Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents.
Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.
Rajanna
B
B
Division of Natural Sciences, Selma University, AL 36701.
Chetty
C S
CS
McBride
V
V
Rajanna
S
S
D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors*; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D003545:Cysteine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D007700:Kinetics; D007854:Lead / Q000494:pharmacology*; D008297:Male; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011188:Potassium / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D007854:Lead; D010744:Phosphoric Monoester Hydrolases; D009597:4-Nitrophenylphosphatase; D005978:Glutathione; D003545:Cysteine; D011188:Potassium; D004229:Dithiothreitol