Atsunobu Yoda

On the reversibility of binding of cardiotonic steroids to a partially purified (Na + K)-activated adenosinetriphosphatase from beef brain☆

Abstract Incubation of a partially purified preparation of NaK ATPase from beef brain with various cardiotonic steroids under conditions of maximum binding of the steroid to the enzyme, followed by dilution, shows that the binding of cardenolide aglycones to the enzyme is reversible but that the binding of bufadienolide aglycones is only partly reversible. The binding of all cardiac glycosides to the enzyme is completely irreversible, suggesting that the sugar in glycosidic linkage with the 3-position of the steroid plays an important role in irreversible binding.

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme.

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.

A new simple preparation method for NaK-ATPase-rich membrane fragments.

Abstract A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel ( Electrophorus electricus ) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.

Irreversible inhibition of adenosine triphosphatases, diglyceride kinase and phosvitin kinase of brain by diisopropylphosphorofluoridate

Abstract High activities of transport ATPase and diglyceride kinase, relative to other preparations, were observed in a deoxycholate-treated particulate fraction from guinea-pig cerebrum. Incubation of the enzyme preparation in various buffers showed an initial inactivation of the diglyceride kinase and a slight activation of the transport ATPase. Both enzymes were irreversibly inhibited on treatment with DFP. In the presence of strophanthidin both enzymes showed similar but not identical rates of inhibition by DFP. The effects of DFP concentration and pH on the inhibitions of both enzymes were similar. ATP protected both enzymes against DFP inactivation, but very much lower concentrations were required for protection of the transport ATPase. Mg 2+ was required for DFP inhibition of transport ATPase but not of diglyceride kinase, even though the latter could be shown to require Mg 2+ for activity. Strophanthidin or K + potentiated the inhibitory effects of DFP on transport ATPase but had no effect on the inhibition of diglyceride kinase. The brain enzyme preparation also contained phosvitin kinase. It was slightly inhibited by DFP during the first few minutes of incubation; no further inhibition was observed on longer incubation. Removal of a sulfhydryl inhibitor from the DFP preparation by fractional distillation did not reduce the inhibitory effect of DFP on the three enzymes. Under conditions in which the rate of inhibition of the transport ATPase by DFP was increased 2.5-fold by strophanthidin there was no increased phosphorylation of the enzyme preparation by [ 32 P]DFP. The possibility that the transport ATPase is a modified form diglyceride kinase, in which water has become the favored phosphate acceptor, is discussed. Tetraethylpyrophosphate showed about the same potency as DFP in inhibiting the transport ATPase.

Characteristics of the Electric Eel Na,K-ATPase Phosphoprotein

Publisher Summary This chapter discusses the characteristics of the electric eel Na, K-ATPase phosphoprotein. The cleavage of ATP by the Na, K-ATPase proceeds through phosphorylated intermediates, the ADP- and K + -sensitive phosphoenzyrnes (E1P and E2P). In all Na, K-ATPase preparations known, E2P is the major component of the phosphorylated form, whereas E1P is the major component only in the presence of high concentrations of Na + or in an enzyme partially inhibited by N-ethylmaleimide (NEM). It is observed that the electric eel Na, K-ATPase prepared without any detergent produced mostly E1P in the presence of 100 mM Na + , but the microsomes of shark rectal gland, a fairly active form of Na, K-ATPase when prepared without detergent, produced mainly E2P. An increase in the Na concentration led to a higher E1P percentage in the shark enzyme. On the other hand, the eel enzyme formed mainly E2P in the presence of 10 mM Na + and did not show the oligomycin effect as in the shark enzyme in the presence of 100 m Na + . Therefore, it is concluded that the differences between eel and shark enzymes result from the different affinities of the two enzymes to Na + .

CCCP activation of the reconstituted NaK-pump

SummaryIn 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 PLs 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 (Vm), we postulated that aVm 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 μm ATP and 20 μm 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 byVm.