Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.     

Molecular weight of (Na+,K+)ATPase from shark rectal gland.

The (Na+,k+)ATPase from the rectal gland of Carcharhinus obscurus has been solubilized in Lubrol WX as an active complex containing 379,900 g of protein and 61 mol of phospholipid. This detergent-lipid-protein complex contains two catalytic subunits of molecular weight 106,400 and four glycopeptide subunits of protein molecular weight 36,600. The latter subunit has a total molecular weight of 51,700 when the carbohydrate is included. Attempts to dissociate this active enzyme complex to smaller size by increasing the detergent concentration led to inactivation. Thus, the smallest active particle in the presence of Lubrol WX contains the two polypeptide subunits in a mole ratio of 2:4 under conditions where the micellar form of detergent is present at a 70:1 molar ratio. This large excess of Lubrol WX eliminates any possibility of artificial togetherness as the result of statistical considerations.     

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.     

Rat brain (Na+-K+)ATPase: modulation of its ouabain-sensitive K+-PNPPase activity by thimerosal

1. The (Na+ + K+) ATPase activity of a rat brain synaptic membrane preparation was inhibited by 10(-5) M thimerosal. 2. The ouabain inhibitable K+-PNPPase activity of thimerosal treated membranes was compared with that of untreated membranes with respect to sensitivity to temperature, ouabain, K+ and ATP. 3. All those kinetic characteristics were substantially altered by treatment with thimerosal.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.     

Interactions of ouabain and vanadate with (Na+,K+)ATPase and isolated cardiac muscle

The interactions of ouabain and vanadate with (Na⁺,K⁺)ATPase were investigated at different potassium concentrations. Also, the contractile effects of a mixture of these two inhibitors were compared to those produced by ouabain or vanadate alone. The results from the enzyme and contractile studies suggested that inhibition of sarcolemmal (Na⁺,K⁺)ATPase was involved in mediating the positive inotropic effect of vanadate.     

Fluorescent properties of (Na+/K+)ATPase

1. The effect of ouabain on the molecular properties of (Na+/K+)-ATPase has been studied in purified preparations of the enzyme, isolated from the microsomal fraction of outer red medulla of porcine kidney, according to a modification of the method described by Jorgensen. 2. Ouabain, a specific inhibitor of (Na+/K+)-ATPase, binds at the potassium site of the enzyme, thus generating an increase in its stability towards the common denaturing agents, such as exposure to different concentration of guanidinium chloride (GdmC1) or to acidic solutions.     

Insulin stimulates both the alpha 1 and the alpha 2 isoforms of the rat adipocyte (Na+,K+) ATPase. Two mechanisms of stimulation.

Results obtained with adipocyte ghosts indicated that the relative pumping activities of the alpha 1 and alpha 2 isoforms of the (Na+,K+) pump depend strongly on intracellular sodium concentration, [Na+]i (McGill, D. (1991) J. Biol. Chem. 266, 15817-15823). Accordingly, [Na+]i was determined in rat adipocytes as a function of ouabain concentration and found to increase gradually as the concentration of ouabain increased. Incubation conditions were therefore designed such that the [Na+]i at 0 M and 10(-5) M ouabain were identical, in order to study the activities of both forms of the pump under identical conditions. Under these conditions, the alpha 2 isozyme accounts for 42% of the total pumping activity; these data prove that the activity of the alpha 2 isozyme is suppressed to a much greater extent than that of the alpha 1 isozyme, in relation to maximally obtainable activities measured in plasma membranes (Lytton J., Lin, J.C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184). Furthermore, insulin stimulation of 86Rb+/K+ uptake in adipocytes results from a 58 and a 128% increase in the activities of the alpha 1 and alpha 2 isozymes (Na+,K+) pump, respectively. In addition, it is shown that under the conditions used to determine the [Na+]i dependence of 86Rb+/K+ uptake into adipocytes (0 mM KCl, various [NaCl]), [Na+]i decreases rapidly upon the addition of KCl/86RbCl for the initiation of the uptake measurement. By making uptake measurements quickly after the addition of KCl to eliminate the effect of a decreasing [Na+]i, we demonstrate that the stimulation of the alpha 1 isozyme is due to a small decrease in the K0.5Na+ whereas the stimulation of the alpha 2 isozyme results from a decrease in the K0.5Na+ and an increase in the Vmax.     

The (Na+, K+)ATPase of rat kidney: purification, biosynthesis, and processing

(Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A- and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests. The endoplasmic reticulum (ER)-rich, and Golgi-rich subfractions were prepared from the rat kidney microsomal fraction by sucrose density gradient centrifugation. On the immunoblot, the molecular weight of the alpha subunit in both fractions was 95 kilodalton (Kd); whereas, that of the beta subunit was 50 Kd in the ER-rich fraction and 54 Kd in the Golgi-rich fraction. When treated with endoglucosidase H, the 50 Kd component was converted to 38 Kd, but the 54 Kd component was endoglucosidase H resistant. These results suggest that the beta subunit (38 Kd) is glycosylated cotranslationally in the ER (50 Kd) then is converted to the mature type subunit (54 Kd) in the Golgi apparatus.     

Characterization of the adipocyte ghost (Na+,K+) pump. Insights into the insulin regulation of the adipocyte (Na+,K+) pump.

The (Na+,K+) ATPase in plasma membranes isolated from rat adipocytes is insensitive to insulin (Lytton J., Lin, J.C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184). For this reason, the characteristics of the (Na+,K+) pump in adipocyte ghosts, prepared by hypotonic lysis of adipocytes (Rodbell, M. (1967) J. Biol. Chem. 242, 5744-5750), were studied. Herein it is demonstrated that the (Na+,K+) pump in ghosts is identical to that described in isolated plasma membranes, sharing the following characteristics: 1) the Ki values for ouabain are 1.3 x 10(-7) M and 4.5 x 10(-5) M for the alpha 2 and alpha 1 isozymes, respectively; 2) the K0.5 values for sodium are 11.4 +/- 1.6 and 7.2 +/- 3.8 mM for the alpha 2 and alpha 1 isozymes, respectively; 3) both forms of the (Na+,K+) pump are insensitive to insulin stimulation, presumably because the activities are already maximal. The ghosts are not in an insulin-stimulated state because the activity of the glucose transporter is not increased as it is in ghosts prepared from insulin-treated cells. In addition, presented evidence demonstrates that ghost internal sodium concentration, [Na+]i, is very sensitive to changes in the activity of the (Na+,K+) pump. If the [Na+]i, of adipocytes is also very sensitive to the activity of the (Na+,K+) pump, the mechanism of insulin stimulation of the adipocyte (Na+,K+) pump requires reexamination.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (2H2O) and dimethyl sulfoxide (Me2SO) on [3H]ouabain binding to (Na+,K+)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg2+ + ATP + Na+). In contrast, both solvents stimulated type II (i.e., Mg2+ + Pi-, Mg2+-, or Mn2+-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg2+ + Na+ + ATP, 75% in the Mg2+ + Pi system, and in the presence of Mn2+ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus 2H2O inhibition of type I ouabain binding was dependent on Na+ concentration in the reaction and was reduced as Na+ was elevated. Contact of the enzyme with the Me2SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na+ + ATP first and then to Me2SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me2SO and 2H2O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H2O, and E1 enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H2O at the active enzyme center and E2 enzyme conformation. This postulation of a role of H2O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.