Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.
We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.
Berberián
G
G
División de Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra, Córdoba, Argentina.
Beaugé
L
L
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D008081:Liposomes; D015636:Magnesium Chloride / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010762:Phosphorylase a / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D008081:Liposomes; D009596:Nitrophenols; D010755:Organophosphates; D009943:Organophosphorus Compounds; D015636:Magnesium Chloride; C008644:nitrophenylphosphate; C011632:acetyl phosphate; D000244:Adenosine Diphosphate; D006003:Glycogen; D005954:Glucosephosphate Dehydrogenase; D010762:Phosphorylase a; D010744:Phosphoric Monoester Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium