Haruo Homareda

Ouabain-sensitive 42K binding to Na+, K+-ATPase purified from canine kidney outer medulla

Abstract Specific and ouabain-sensitive potassium binding to Na + , K + -ATPase was directly observed by centrifugation method with the purified enzyme and 42 K. The specific binding reached to saturation level at concentrations more than 0.2 mM KCl and the level was 6.2 nmol per mg ATPase with specific activity of 1470 μmol Pi/h·mg. The binding level, however, was proportional to the enzyme unit used. Simultaneous determination of 42 K binding and [ 3 H]ouabain binding showed that two mol of potassium binding were blocked by one mol of ouabain binding per 3.2×10 5 g enzyme. Although the apparent dissociation constant of the specific potassium binding was estimated at about 50 μM, Scatchard plot of the binding revealed non-linear relationship suggesting that the two potassium sites existed on one catalytic unit of enzyme would be not equivalent but cooperative.

Purification and characteristics of (Na+, K+)-ATPase from canine kidney by zonal centrifugation in sucrose density gradient.

Microsomes were prepared from the outer medulla of canine kidney. Partially purified preparation of (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was obtained by solubilization of microsomes with sodium deoxycholate, and by precipitation with dilution. The deoxycholate-enzyme thus obtained was further purified by incubation with sodium dodecyl sulphate in the presence of ATP followed by a single zonal centrifugation in a sucrose density gradient by the method of Jørgensen [(1974) Biochim. Biophys. Acta 356, 36-52]. The (Na+, K+)-ATPase was purified to a specific activity of 1600--1800 micronmol Pi - h-1 - mg-1 protein. The yield was 20 mg per single centrifugation with a zonal rotor. Electron microscopy showed that the sectioned pellet of the purified enzyme contained exclusively membranous fragments in contrast with membranous vesicles of starting microsomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that almost all proteins were accounted for by two polypeptides with molecular weights of 105 000 and 58 000, and that the mass ratio of the large to the small polypeptide was 82 : 18.

Binding domain of oligomycin on Na+,K+-ATPase

Oligomycin inhibits Na(+),K(+)-ATPase activity by stabilizing the Na(+) occlusion but not the K(+) occlusion. To locate the binding domain of oligomycin on Na(+),K(+)-ATPase, the tryptic-digestion profile of Na(+),K(+)-ATPase was compared with the profile of Na(+) occlusion within the digested Na(+),K(+)-ATPase in the presence of oligomycin. The Na(+) occlusion profile is responsible for the digestion profile of the alpha-subunit, which is the catalytic subunit of the ATPase. The effect of oligomycin on chimeric Ca(2+)-ATPase activity was examined. The chimera used, in which the 163 N-terminal amino acids of chicken sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 were replaced with the 200 N-terminal amino acids of the chicken Na(+),K(+)-ATPase alpha1-subunit, partially retains the Na(+)-dependent characteristics of Na(+), K(+)-ATPase, because the chimeric Ca(2+)-ATPase activity is activated by Na(+) but inhibited by ouabain, a specific inhibitor of Na(+),K(+)-ATPase (Ishii, T., Lemas, M.V., Takeyasu, K., 1994, Proc. Natl. Acad. Sci. U. S. A., 91, 6103-6107). Oligomycin depressed the activation by Na(+) of the chimeric Ca(2+)-ATPase activity. These findings suggest that the 200 N-terminal amino acids of the Na(+), K(+)-ATPase alpha-subunit include a binding domain for oligomycin.

Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.

To study the membrane insertion of the Na+,K+-ATPase (EC 3.6.1.37) alpha subunit with six to eight transmembrane segments, mRNAs encoding the entire alpha subunit and its four different domains were prepared and translated in rabbit reticulocyte lysate with rough microsomal membranes. On the basis of the resistance of the membrane-inserted products to alkali extraction and the failure to insert the translation products into N-ethylmaleimide-treated membranes, it is suggested that at least two signal sequences are contained within the four transmembrane segments from the amino terminus of the alpha subunit.

