Dynamics of Na,K-ATPase sites in lateral cochlear wall tissues of the rat

In order to study vertebral fractures in various study populations, we earlier prepared a database of vertebral dimensions derived from spinal radiographs of 191 normal women seen regularly over 25 years. In this report we have expanded the range of measurements to include vertebral levels T3 to L5. We report means and standard deviations on anterior and posterior heights, on wedge shape and on heights relative to adjacent vertebrae. When one or both of the latter two quantities are 'far' below the mean, a vertebra is called deformed. We also describe a more flexible way of expressing damage using the number of deformed vertebrae, the degree of deformity of individual vertebrae, or the total damage to the entire spine. In assessing damage we use criteria for deformity adjusted to the limits detected by an experienced diagnostician, replacing an earlier approach based on 95% probability limits of normal variation. The normal women from whom these variations are ascertained are a low-prevalence group with respect to vertebral deformity, with prevalence of 2.8%. When the criteria developed from these women were applied to a moderate-prevalence group (37%) the model had a sensitivity of 97%, a specificity of 89% and an accuracy of 92% as regards the identification of subjects with damaged vertebrae. When used epidemiologically for a moderate-prevalence group the model has a known overestimation of 15%. the model is compared with other schemes for identifying vertebral deformities.     

The gamma subunit of the Na,K-ATPase induces cation channel activity

The gamma subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The gamma subunit was expressed in Sf-9 insect cells and Xenopus oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the gamma subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human gamma subunit. Confocal microscopy demonstrates that the gamma subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that gamma colocalizes with alpha1 when these proteins are coexpressed. When Sf-9 cells were coinfected with alpha1 and gamma, antibodies to the gamma subunit were able to coimmunoprecipitate the alpha1 subunit, suggesting that gamma is able to associate with alpha1. The gamma subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in Xenopus oocytes. Evidence that the gamma subunit is a functional component was supported by experiments showing gamma-induced cation channel activity when expressed in oocytes and increases in Na+ and K+ uptake when expressed in Sf-9 cells.     

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.     

E2P phosphoforms of Na,K-ATPase. II. Interaction of substrate and cation-binding sites in Pi phosphorylation of Na,K-ATPase

In this investigation the effects of alkali cations on the transient kinetics of Na,K-ATPase phosphoenzyme formation from either ATP (E2P) or Pi (E'2P) were characterized by chemical quench methods as well as by stopped-flow RH421 fluorescence experiments. By combining the two methods it was possible to characterize the kinetics of Na, K-ATPase from two sources, shark rectal glands and pig kidney. The rate of the spontaneous dephosphorylation of E2P and E'2P was identical with a rate constant of about 1.1 s-1 at 20 degreesC. However, whereas dephosphorylation of E2P formed from ATP was strongly stimulated by K+, dephosphorylation of E'2P formed from Pi in the absence of alkali cations was K+-insensitive, although in pig renal enzyme K+ binding to E'2P could be demonstrated with RH421 fluorescence. It appears, therefore, that in pig kidney enzyme the rapid binding of K+ to E'2P was followed by a slow transition to a nonfluorescent form. For shark enzyme the K+-induced decrease of RH421 fluorescence of Pi phosphorylated enzyme was due to K+ binding to the dephosphoenzyme (E1), thus shifting the equilibrium away from E'2P. When Pi phosphorylation was performed with enzyme equilibrated with K+ or its congeners Tl+, Rb+, and Cs+ but not with Na+ or Li+, both the phosphorylation and the dephosphorylation rates were considerably increased. This indicates that binding of cations modifies the substrate site in a cation-specific way, suggesting an allosteric interaction between the conformation of the cation-binding sites and the phosphorylation site of the enzyme.     

Tissue-specific distribution and modulatory role of the gamma subunit of the Na,K-ATPase

The Na,K-ATPase comprises a catalytic alpha subunit and a glycosylated beta subunit. Another membrane polypeptide, gamma, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with alpha and beta in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-gamma antiserum to define the tissue specificity and topology of gamma and to address the question of whether gamma has a functional role. The trypsin sensitivity of the amino terminus of the gamma subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that gamma subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, alpha1-transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into gamma function, the effects of the anti-gamma serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that gamma stabilizes E1 conformation(s) of the enzyme and that anti-gamma counteracts this effect: (i) anti-gamma inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) --> E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-gamma indicating that the antibody shifts the E1 left and right arrow left and right arrow E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+ concentration reduced to levels ( --> E2P transition, anti-gamma is stimulatory. These observations taken together with evidence that the pig gamma subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb+ ions partially protect it against this effect, indicate that the gamma subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-gamma disrupts alphabeta-gamma interactions and counteracts these modulatory effects of the gamma subunit.     

The role of Na,K-ATPase inhibitors in hypertension and end-stage renal disease

In Part I of this two-part study, the authors investigated heat production during osteotomy drilling at three different speeds, and determined that high-speed drilling produced the least heat when using 700 XL carbide burs. Part II of the study histologically examines the rate and quality of healing after drilling osteotomies at the three speeds in the mandible. Osteotomies were histologically examined 2, 4, and 6 weeks postoperatively. Histologic findings suggested that in the initial 6 weeks, the rate of healing and quality of new bone formation were higher after high-speed drilling than after low- or intermediate-speed drilling. These results, when considered with the results reported in Part I in which a 4.3 degrees C difference in heat production was observed between the speeds, seem to imply a relationship between heat production and healing for osteotomy drilling.     

Increased hepatic Na,K-ATPase activity during hepatic regeneration is associated with induction of the beta1-subunit and expression on the bile canalicular domain

Cellular and molecular mechanisms regulating the activity of the sodium pump or Na,K-ATPase during proliferation of hepatocytes following 70% liver resection have not been defined. Na,K-ATPase may be regulated by synthesis of its alpha- and beta-subunits, by sorting to either the sinusoidal or apical plasma membrane domains, or by increasing membrane lipid fluidity. This study investigated the time course of changes during hepatic regeneration for Na, K-ATPase activity, lipid composition and fluidity, and protein content of liver plasma membrane subfractions. As early as 4 h after hepatic resection, Na,K-ATPase activity was increased selectively in the bile canalicular fraction. It reached a new steady state at 12 h and remained elevated for 2 days. Although hepatic regeneration was associated with a reduced cholesterol/phospholipid molar ratio and increased fluidity, measured with two different probes, these changes in lipid metabolism were in the sinusoidal membrane domain. The Na,K-ATPase beta1-subunit, but not the alpha1-subunit, was increased selectively at the bile canalicular surface as shown by immunoblotting of liver plasma membrane subfractions and the morphological demonstration at both the light and electron microscopic levels. Furthermore, cycloheximide blocked the rise in beta1-subunit mRNA levels. Since the time course for beta1-subunit accumulation was similar to that for activation of Na,K-ATPase activity, this change implicated the beta1-subunit in activating sodium pump activity.     

Topology of the alpha-subunit of Na,K-ATPase based on proteolysis. Lability of the topological organization

Topology of the alpha-subunit of Na,K-ATPase has been analyzed utilizing proteolytic digestion. Evidence is presented for a model with 10 transmembrane segments and lability of the C-terminal domain (M7-M10). Using reconstituted proteoliposomes, inside-out oriented pumps were digested with trypsin at the cytoplasmic surface. Evidence was obtained for the M7/M8 pair and cytoplasmic splits between M8 and M9 and between M9 and M10. Because an extracellular split between M9 and M10 was also observed, using right-side-out oriented renal microsomes, we propose that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile. Using renal microsomes, extracellular digestion of the alpha-subunit by trypsin, chymotrypsin, or an endogenous protease has been observed, after incubation at 55 or at 45 degrees C with beta-mercaptoethanol (beta-ME) and n-butanol. Both perturbations inactivate enzyme activity. Rb ions protect against inactivation and digestion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymotrypsin cut between M7 and M8 and between M9 and M10, consistent with the 10-segment model. At 55 degrees C, the topological organization is altered, the M8/M9 connecting loop is exposed at the extracellular surface, and an additional split between M8 and M9 is observed. Extracellular digestion of the alpha-subunit is associated with digestion of the beta-subunit near the first extracellular S-S bridge. Rb ions protect the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interactions.     

Explaining on request a correlation between membrane Na,K-ATPase and K+ content in erythrocytes and other findings in the preceding paper

Epidemiological features of meningococcal disease described as from the second half of the 80's inclusive, have motivated a revision of current guidelines for sporadic disease and outbreak control. The increase of disease among teenagers and linked cases involving schools are the two most significant aspects that have prompted the revision of control measures. Vaccination routines and advice for the disease management of clusters are also relevant features recently revised. This present paper describes the management and some epidemiological features of secondary cases.     

Changes in steady-state conformational equilibrium resulting from cytoplasmic mutations of the Na,K-ATPase alpha-subunit

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.     

Functional activity of Na+,K(+)-pump in normal and pathological tissues

Na+,K(+)-ATPase, supporting the ionic homeostasis of the cell, is under control of Na+, K+, Mg2+, and ATP. The regulating effect of Mg2+ is rather unclear, whereas the Na+/K+ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na+,K(+)-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na+,K(+)-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na+,K(+)-ATPase--an integral protein of the outer cell membranes.     

Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

Distribution of Na,K-ATPase is normal in the inner ear of a mouse with a null mutation of the glucocorticoid receptor

This study was performed in order to test the hypothesis that the glucocorticoid hormone stimulates the formation of Na,K-ATPase in the inner ear of the mouse. An immunohistochemical study with respect to the presence and distribution of glucocorticoid receptors (GR) and Na,K-ATPase in the vestibular and cochlear regions of the inner ear was performed on a C57BL mouse with a null mutation of the glucocorticoid receptor (GR mutant mouse). The wild type C57BL mouse and the CBA mouse served as normal controls. As expected, the homozygous GR mutant mouse showed no specific staining for GR in the inner ear. The heterozygous GR mutant mouse showed faint staining of GR in the spiral limbus, the spiral ganglion, the organ of Corti and the utricle. This staining was markedly less than in the wild type C57BL mouse. Antibody labelling of Na,K-ATPase in the inner ear showed no significant difference between the homozygous and the heterozygous GR mutant mouse as compared to the control wild type C57BL mouse or the CBA mouse. Although earlier studies have shown a positive correlation between levels of glucocorticoid hormone in serum and the concentration of Na,K-ATPase in the inner ear, the hypothesis that glucocorticoid hormones alone stimulate the formation of Na,K-ATPase in the inner ear could not be confirmed by this study. Thus other regulating substances must be considered.     

Influence of inhaled nitric oxide and hyperoxia on Na,K-ATPase expression and lung edema in newborn piglets

This study was undertaken to examine the combined effect of nitric oxide (NO) and hyperoxia on lung edema and Na,K-ATPase expression. Newborn piglets were exposed to room air (FiO2 = 0.21), room air plus 50 ppm NO, hyperoxia (FiO2 >/= 0.96) or to hyperoxia plus 50 ppm NO for 4-5 days. Animals exposed to NO in room air experienced only a slight decrease in Na,K-ATPase alpha subunit protein level. Hyperoxia, in the absence of NO, induced both the mRNA and the protein level of Na,K-ATP-ase alpha subunit and significantly increased wet lung weight, extravascular lung water, and alveolar permeability. NO in hyperoxia decreased the hyperoxic-mediated induction of Na,K-ATPase alpha subunit mRNA and protein while wet lung weight, extravascular lung water, and alveolar permeability remained elevated. These results suggest that 50 ppm of inhaled NO may not improve hyperoxic-induced lung injury and may interfere with the expression of Na,K-ATPase which constitutes a part of the cellular defense mechanism against oxygen toxicity.     

Role of beta-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase

The beta-subunit of Na,K-ATPase (betaNK) interacts with the catalytic alpha-subunit (alphaNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing alphaNK in Xenopus oocytes along with N-terminally modified betaNK or with chimeric betaNK/betaH,K-ATPase (betaHK). Complete truncation of the betaNK N terminus allows for cell surface-expressed, functional Na,K-pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the beta N terminus with the alphaNK but rather that N-terminal truncation induces a conformational change in another functionally relevant beta domain. Comparison of the functional properties of alphaNK.betaNK, alphaNK.betaHK, or alphaNK. betaNK/betaHK complexes shows that the effect of the betaNK on K+ binding is mainly mediated by its ectodomain. Finally, betaHK/NK containing the transmembrane domain of betaHK produces stable but endoplasmic reticulum-retained alphaNK.beta complexes, while alphaNK/betaHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of betaNK with alphaNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the beta N terminus plays a role in the beta-subunit's folding necessary for correct interactions with the alpha-subunit.     

Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin

Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.     

Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia

1. The benzoylguanidine derivative Hoe 694 ((3-methylsulphonyl-4- piperidino-benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA). 2. To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10(-7) M Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts. 3. Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively. 4. The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts. 5. Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose-dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg-1, i.v. Blood pressure and heart rate remained unchanged. 6. We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.     

The effect of ionic strength and specific anions on substrate binding and hydrolytic activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3-, eosine2-, p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0-0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl approximately Na2SO4 approximately Na-acetate < NaNO3 < NaSCN < NaCl04. We treated the influence of NaCl and Na2SO4 on K(diss) for E x ADP as a "pure" ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3- effect was compatible with competitive binding of NO3- and ADP in addition to the general I-effect. K(diss) for E x NO3 was approximately 32 mM. Analysis of V(max)/K(m) for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.     

The Drosophila Na,K-ATPase alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.