Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Scaling of Na+,K+-ATPase molecular activity and membrane fatty acid composition in mammalian and avian hearts

We have examined Na(+),K(+)-ATPase molecular activity and membrane fatty acid composition in the heart of six mammalian and eight avian species ranging in size from 30 g in mice to 280 kg in cattle and 13 g in zebra finches to 35 kg in emus, respectively. Na(+),K(+)-ATPase activity scaled negatively with body mass in both mammals and birds. In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and molecular activity (turnover rate) of Na(+),K(+)-ATPase enzymes, while in small birds, higher Na(+),K(+)-ATPase activity appeared to result primarily from an increased molecular activity of individual enzymes. The unsaturation index of cardiac phospholipids scaled negatively with body mass in both groups, while a significant allometric increase in monounsaturate content was observed in the larger mammals and birds. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid (22:6n-3) displayed the greatest variation, scaling negatively with body mass and varying greater than 40-fold in both mammals and birds. Membrane fatty acid profile was correlated with Na(+),K(+)-ATPase molecular activity in both mammals and birds, suggesting a potential association between membrane lipid composition and the activity of membrane-bound enzymes in the hearts of endotherms.     

Intracellular Na+, K+ and Cl- activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (Vm) and intracellular Na+, K+ and Cl- activities were measured under steady-state conditions in normal saline medium (Na+ = 154, K+ = 6, Cl- 150 mequiv./l). Membrane potential was estimated to be -23.3 +/- 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of Vm and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na+, K+ and Cl- activities are 4.6 +/- 0.5; 68.3 +/- 8.0; and 43.6 +/- 2.1 mequiv./l, respectively. The apparent activity coefficients for Na+ and K+ are 0.18 +/- 0.02 and 0.41 +/- 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl- (0.67 +/- 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl- is not different from zero. The results establish that the energy available from the Na+ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.     

Quinine inhibits multiple Na+ and K+ transport mechanisms in Ehrlich ascites tumor cells

The interaction of quinine with K+ and Na+ transport mechanisms has been investigated in Ehrlich ascites tumor cells. Quinine affects both Ca2+-dependent K+ channel and total K+ influx. Activation of Ca+-dependent K+ channels by propranolol is abolished by quinine (1 mM). In addition, quinine inhibits the ouabain-sensitive component of K+ influx with an apparent Ki of 0.32 +/- 0.02 mM and the furosemide-sensitive component with a Ki of 0.24 +/- 0.01 mM. Furthermore, a significant fraction (52%) of Na+ influx is inhibited by quinine. The same component is sensitive to amiloride, suggesting that it represents Na+/H+ antiport. Concomitant with the inhibition of K+ and Na+ transport, quinine stimulates ATP hydrolysis by 57%. The results suggest that quinine exerts broad, nonspecific effects on cellular mechanisms which serve to regulate cation transport in Ehrlich cells.     

Activation of (Na+ + K+)-ATPase

Enzymes catalyze essential chemical reactions needed for living processes. (Na+ +K+)-ATPase (NKA) is one of the key enzymes that control intracellular ion homeostasis and regulate cardiac function. Little is known about activation of NKA and its biological impact. Here we show that native activity of NKA is markedly elevated when protein-protein interaction occurs at the extracellular DVEDSYGQQWTYEQR (D-R) region in the alpha-subunit of the enzyme. The apparent catalytic turnover of NKA is approximately twice as fast as the controls for both ouabain-resistant and ouabain-sensitive enzymes. Activation of NKA not only markedly protects enzyme function against denaturing, but also directly affects cellular activities by regulating intracellular Ca2+ transients and inducing a positive inotropic effect in isolated rat cardiac myocytes. Immunofluorescent labeling indicates that the D-R region of NKA is not a conventional digitalis-binding site. Our findings uncover a novel activation site of NKA that is capable of promoting the catalytic function of the enzyme and establish a new concept that activating of NKA mediates cardiac contraction.     

Determination of total (Na+ + K+)-ATPase activity of isolated or cultured cells

The aim of this work was to determine if the total (Na+ + K+)-ATPase of the plasma membrane of a cell population could be assayed without cell homogenization and partial purification of the enzyme. Several types of intact cells that were placed in an assay medium containing MgATP, Na+, and K+ hydrolyzed little or none of the added ATP. When the cells were pretreated with the ionophore alamethicin and then placed in the assay medium, they exhibited an ouabain-sensitive (Na+ + K+)-ATPase activity that increased and reached a limiting value with increasing alamethicin concentration. Since alamethicin did not increase the activity of the purified membrane-bound (Na+ + K+)-ATPase, its effects on the intact cells are probably due to the formation of large channels within the plasma membrane that allow the free access of the components of the assay medium to the intracellular domains of (Na+ + K+)-ATPase. Utilizing whole cells treated with alamethicin, total (Na+ + K+)-ATPase activity was determined in clonal pheochromocytoma cells (PC12), neuroblastoma x glioma hybrid cells (NG108-15), and myocytes isolated from adult and neonatal rat hearts. With the use of this whole-cell assay, the ouabain sensitivities of the enzymes in adult and neonatal rat heart myocytes were determined and found to be the same as those that have been determined with the use of partially purified enzymes.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.     