Immunochemical identification of exposed regions of the Na+,K+‐ATPase α‐subunit

A polyclonal antibody against the Na+,K+‐ATPase holoenzyme was prepared. This antibody recognized native Na+,K+‐ATPase and inhibited its activity. The peptide fragments corresponding to various regions of the Na+,K+‐ATPase α‐subunit, which were synthesized from the cDNA, were immunoprecipitated with the antibody, and the M32‐D75, M158‐D197 and M470‐V552 fragments (the latter included K508, a putative ATP binding site) were identified as the epitopes. The M267‐I442 fragment, which included a phosphorylation site at D376, and the C‐terminal one‐third of the α‐subunit from M615 to the C‐terminus, were not detected using this antibody. These results suggest that at least three regions on the Na+,K+‐ATPase α‐subunit, M32‐D75, M158‐D197 and M470‐V552, cover its exposed regions, and that some of them are essential for ATPase activity.

Stoichiometrical Binding of Ligands to Less than 160 KilodaltonsofNa.K-ATPase

Publisher Summary This chapter discusses the stoichiometrical binding of ligands to less than 160 kilodattons of Na, K-ATPase. Na, K-ATPase was purified from the outer medulla of dog kidney by the method of Jorgensen modified by Y. Hayashi and R.L. Post. About 15 mg of enzyme protein with specific activity of more than 40 μmoles P i /mg protein/min at 37°C was obtained per one zonal centrifugation with Beckman Ti 15 rotor. The highest active fraction reached a specific activity of 45. As the activity of the present preparation was almost twice as much as that of previous preparation to which ouabain binding was 3 nmoles/mg protein, it was examined whether this preparation could bind 6 nmoles ouabain/mg protein in proportion to the increased specific activity. The chapter also graphically presents the titration profile of [ 3 H] ouabain binding to the purified Na, K-ATPase. The amounts of ouabain, ATP, K + , and Na + specifically bound to the same enzyme preparation were 6.1, 5.5, 11.3, and 14.7 nmoles/mg protein, respectively. The maximal value of ouabain binding, 6.3 nmoles/mg protein, corresponds to one molecule of ouabain bound per 160 kilodaltons of protein. The results obtained suggest that the main protein components for the functional unit of the enzyme consist of one α and one β chain.

Localization of Na+/K+-ATPase in silkworm brain: A possible mechanism for protection of Na+/K+-ATPase from Ca2+

In mammalian blood, the Na(+) concentration is higher than the K(+) concentration, whereas in hemolymph of lepidopterous insects, the K(+) concentration is higher than the Na(+) concentration. Na(+)/K(+)-ATPase regulates Na(+) and K(+) concentrations in mammalian blood. Therefore, the absence of Na(+)/K(+)-ATPase in lepidopterous insects might be expected. However, we have observed that Na(+)/K(+)-ATPase is abundant in nerve tissues of larvae of silkworm, a lepidopterous insect. Furthermore, we found that silkworm Na(+)/K(+)-ATPase was completely inhibited by 3 mM Ca(2+)in vitro (Homareda, 2010), although the Ca(2+) concentration is very high (30-50 mM) in the hemolymph of silkworm larvae. To investigate the reason why silkworm Na(+)/K(+)-ATPase is not inhibited by Ca(2+)in vivo, we observed the localization of Na(+)/K(+)-ATPase in nerve tissues using immunohistochemical techniques. Na(+)/K(+)-ATPase was distributed in the cortex and neuropile but not in the perineurium of the silkworm brain, while plasma membrane Ca(2+)-ATPase appeared to distribute in the perineurium as well as in the cortex and neuropile. These results support a possibility that neuronal Na(+)/K(+)-ATPase is protected from a high Ca(2+) concentration by the blood-brain barrier consisting of perineurial glial cells with plasma membrane Ca(2+)-ATPase.

