Increased (Na+,K+)-ATPase concentrations in various tissues of rats caused by thyroid hormone treatment.

Effects of triiodothyronine treatment on (Na+,K+)-ATPase in the brain, liver, kidney, and skeletal muscle were studied in the rat. The number of (Na+,K+)-ATPase units in the particulate fractions obtained from deoxycholate-treated homogenates was estimated from the concentration of [3H]ouabain binding sites assayed with a labeled drug-displacement method. The concentration of [3H]ouabain binding sites was highest in the brain tissue, intermediate in the kidney, and relatively low in the liver and skeletal muscle. The affinity of the binding sites for ouabain was highest in the brain, intermediate in the skeletal muscle, low in the kidney, and lowest in the liver. Triiodothyronine treatment increased the [3H]ouabain binding site concentration in the liver, kidney, and skeletal muscle but failed to affect it in the brain. Affinity of the binding sites for ouabain was unchanged by the triiodothyronine treatment in all tissues studied. These data indicate that triiodothyronine treatment of rats results in an increased tissue concentration of (Na+,K+)-ATPase in the liver, kidney, and skeletal muscle, but not in the brain. These changes do not accompany marked changes in the characteristics of the enzyme.     

Free fatty acids and (Na+,K+)-ATPase: effects on cation regulation, enzyme conformation, and interactions with ethanol

Effects of free fatty acids on parameters of (Na+,K+)-ATPase regulation related to enzyme conformation were examined. Sensitivity to inhibition by free fatty acid increased as the number of double bonds increased. Free fatty acids reduced affinity for K+ or Na+ at their regulatory sites without altering apparent K+ affinity at its high-affinity site, and increased apparent affinity for ATP. The apparent E2/E1 ratio and apparent delta H and delta S for the E1-E2 transition were reduced by fatty acid. High K+ or low temperature reduced the sensitivity of enzyme to inhibition by free fatty acid. In the presence of low K+, arachidonic acid potentiated inhibition of phosphatase activity by ethanol. Arachidonic acid alone had little effect on the rate of ouabain binding, but accelerated ouabain binding in the presence of K+. These data suggest that fatty acids alter (Na+,K+)-ATPase by preventing the univalent cation-mediated transition to E2, the K+-sensitive form of enzyme. (Na+,K+)-ATPase could potentially be influenced in vivo by free fatty acids released by phospholipases or during hypoxia, or by changes in membrane lipid saturation.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Characterization of ouabain-resistant mutants of a canine kidney cell line, MDCK

Madin-Darby canine kidney (MDCK) cells were mutagenized and variants resistant to 10, 160, and 2000 times the ouabain lethal dose for wild type cells selected. The phenotypes were stable in the absence of selection. The frequencies with which variants were recovered were consistent with genetic alterations being responsible for drug resistance. It was shown that 50% of the (Na+, K+)-ATPase activity present in mutant cells had a higher Kd for ouabain than normal while 50% remained wild type for ouabain binding. Wild type MDCK cells were measured to have 2 X 10(6) ouabain binding sites per cell with a Kd for the drug of 0.6-1.0 X 10(-7) M. The novel (Na+, K+)-ATPase activities in the mutants demonstrated Kd values for ouabain of 10(-5) M, 3 X 10(-4) M, or 3 X 10(-3) M for the different mutant classes tested. The rate of synthesis of the (Na+, K+)-ATPase as well as the total amount of enzyme per unit of cell protein was unaltered in the mutants. Comparison of the alpha subunit of the enzyme, known to contain the ouabain-binding site, by sodium dodecyl sulfate-gel electrophoresis did not reveal any difference in the size of this subunit in mutant versus wild type cells.     

Modification of (Na+,K+)-ATPase function by purified antibodies to the holoenzyme. Effects on enzyme activity and (3H)ouabain binding.

Antibodies (abys) raised to (Na+,K+)-ATPase were purified by elution methods and shown to be cross-reactive with anti-gamma-globulin and the original antigen. Abys were isolated from two different antisera and the effects on (Na+,K+)-ATPase hydrolytic activity and [3H]ouabain binding were measured. The antisera fractions differed as to their maximum level of inhibition of hydrolytic activity and maximal [3H]ouabain binding, but both fractions caused inhibition of maximal [3H]ouabain binding to the same quantitative extent as inhibition of hydrolytic activity. Variable effects on the rate of [3H]ouabain binding were noted which were highly dependent on ligand conditions. During the "turnover state conditions" of the enzyme, the abys stimulated the rate of [3H]ouabain binding to the (Na+,K+)-ATPase. We conclude that effects of aby-(Na+,K+)-ATPase interaction depend upon degree of purity of aby, specificity, aby/enzyme ratios, and ligand conditions.     

