Inhibition by lead of human erythrocyte (Na+ + K+)-adenosine triphosphatase associated with binding of 210Pb to membrane fragments

Abstract

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.

In vitro Inhibition of Na-K-ATPase by Trace Metals: Relation to Renal and Cardiovascular Damage

“…We found a 50% inhibition of renal Na-K-ATPase at a con centration o f 7 x 10 5M, which is similar to that described for human kidney Na-K-ATPase [48]. Pb causes a very slowly reversible inhibition of the enzyme [31] which is reduced when the concentration of ATP is increased [48], i.e. a competitive inhibition with regard to its substrate as found in the present study.…”

“…Many of the trace metals investigated in the present study accumulate pre ferentially in the kidney cortex, e.g., Cd [17], Cu [18], Hg [19], Ni [20], Pb [21], Ur [22,23], and may thereby cause metabolic a n d /o r morphological alterations at structures located within this region of the kidney. They may cause damage to mitochondria, e.g., Cd [17], Cu [24][25][26], Hg [19], Pb [27], Zn [28], or accumulate in ATPase-rich microsomes, e.g., Zn [29], Cu [30], Pb [31], and in the cyto-plasma, e.g. Ni [20], Pb [21], Zn [29], of the proximal tubular epithelium.…”

Control of the Na+, K+-ATPase under normal and pathological conditions

“…PbC1 2 inhibits the phosphatase activity of the human erythrocyte Na + ,K+-ATPase (Caspers and Siegel, 1980), as well as that of rat brain (Siegel et al, 1977). This metal ion can inhibit [ 3H]ouabain binding to the high affinity forms of the cerebromicrovascular Na + ,K+ -ATPase (Caspers et al, 1990) in the same concentration range as that found in the cerebral endothelium during experimental lead intoxication (Toews et al, 1978).…”

Low Lead Doses and Atherogenic Diet in Rabbits: Biochemical Results in Blood

“…The drop in erythrocyte magnesium from the 42nd day on may be explained by a change in the activity of mem brane adenosine triphosphatases (ATPases): Na+-K+-ATPase and Mg2+-ATPase [36], In effect, Torkhovskaya et al [37] have recently demonstrated that an atherogenic diet in rab bits results in decreased activity of erythro cyte membrane Na+-K+-ATPase. Moreover, Caspers and Siegel [38], in a study of the effect of lead in vitro on fragmented human erythrocyte membranes, have indicated that lead acts as an irreversible or very slowly reversible inhibitor of Na+-K+-ATPase and Mg2+-ATPase activities.…”