Stimulation of p‐nitrophenylphosphatase activity of Na+/K+‐ATPase by NaCl with oligomycin or ATP

It is known that the addition of NaCl with oligomycin or ATP stimulates ouabain‐sensitive and K+‐dependent p‐nitrophenylphosphatase (pNPPase) activity of Na+/K+‐ATPase. We investigated the mechanism of the stimulation. The combination of oligomycin and NaCl increased the affinity of pNPPase activity for K+. When the ratio of Na+ to Rb+ was 10 in the presence of oligomycin, Rb+‐binding and pNPPase activity reached a maximal level and Na+ was occluded. Phosphorylation of Na+/K+‐ATPase by p‐nitrophenylphosphate (pNPP) was not affected by oligomycin. Because oligomycin stabilizes the Na+‐occluded E1 state of Na+/K+‐ATPase, it seemed that the Na+‐occluded E1 state increased the affinity of the phosphoenzyme formed from pNPP for K+. On the other hand, the combination of ATP and NaCl also increased the affinity of pNPPase for K+ and activated ATPase activity. Both activities were affected by the ligand conditions. Oligomycin noncompetitively affected the activation of pNPPase by NaCl and ATP. Nonhydrolyzable ATP analogues could not substitute for ATP. As NaE1P, which is the high‐energy phosphoenzyme formed from ATP with Na+, is also the Na+‐occluded E1 state, it is suggested that the Na+‐occluded E1 state increases the affinity of the phosphoenzyme from pNPP for K+ through the interaction between α subunits. Therefore, membrane‐bound Na+/K+‐ATPase would function as at least an (αβ)2‐diprotomer with interacting α subunits at the phosphorylation step.

Properties of silkworm Na+/K+-ATPase

The high Na(+) and low K(+) concentrations in mammalian blood are maintained by Na(+)/K(+)-ATPase. In contrast, the K(+) concentration is higher than the Na(+) concentration in the hemolymph of the silkworm Bombyx mori, a Lepidopterous insect. Although Na(+)/K(+)-ATPase, therefore, appears not to be in silkworm, we confirmed the presence of Na(+)/K(+)-ATPase in nerve tissues of silkworm but not in skeletal muscle or the dorsal vessel. The enzymatic properties of silkworm Na(+)/K(+)-ATPase were characterized in detail and compared with those of dog Na(+)/K(+)-ATPase. Silkworm Na(+)/K(+)-ATPase had a much lower affinity for K(+) and a somewhat higher affinity for Na(+) than dog Na(+)/K(+)-ATPase. The optimal temperature of silkworm Na(+)/K(+)-ATPase activity was lower than that of dog Na(+)/K(+)-ATPase. The optimal Mg(2+) concentration, pH and sensitivities to Ca(2+) and ouabain, a specific inhibitor of Na(+)/K(+)-ATPase, of the two ATPases were identical. These results indicate that the enzymatic properties of the silkworm Na(+)/K(+)-ATPase are suitable for its growth, despite the differences between dog and silkworm Na(+)/K(+)-ATPases. Antisera raised against dog Na(+)/K(+)-ATPase recognized only the α-subunit of silkworm Na(+)/K(+)-ATPase.

Increase in Na Pump Activity of Brain-type Isoforms via Increased Turnover Rate after Glutamate Excitation of Cerebral Neurons

Na+/K+-ATPase (=Na pump) is abundant in nervous tissue and plays an important role in neuronal excitation by producing sodium and potassium ion-gradients across the cell membrane. Isoforms of Na+/K+-ATPase are known to exist in brain. Highly ouabain-sensitive isoform (brain-type; α2 and α3) appeared and increased during the maturation of primary cultured rat cerebral neurons besides the weakly ouabain-sensitive isoform pre-existing (common-type; α1) (1). We demonstrated that the Na pump activity of brain-type isoform in the matured neurons increased several folds after glutamate excitation while the activity of common-type isoform decreased slightly (2,3). To discriminate whether the increase in Na pump activity is due to increase in number or turnover rate of the pump, the number of Na pump was determined by [3H]ouabain binding experiment.