Ligand binding sites of the ouabain-complexed (Na+ + K+)-ATPase

To clarify the mechanism of inhibition of (Na+ + K+)-ATPase by cardiac glycosides, we tried to see if ouabain binding alters the properties of the binding sites for Na+, K+, and ATP. Ouabain was bound in the presence of either Na+ + MgATP or MgPi. Ligand-induced changes in the rate of release of ouabain from the two resulting complexes were used as signals to determine the affinities, the numbers, and the interactions of the ligand binding sites. Because the two complexes showed differences in the properties of their ligand binding sites, and since neither complex could be converted to the other, it is concluded that either the enzyme has two dissimilar but mutually exclusive ouabain sites or that it can be frozen in two distinct conformations by ouabain. The following ligand sites were identified on the two complexes: 1) two coexisting ATP sites (K0.5 values, 0.1 and 2 mM) representing altered states of the catalytic and the regulatory sites of the native enzyme; 2) mutually exclusive Na+ and K+ sites whose affinities (K0.5 values, 1.3 mM Na+ and 0.1 mM K+) suggested their identities with the high affinity uptake sites of the native enzyme; and 3) coexisting low affinity Na+ and K+ sites (K0.5 values, 0.2-0.6 M) representing either the discharge sites, or the regulatory sites, or the access channels of the native enzyme. The data suggest that the inability of the ouabain-complexed enzyme to participate in the normal reaction cycle is not because of its lack of ligand binding sites but most likely due to ouabain-induced disruptions of interprotomer site-site interactions.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Antigenic differences in (Na+, K+)-ATPase preparations isolated from various organs and species.

Antisera to purified (Na+, K+)-ATPase raised in rabbits and in sheep were purified by an absorption procedure employing purified canine kidney (Na+, K+)-ATPase. The antibodies were fractionated into two components, one which inhibited catalytic activity, and a second which inhibited ouabain binding. Under certain conditions, the fraction that inhibited ouabain binding also inhibited catalytic activity, and the effectiveness of both was dependent to some extent on the ligands present in the incubation medium. Thus, both antibody fractions appeared to detect conformations of the enzyme that depended upon ligand-induced perturbations. When the antibody raised against catalytic activity was incubated with erythrocyte membrane fragments, an inhibition of the (Na+, K+)-ATPase occurred, but only minimal or no effect on potassium influx or on digoxin-induced inhibition of potassium flux in intact erythrocytes was noted. In a similar experiment, however, the antibody against ouabain binding significantly inhibited potassium influx, suggesting specificity in terms of the macromolecular surfaces of the pump which were exposed to the external medium. We concluded that there may be organ and species differences among (Na+, K+)-ATPase preparations. Antibodies prepared in rabbits and sheep were fractionated by absorption to dog brain enzyme. Both the antibody fraction which bound to the brain enzyme and that which did not bind inhibited the dog kidney (Na+, K+)-ATPase, but only the former inhibited dog brain (Na+, K+)-ATPase. When the two fractions were recombined, inhibition was restored to the extent of the unfractionated antibody.     

Modification of the Rate of Ouabain Binding to (Na++ K+)ATPase by Lithium Ions

We report on the interactions of Li+, a congener of K+ with the (Na+ + K+)-ATPase from E Electricus as measured by their effects on the rate of [3H]-ouabain binding to this enzyme. Like K+, Li+ slows ouabain binding under both Type I (Na+ + ATP) and Type II (P1) conditions, but with lower affinity. In contrast to K+, the Li+ inhibition curve is hyperbolic, suggesting interaction at an uncoupled site. Also differing from the complete inhibition by high K+, a residual ouabain-binding rate persists at high Li+. The interactions of Li+ and K+ are synergistic: the apparent K+ affinity increases 3 to 4-fold in presence of Li+. These results are consistent with the conclusion that Li+ interacts with only one of the two K+ sites and may be of interest in interpreting lithium pharmacology.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.     

