It is now fairly well established that an adenosine triphosphatase (ATPase) which is present in a variety of membrane preparations is closely related to coupled Na+ and K+ transport.1-12 This is based in part on its requirement for the simultaneous presence of Na+ and K+ and its inhibition by cardioactive steroids such as ouabain. It is not known whether the ATPase reflects the actual operation of coupled Na+ and K+ transport in the membrane fragments or whether it is uncoupled from the overall Na+ and K+ transport system. It has generally been thought that the "transport ATPase" involves the formation of one or more phosphorylated intermediates. Evidence has been presented which argues against phosphatidic acid,13' 15polyphosphoinositides,14' 15 and peptidically bound phosphoserinel3 serving as intermediates. Recent studies'6-19 have indicated the presence of a phosphorylated intermediate which remains bound to protein in trichloroacetic acid precipitates of the ATPase preparation.An important question to be answered is the chemical nature of this phosphorylated intermediate. This paper is a report of preliminary studies on its characterization. A brain ATPase preparation was found to maintain high steady-state levels of the radioactive phosphorylated intermediate on incubation with ATP--y-P32 and Na+. Digestion of the denatured ATPase preparation with pepsin liberated radioactive compounds which migrated toward the cathode on electrophoresis on paper at pH 3.4. The radioactivity in these peptides was released as inorganic phosphate on incubation of pepsin digests with either hydroxylamine at pH 5.3 or with acyl phosphatase, suggesting that the phosphate bond in the phosphorylated intermediate is an acyl phosphate. The stability of the intermediate at various pH's is also compatible with its being an acyl phosphate compound.Experimental.-Preparation of ATPase: Eight guinea pig brains were each homogenized in 10 ml of sucrose-EDTA.E0 The pooled homogenates were centrifuged at 13,600 X g for 10 min. The supernatant fluid and the loosely packed buff-colored layer were decanted into a flask and mixed with an equal volume of 1 M NaCl and 0.2 vol of 0.75% Tris deoxycholate. The mixture was homogenized briefly and then centrifuged at 78,500 X g for 1 hr. The residue was resuspended in 160 ml of sucrose-EDTA and centrifuged again for 1 hr at 78,500 X g. The pellet was suspended in 10-20 ml of sucrose-EDTA, and the enzyme was stored in a dry-ice chest where it retained activity for at least one month. The "transport ATPase" was assayed as described previously;20 it averaged about 75 ,moles of ATP hydrolyzed per mg protein per hour at 37°C. The "nontransport" ATPase activity was about 10-20% of that of the "transport ATPase."Large-scale labeling of the enzyme with ATP-y-P32: A stock incubation solution was prepared by mixing together 19 ml of 3.8 mM ATP--y-P32, 7.2 ml of 10 mM MgCk, 72 ml of a buffer containing 35 mM Tris and 1.5 mM phosphoric acid,'7 72 ml of 0.28 M NaCl, and water to make a final volume of 180 ml. Thawe...
53 706 binations, the followings have been reported: Mg,3 Mg + ATP,3 Mg + Pi,3v6Mg + Na + ATP,2*3 Mg + a~e t a t e ,~~~ Mg + a r~e n a t e ,~ Mg + Na + nucleotide,69" Mn++,3 Na + Sr,3 Mg + p-nitrophenylphosphate,8>12 and Mg + acetyl phosphate.I2 The two ligand systems that are most effective are the Mg and Pi system and the Na, Mg, and ATP system.
In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na+ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanide m-chlorophenyl hydrazone (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 degrees 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 (Vm), we postulated that a Vm 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 complete with cytoplasmic K+ when cytoplasmic Na+ was saturated. When the PLs were phosphorylated with 20 microM ATP and 20 microM 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 by Vm.
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