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The (Na(+)+K+)-ATPase is responsible for maintenance of the ionic milieu of cells. The objective of this study is to investigate the effect of aluminum, an ion implicated in several neurological disorders, on ATP hydrolysis catalyzed by the rat brain synaptosomal (Na(+)+K+)-ATPase and on the binding of [3H]ouabain to this enzyme. AlCl3 (25-100 microM) inhibits the phosphatase activity of the (Na(+)+K+)-ATPase in a dose-dependent manner. AlCl3 appears to act as a reversible, noncompetitive inhibitor of (Na(+)+K+)-ATPase activity by decreasing the maximum velocity of the enzyme without significantly affecting the apparent dissociation constant with respect to ATP. AlCl3 may affect Mg2+ sites on the (Na(+)+K+)-ATPase but does not appear to interact with Na+ or K+ sites on the enzyme. In contrast to this inhibitory effect on the phosphatase function of the enzyme, AlCl3 (1-100 microM) stimulates the binding of [3H]ouabain to the (Na(+)+K+)-ATPase. This effect is due to an increase in the maximum [3H]ouabain binding capacity of the enzyme with no change in the [3H]ouabain binding affinity. These data support the hypothesis that AlCl3 may stabilize the phosphorylated form of the synaptosomal (Na(+)+K+)-ATPase which increases [3H]ouabain binding while inhibiting the phosphatase activity of the enzyme.

Fragmented human erythrocyte membranes were exposed to PbC12 for 10-40 min at 23°C prior to (Na÷+ K+)-ATPase assay. Inhibition increased with exposure time. Enzyme activity in 5 pg membrane protein was inhibited 50% after a 10-min exposure to 1.0 nmol PbC12 (25 pM final concentration) and was inhibited 100% after 40 rain. When membranes at various concentrations were exposed to PbC12 for 40 min, inhibition was linear with the ratio of PbC12 to protein. Inhibition of 100% was obtained at 0.2 nmol PbC12/pg protein. A graph of activity vs. [protein] in the presence of PbC12 intercepted the abscissa to the right of the origin, indicating that lead acts as an irreversible or very slowly reversible inhibitor. Addition of 1 mM 2,3-dimercaptopropanol, 1,3dithiothreitol, DL-penicillamine or EDTA after 40 min exposure to 100 pM PbC12 restored 45, 64, 81 and 92% of the (Na ÷ + K+)-ATPase, respectively. These chelators, excluding EDTA, prevented inhibition when added before PbC12. Two washings of the membrane fragments with water or 10 mM imidazole-HC1 (pH 7.4) did not restore activity. 21°Pb bound tightly to membrane fragments and beginning of saturation was observed at 0.19 nmol Pb 2÷ bound/ pg protein. This corresponded to 200 pM final concentration of PbC12 in the ATPase assay. At 0.2 nmol PbC12/pg protein {100% inhibition of ATPase), from 0.10-0.17 nmol of lead was bound per pg protein. Under the same conditions, 1 mM DL-penicillamine removed 80% of the bound lead which correlated with its restoration of ATPase activity. Pb 2÷ does not appear to * This work was presented to the Federation of American Societies for Experimental Biology, April 1-10, 1979, Dallas, TX, U.S.A. (Fed. Proc. (1979) 38,321.) 28 denature the enzyme. The irreversible kinetics may be related to sequestration of Pb 2÷ within vesicles that interfere with the accessibility of chelators to Pb 2÷ binding sites.

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