CCCP activation of the reconstituted NaK-pump |
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Authors: | Atsunobu Yoda Shizuko Yoda |
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Institution: | (1) Department of Pharmacology, University of Wisconsin Medical School, 53706 Madison, Wisconsin |
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Abstract: | Summary In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15°C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (V
m
), we postulated that aV
m
build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K+ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London)
387:331–355). Although cytoplasmic K+ accelerated the change of ADP-and K+-sensitive EP (E*P) to K+-sensitive ADP-insensitive EP (E2P), CCCP did not compete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 m ATP and 20 m palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E*P content and decreased the ADP-sensitive K+-insensitive EP (E1P). The results described above suggest that CCCP affects the E1P to E*P change in the E1P E*P E2P conversion and that this reaction step is inhibited byV
m
. |
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Keywords: | NaK-pump CCCP membrane potential NaK-ATPase proteoliposome voltage-sensitive step EP conversion ionophore |
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