Two different phosphorylation-dephosphorylation cycles of Na,K-ATPase proteoliposomes accompanying Na+ transport in the absence of K+

The phosphorylated intermediate (EP) of the Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme is composed of an ADP-sensitive K+-insensitive form (E1P), an ADP- and K+-sensitive form (E*P), and a K+-sensitive ADP-insensitive form (E2P). The composition of the intermediate varied with the cholesterol content of the lipid bilayer. The PL containing less than 30 mol % cholesterol (LCPL) formed E2P-rich EP in the presence of 10 mM Na+ on both sides at 15 degrees C, while the PL containing more than 35 mol % cholesterol (HCPL) formed E*P-rich EP under the same condition. In the presence of ionophore (monensin, nigericin, A23187), the HCPL formed E2P-rich EP as reported in the preceding paper. The turnover rate of Na-ATPase activity (the ratio of Na-ATPase to the EP level) in the LCPL was lower than that in the HCPL, and the addition of 20 microM monensin or A23187 to the HCPL reduced the Na-ATPase activity. The coupling ratio of Na+ influx (cellular efflux):Na+ efflux (cellular influx):ATP hydrolysis was 2.8:1.8:1 in the LCPL, although 1.6:0.6:1 in the HCPL. The coupling ratio of Na+ influx:ATP hydrolysis in the HCPL increased to 2.8:1 in the presence of A23187. Moreover, the increase of ATP concentration enhanced not only the Na-ATPase activity in the LCPL and HCPL with monensin but also the Na+ influx in the LCPL. This ATP enhancement was not found, however, in the HCPL without ionophores. The ADP enhancement of the Na+ influx was not observed in either the HCPL or the LCPL. We conclude from these observations that there are at least two different phosphorylation-dephosphorylation cycles (an E2P cycle and an E*P cycle) in the PL in the absence of K+. The E2P cycle transports three Na+ from the extravesicular (cytoplasmic) to the intravesicular (extracellular) side and two Na+ in the opposite direction per cycle and is similar to the ATP-dependent Na+-Na+ exchange system already reported (Blostein, R. (1983) J. Biol. Chem. 258, 7948-7953; Cornelius, F., and Skou, J. C. (1985) Biochim. Biophys. Acta 818, 211-221). However, the E*P cycle transports one Na+ from the extravesicular to the intravesicular side/cycle and has not yet been previously reported.