Abstract
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.
Highlights
The phosphorylated intermediate( E P ) of t h e Na,K- The Na-ATPase reaction of Na,K-ATPaseis believedto be ATPase proteoliposomes (PL) prepared fromthe elec- the phosphorylation-dephosphorylationcycle wherethe phostric eel enzyme is composed of a n ADP-sensitive K+- phorylated enzyme (EP)' is the reaction intermediate (1,2)
Na'-Na' exchange has been seen in the red cells and ghosts
Na+ transport was not observed in any systems containing. This result indicates that E*P, the main EP component in E*P-rich EP or E2P-rich EP, even though HCPL forms the HCPL, dephosphorylates through a pathway
Summary
For determination of the ADP-insensitive EP and K+-insensitive EP contents, the phosphorylation was terminated by the addition of 0.5 ml of the 180 mM Tris3/CDTA with or without 1.5 mM ADP and/or 15 mM KC1 plus 60 p~ nigericin, and~after s the the value of the control experiments within the standard error (+ 6%).For the influx measurement, the same aliquot (0.5 ml) of the original PL suspension was incubated overnight at 0 "C without [22Na]NaC1and diluted 10-foldwith the reaction medium containing 25 pCi of [22Na]NaC1T. For the ATP hydrolysis (= Na-ATPase activity) measurement, the same reaction medium as that for the influx measurement was used without phosphocreatine and creatine kinase, and concentration of the PL was one-twentieth of that for the flux assay. The other assay procedures are the same as described above
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