) lanthanides tively with respect to Na+, in that Na' affinity is reduced in compete with Mg2+at the (M$+)-ATPbinding site
Summary
FITC labeling of the (Na+,K+)-ATPasweas as described previously (Rephaeli et al, 1986; Karlish, 1980). Equilibrium fluorescence measurements were performed at room temperature using a Perkin-Elmer MPF 4A spectrofluorimeter.Excitation and emission wavelengths were 495 and 520 nm, respectively; the slit width was 10 nm and the time constant was 0.3 s. Excitation of the FITC-labeled enzyme was at 480 nm (slit width, 2 nm),andemitted light was recorded after passage through a Schott cut-off filter OG 513. The ADS1 software for signal recording, saving, averaging, and nonlinear regression analysis was used to handle the data. Rb' occlusion at different Rb' concentrations in the absence or presence of La3+. To enzyme (0.014 mg/ml) in Tris-HC1, 150 mM, pH 7.0, 2 p M of Tb:" were added in the presence or absence of 4 mM Rb' a t 4 "C for 10 min.
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