The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase.
Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.
González-Lebrero
Rodolfo M
RM
Instituto de Química y Fisicoquímica Biológicas y Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina. rcr@mail.retina.ar
Kaufman
Sergio B
SB
Garrahan
Patricio J
PJ
Rossi
Rolando C
RC
D000818:Animals; D001665:Binding Sites; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D008024:Ligands; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D012413:Rubidium; D011188:Potassium