Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Potassium blocks barium permeation through a calcium-activated potassium channel

Single high-conductance Ca2+-activated K+ channels from rat skeletal muscle were inserted into planar lipid bilayers, and discrete blocking by the Ba2+ ion was studied. Specifically, the ability of external K+ to reduce the Ba2+ dissociation rate was investigated. In the presence of 150 mM internal K+, 1-5 microM internal Ba2+, and 150 mM external Na+, Ba2+ dissociation is rapid (5 s-1) in external solutions that are kept rigorously K+ free. The addition of external K+ in the low millimolar range reduces the Ba2+ off-rate 20-fold. Other permeant ions, such as Tl+, Rb+, and NH4+ show a similar effect. The half-inhibition constants rise in the order: Tl+ (0.08 mM) less than Rb+ (0.1 mM) less than K+ (0.3 mM) less than Cs+ (0.5 mM) less than NH4+ (3 mM). When external Na+ is replaced by 150 mM N-methyl glucamine, the Ba2+ off-rate is even higher, 20 s-1. External K+ and other permeant ions reduce this rate by approximately 100-fold in the micromolar range of concentrations. Na+ also reduces the Ba2+ off-rate, but at much higher concentrations. The half-inhibition concentrations rise in the order: Rb+ (4 microM) less than K+ (19 microM) much less than Na+ (27 mM) less than Li+ (greater than 50 mM). The results require that the conduction pore of this channel contains at least three sites that may all be occupied simultaneously by conducting ions.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of [3H]DAGO1 binding to rat brain by low concentrations of monovalent cations

The effects of mono- and di-valent cations and the nonhydrolyzable guanyl nucleotide derivative 5'-guanylimidodiphosphate (Gpp(NH)p) on the binding of the selective, high affinity mu-opiate receptor agonist, [3H]DAGO ([3H]Tyr-D-Ala-Gly-Mephe-Gly-ol), to rat brain membranes were studied in a low ionic strength 5 mM Tris-HCl buffer. Na+ and Li+ (50 mM) maximally increased [3H]DAGO binding (EC50 values for Na+, 2.9 mM and Li+, 6.2 mM) by revealing a population of low affinity binding sites. The density of high affinity [3H]DAGO binding sites was unaffected by Na+ and Li+, but was maximally increased by 50 mM K+ and Rb+ (EC50 values for K+, 8.5 mM and Rb+, 12.9 mM). Divalent cations (Ca2+, Mg2+; 50 mM) inhibited [3H]DAGO binding. Gpp(NH)p decreased the affinity of [3H]DAGO binding, an effect that was enhanced by Na+ but not by K+. The binding of the mu-agonist [3H]dihydromorphine was unaffected by 50 mM Na+ in 5 mM Tris-HCl. In 50 mM Tris-HCl, Na+ (50 mM) inhibited [3H]DAGO binding by decreasing the density of high affinity binding sites and promoting low affinity binding. The effects of Na+ in 5 mM and 50 mM Tris-HCl were also investigated on the binding of other opiate receptor agonists and antagonists. [3H]D-Ala-D-Leu-enkephalin binding was increased and inhibited. [3H]etorphine binding increased and was unchanged, and both [3H]bremazocine and [3H]naloxone binding increased by 50 mM Na+ in 5 mM and 50 mM Tris-HCl, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Duplex-tetraplex equilibrium between a hairpin and two interacting hairpins of d(A-G)10 at neutral pH.

d(A-G)10 forms two helical structures at neutrality, at low ionic strength a single-hairpin duplex, and at higher ionic strength a double-hairpin tetraplex. An ionic strength-dependent equilibrium between these forms is indicated by native PAGE, which also reveals additional single-stranded species below 0.3 M Na+, probably corresponding to partially denatured states. The equilibrium also depends upon oligomer concentration: at very low concentrations, d(A-G)10 migrates faster than the random coil d(C-T)10, probably because it is a more compact single hairpin; at high concentrations, it co-migrates with the linear duplex d(A-G)10 x d(C-T)10, probably because it is a two-hairpin tetraplex. Molecular weights measured by equilibrium sedimentation in 0.1 M Na+, pH 7, reveal a mixture of monomer and dimer species at 1 degree C, but only a monomer at 40 degrees C; in 0.6 M Na+, pH 7, only a dimer species is observed at 4 degrees C. That the single- and double-stranded species are hairpin helices, is indicated by preferential S1 nuclease cleavage at the center of the oligomer(s), i.e., the loop of the hairpin(s). The UV melting transition below 0.3 M Na+ or K+, exhibits a dTm/dlog[Na+/K+] of 33 or 36 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex to a single-hairpin duplex with extrahelical residues. When [Na+/K+] > or = 0.3 M, dTm/dlog [Na+/K+] is 19 or 17 degrees C, respectively, consistent with conversion of a two-hairpin tetraplex directly to single strands. A two-hairpin structure stabilized by G-tetrads is indicated by differential scanning calorimetry in 0.15 M Na+/5 mM Mg2+, with deltaH of formation per mole of the two-hairpin tetraplex of -116.9 kcal or -29.2 kcal/mol of G-tetrad.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center

The effect of metal substitution on the activity and structure of the aminopeptidase P (APPro) from Escherichia coli has been investigated. Measurements of activity in the presence of Mn2+, Mg2+, Zn2+, Na+, and Ca2+ show that significant activity is seen only in the Mn-bound form of the enzyme. The addition of Zn2+ to [MnMn(APPro)] is strongly inhibitory. Crystal structures of [MnMn(APPro)], [MgMg(APPro)], [ZnZn(APPro)], [ZnMg(APPro)], [Ca_(APPro)], [Na_(APPro)], and [apo(APPro)] were determined. The structures of [Ca_(APPro)] and [Na_(APPro)] have a single metal atom at their active site. Surprisingly, when a tripeptide substrate (ValProLeu) was soaked into [Na_(APPro)] crystals in the presence of 200 mM Mg2+, the structure had substrate, but no metal, bound at the active site. The structure of apo APPro complexed with ValProLeu shows that the N-terminal amino group of a substrate can be bound at the active site by carboxylate side chains that normally bind the second metal atom, providing a model for substrate binding in a single-metal active enzyme. Structures of [MnMn(APPro)] and [ZnZn(APPro)] complexes of ProLeu, a product inhibitor, in the presence of excess Zn reveal a third metal-binding site, formed by two conserved His residues and the dipeptide inhibitor. A Zn atom bound at such a site would stabilize product binding and enhance inhibition.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.     

A monoclonal antibody specific for the duplex DNA poly[d(TC)].poly[d(GA)]

Although most duplex DNAs are not immunogenic some synthetic DNAs such as poly[d(Tm5C)].poly[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid-phase radioimmune assay. Jel 172 bound well to poly[d(TC)].poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)].poly[d(GAA)] and poly[d(TCC)].poly[d(GGA)]. The binding to poly[d(Br5UC)].poly[d(GA)] was enhanced while that to poly[d(TC)].poly[d(IA)] was decreased compared to poly[d(TC)].poly[D(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.