Investigation of Na(+),K(+)-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism.
Charge translocation by Na(+),K(+)-ATPase was investigated by adsorbing membrane fragments containing Na(+),K(+)-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na(+),K(+)-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na(+) and K(+) ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme alpha-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na(+),K(+)-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na(+) but not of K(+), the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na(+),K(+)-ATPase.
Tadini-Buoninsegni
Francesco
F
Department of Chemistry, Florence University, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy.
Nassi
Paolo
P
Nediani
Chiara
C
Dolfi
Andrea
A
Guidelli
Rolando
R
D017766:Acid Anhydride Hydrolases / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002412:Cations; D002918:Chymotrypsin; D025802:Electric Capacitance; D004800:Enzymes, Immobilized / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D011189:Potassium Chloride; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013552:Swine; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate; D002412:Cations; D002918:Chymotrypsin; D004800:Enzymes, Immobilized; D011189:Potassium Chloride; D012965:Sodium Chloride; D000254:Sodium-Potassium-Exchanging ATPase; D000097024:Acylphosphatase; C117608:alpha-chymotrypsin