Effects of lead and selected pyrethroids/piperonyl butoxide alone and in combination on Na, K‐ATPase

Abstract

In vitro effects of Pb2+, the pyrethroid insecticides cypermethrin, fenvalerate and the syner‐gist piperonyl butoxide on sodium‐potassium‐activated adenosine triphosphatase (Na,K‐ATPase) from dog kidney were determined. Pb2+ with an estimated IC50 value of 5.2 μM was found to be a potent inhibitor of Na,K‐ATPase activity, whereas Na,K‐ATPase was less sensitive to the pyrethroids tested and piperonyl butoxide. Investigation with circular dichroism (CD) spec‐troscopy showed that inhibition occurs through conformational changes of the α‐subunit of the enzyme. The kinetic characteristics of inhibition of Na,K‐ATPase with varying substrate (ATP) concentrations as well as with varying Na+ concentrations exhibited a competitive type of inhibition with Pb2+ in the μM range. With Pb2+ alone in the enzyme assay no conformational changes of the protein could be observed which confirmed the assumption that Pb2+ can bind to the Na+ binding site of the α‐subunit. Uncompetitive type of inhibition occurred with varied K+ concentrations demonstrating that this cation binding site is not affected directly by Pb2+. Complete reversal of Pb2+ by DTT confirms that a possible target for interaction of this heavy metal ion with Na, K‐ATPase are specific SH groups. Synergistic effects could only be determined with higher Pb2+ concentrations of 3, 5 and 7 μM plus piperonyl butoxide while all other combinations with this heavy metal plus organic substances where of the additive type. With CD spectroscopy also only additive effects were observed. These results demonstrate that higher concentrations of piperonyl butoxide favor the binding of Pb2+ to the Na+ binding site by conformational changes of the protein.