Effects of polyamines on partial reactions of membrane (Na+ + K+)-ATPase.

Spermine and spermidine inhibit the (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) reaction so that the effect increases as the ionic content due to Na+ and K+ in the reaction is reduced. Several other amines inhibit (Na+ + K+)-ATPase to varying degress and methylglyoxal-bis-(guanylhydrazone) was the most potent inhibitor among those tested. The inhibition by polyamines of the ATPase is uncompetitive with respect to Mg2+ and ATP activation of the reaction. Various naturally occurring polyamines and other amines inhibited Na+ activation of (Na+ + K+)-ATPase as well as Na+-dependent phosphoenzyme formation in an apparently competitive manner with respect to Na+. Likewise, K+-activation of (Na+ + K+)-ATPase as well as K+-p-nitrophenyl phosphatase was inhibited in an apparently competitive manner with respect to K+. Both the cation charge and structure (e.g., aliphatic chain length) may contribute to the inhibitory effects of the amines; however, Na+ sites appear to be more sensitive to cation charge than the aliphatic chain length of the amine, whereas the opposite appears to be true for K+ sites. The results do not indicate a specific effect of polyamines on (Na+ + K+)-ATPase or its partial reactions.     

Lipid composition and (Na+ + K+)-ATPase activity in rat lens during triparanol-induced cataract formation

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.     

Comparative temperature dependence of (Na+ + K+)-ATPase.

The temperature dependence of (Na+ + K+)-ATPase was measured, utilizing preparations of enzyme from heat and kidney of rats, hamsters, guinea pigs, ground squirrels, turtles, chickens, and ducks. The two hibernating species, hamsters and ground squirrels, were studied awake at normothermia and hibernating at 4 degrees C. The results for every species except the turtles showed the same temperature dependence established for (Na++K+)-ATPase from rabbit kidney with a quasi-linear dependence above 15 degrees C and little or no activity below 15 degrees C. Turtle enzymes showed a broad activity versus temperature curve with a fall-off at high and low temperatures. The data in all cases, including the turtle data, may be fitted by a previously described thermodynamic kinetic model. Further, the model will fith the turnover or decrease in enzyme activity at higher temperatures observed in a number of cases. These results do not support the widely imputed ion pumping role for (Na++K+)-ATPase.     

Alteration of the fatty acid composition of Ehrlich ascites tumor cell lipids.

The fatty acid composition of Ehrlich ascites tumor lipids was altered markedly $\text{in vivo}$ by changing the type of fat fed to the tumor-bearing mice. As compared with regular chow, large differences were produced in polar and neutral lipid fatty acyl groups when the tumor cells were grown in mice fed coconut oil, sunflower oil or fat deficient diets. Subcellular membrane fractions obtained from these cells exhibited similar variations in fatty acyl composition. This experimental system provides large quantities of malignant cells for study of the relationships between membrane lipid structure and function.     

Isolation of plasma membranes from Ehrlich ascites tumor cells Influence of amino acids on (Na + K)-ATPase and K-stimulated phosphatase

A method was developed for isolating plasma membranes from Ehrlich ascites tumor cells. The plasma membranes appeared as highly irregular shrunken sacs or ghosts. Enzymatic characterization of the plasma membranes showed them to be high in (Na⁺ + K⁺-ATPase activity and K⁺-stimulated phosphatase activity. A detailed study showed that both of these latter enzymic functions were stimulated by various amino acids. Such stimulation occurred in the 1–15 mM range of amino acids and was most effective for aromatic species, e.g. phenylalanine and histidine. The amino acid stimulation, which appeared to show little or no stereospecificity, was eliminated by a one carbon separation of NH₂ and COOH groups. Since the metal chelating agent EDTA was also effective in mimicking the stimulation by amino acids, and since a mild washing procedure did not render membranes insensitive to subsequent amino acid or EDTA stimulation, it is proposed that the operation of the (Na⁺ + K⁺)-ATPase (and K⁺-stimulated phosphatase) is to some extent controlled by a tightly bound metal. The possible physiological function of an amino acid-regulated transport ATPase is discussed.