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.     

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.     

Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Affinity chromatography for human Na+, K+-ATPase inhibitors in plasma and urine

Semi-purified dog kidney Na+,K+-ATPase cross-linked with ovalbumin was used in batch-wise affinity chromatography for the detection of endogenous Na+,K+-ATPase inhibitor in human plasma and urine. Ammonium acetate 1 M washed off the endogenous inhibitor from the immobilized enzyme. The inhibitory activity of the eluate from hypertensive plasma and urine was significantly higher (p less than 0.0025, n = 5 and p less than 0.005, n = 6 respectively) than that of normotensive. This latter was correlated with the ability of plasma from the same subjects to compete with ouabain binding to erythrocytes. Plasma and urine extracts inhibited the activity of Na+, K+-ATPase in a dose-dependent manner as ouabain does and were shown to contain 3 or 4 active compounds by high pressure liquid chromatography. The activity of some of these compounds was lost after peptidase treatment. These data support the heterogeneity of endogenous inhibitors of Na+,K+-ATPase activity in plasma and urine.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Membrane (Na+ + K+)-ATPase of canine brain, heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures.

Effects of commonly used purification procedures on the yield and specific activity of (Na+ + K+)-ATPase (Mg2+-dependent, Na+ + K+-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na+ + K+)-ATPase and the ratio between (Na+ + K+)-ATPase and Mg2+-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na+ + K+)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na+ + K+)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzymes preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain.enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purfication procedure. These results indicate that the presentyl used purification procedures may alter the properties of membrane (Na+ + K+)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na+ + K+)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.     

Immunochemical characterization of a functional site of (Na+,K+)-ATPase

Several hybridoma cell lines secreting antibodies specific to the membrane (Na+,K+)-dependent ATPase from lamb kidney medulla have been isolated by using the methods developed by Kohler and Milstein. One of these antibodies (designated M7-PB- E9 ) has been shown to be directed against a functional epitope or antigenic site of the catalytic (alpha) subunit of the enzyme. Although this antibody was raised to the "native" holoenzyme, it has a higher apparent affinity toward the isolated, delipidated, and inactive alpha subunit than toward the holoenzyme. This antibody shows a 10-fold faster initial rate of binding to the alpha subunit than to the holoenzyme. The antibody dissociation rates from both isolated alpha subunit and holoenzyme are similarly slow, and the binding can be considered a pseudoirreversible reaction. By binding at this site, the antibody, however, acts like a "partial competitive inhibitor" with respect to ATP and acts as an uncompetitive or mixed competitive inhibitor with respect to the Na+ and K+ dependence of ATPase hydrolysis. This antibody also does not alter the cooperativity at either the Na+ or the K+ sites. The antibody causes a partial inhibition of the Na+- and MgATP-dependent phosphoenzyme intermediate formation but has no effect on either ADP in equilibrium ATP exchange or the K+-stimulated dephosphorylation step. In addition, the K+-dependent p-nitrophenylphosphatase activity of the enzyme was not affected. In the presence of Mg2+, the antibody stimulates the rate of cardiac glycoside binding [( 3H]ouabain) to the (Na+,K+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group

A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent dephosphorylation of the enzyme without inhibiting either (Na+)-ATPase activity or Na+-dependent phosphorylation. Mg2+ also induced a 5 to 6% enhancement in the fluorescence intensity of enzyme labeled with the fluorescent sulfhydryl reagent, 2-(4-maleimidylanilino)naphthalene-6-sulfonate. As in the case of Mg2+ inhibition of activity, the affinity for Mg2+ as an inducing agent for this effect was significantly reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced by both Na+ and ATP, suggesting that the same change was being monitored in both cases. The Mg2+ effect was reduced in magnitude by ouabain and prevented by oligomycin, specific inhibitors of the enzyme. In addition, K+ (and cations that substitute for K+ in supporting activity) induced a 3 to 4% enhancement in fluorescence intensity in the presence of Na+, Mg2+, and ATP, although the K+ and Mg2+ effects appeared to be different on the basis of their excitation spectra. The K+ effect was inhibited by ouabain and occurred with a rate greater than the rate of turnover of the enzyme, permitting its involvement in the catalytic cycle.