The terminal phosphate in d(pGpG) reduces self-association

An investigation of the self-association behavior of 2'-deoxy[5'-phosphate-guanylyl-(3'-5')-guanosine] (d(pGpG)) in the presence of Na+ and K+ ions has been carried out by 1H and 31P NMR and FTIR spectroscopy. A comparison has been made of the self- association behavior of d(pGpG) with that of the related dinucleotide d(GpG), which has been shown to form extended structures based on stacked G-tetrads. Chemically, d(pGpG) monomer differs from d(GpG) only by the addition of a phosphate at the 5'-OH of the sugar residue. It was found that the addition of the second phosphate interferes with self-association. A suitable counterion is all that is required by d(GpG) to induce the formation of large super structures, but for d(pGpG) a large excess of salt is needed to produce the same effect. However, once self-association occurs, d(pGpG) forms similar structures to d(GpG) and has nearly the same properties. For both compounds, the K+ ion induces a more stable structure than the Na+ ion. The 31P NMR chemical shift ranges of d(pGpG) were consistent with the reported data for a phosphodiester and terminal phosphate. The small change in the chemical shift of the terminal phosphate with increasing temperature suggests that no major change in the terminal phosphate conformation occurred upon self-association. It was concluded that the terminal phosphate did not result in steric hindrance to self-association, but that interference to self-association was due to electrostatic repulsion effects.     

The effect of monovalent cations on the association behavior of guanosine 5'-monophosphate, cytidine 5'-monophosphate, and their equimolar mixture in aqueous solution

¹H- and ³¹P-nmr spectroscopy have been used to investigate the self-association of M₂(5′-CMP) [M = Li⁺, Na⁺, K⁺, Rb⁺, or (CH₃)₄ N⁺; 5′-CMP = cytidine 5′-monophosphate], the self-association of Li₂(5′-GMP) (5′-GMP = guanosine 5′-monophosphate), and the heteroassociation of 5′-GMP and 5′-CMP (1 : 1 mole ratio) in aqueous solution as a function of the nature of the monovalent cation. Proton spectral differences for the different 5′-CMP salts exhibit a cation-size dependence and have been ascribed to a change in the stacking geometry. An average stacking association constant of 0.63 ± 0.24M^?1 at 1°C, consistent with the weak stacking interactions of the cytosine bases, was determined for the 5′-CMP salts. Heteroassociation of 5′-GMP and 5′-CMP follows the reverse of the cation order for the formation of ordered aggregates of 5′-GMP. Heteroassociation occurs in the presence of Li⁺, Na⁺, and Rb⁺ ions, but only self-association occurs for the K⁺ nucleotides. Li₂(5′-GMP), which does not form ordered species, self-associates to form disordered base stacks with a stacking constant of 1.63 ± 0.11M^?1 at 1°C.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

Self-assembly of dideoxyguanosine (3′,3′) and (5′,5′)-monophosphates

The title compounds show a pronounced cation-directed ability to self-assemble in water and to gives columnar structures similar to four-stranded helices; for compound (5′→5′)-d(GpG), this leads to the formation of cholesteric and hexagonal liquid crystalline phases. Both phases are columnar and the cholesteric phase is left-handed. This behaviour is a further confirmation of the tendency of guanine derivatives to self-assemble to give stacked columnar structures whenever not impossible for structural reasons. The CD spectra of the aggregates in isotropic solutions are dominated by a negative exciton couplet centred around 250 nm associated to a left-handed columnar chirality. The shapes of the profiles, in the 220–300-nm region, for (5′→5′)-d(GpG) (in water or in saline solutions) and for (3′→3′)-d(GpG) (in KCl solution) are quasi-mirror images of those of poly(G) and (3′→5′)-d(GpG). The appearance of relatively intense CD signals around 280–300 nm in solution of (3′→3′)-d(GpG) in the presence of NaCl resembles that of (3′→5′)-d(GpG) in the presence of Rb⁺ or Na⁺. In the compounds investigated in this work, which present two equivalent ends, one observes the two CD features that have been associated, in the current literature, with the signature of four-stranded parallel and antiparallel structures: hence the origin of these CD bands cannot be found in the polarity of the strands. Self-assembly is favoured by the addition of extra salt and the stabilising effect of K⁺ is greater than that of Na⁺, in the case of (3′→3′)-d(GpG), an assembled species could be detected by CD only in the presence of extra salt. Chirality 10:734–741, 1998. © 1998 Wiley-Liss, Inc.     

A UV resonance Raman investigation of poly(rI): Evidence for cation-dependent structural perturbations

Poly(rI) stabilized by either Na⁺ or K⁺ was investigated using uv resonance Raman (UVRR) spectroscopy. Raman excitation profiles of inosine 5′-monophosphate demonstrated the 250 nm excitation selectively enhances base stacking interactions, while ribose and carbonyl stretching vibrations are preferentially enhanced with 210 nm excitation. These wavelengths were used to examine the structure of poly(rI) in the presence of either K⁺ or Na⁺ as a function of temperature. UVRR studies revealed that the K⁺ stabilized form is more thermally stable, yielding a T_m of ∼ 47°C compared to a T_m of ∼ 30°C for the Na⁺ stabilized form. We observed that both the ribosyl conformation and the coordination of the carbonyl groups depend on the nature of the cation. The C₆O stretching frequency indicates that Na⁺ coordinates much more strongly to the carbonyl groups than K⁺ (1672 cm⁻¹ Na⁺ vs 1684 cm⁻¹ K⁺ at 4°C). Conformationally sensitive modes of the phosphate backbone and the ribosyl ring indicate that Na⁺ stabilized poly(rI) predominantly exists in a C_3′-endo ribose conformation, whereas K⁺ stabilized poly(rI) adopts a C_2′-endo conformation possibly as a consequence of the larger ionic radius of the K⁺ ion. © 1998 John Wiley & Sons, Inc. Biopoly 46: 475–487, 1998     

K+ and Na+-Induced Self-Assembly of Telomeric Oligonucleotide d(TTAGGG)n

Abstracts

The telomeric DNA oligomers, d(TTAGGG)_n where n=1, 2, 4, could self-associate into the multi-stranded structures in appropriate condition and exhibited a different CD spectra. The present of Na⁺ was more advantage to facilitate the formation of anti-parallel conformation, but the present of K⁺ enhanced their thermal stability. Spectroscopic analysis of 3, 3′- diethyloxadicarbocyanine (DODC) showed the formation of hairpin quadruplex structures for d(TTAGGG)₂ and d(TTAGGG)₄, but d(TTAGGG) could not. The four-stranded tetraplexes and branched nanowire formed in the present of K⁺ or Na⁺ alone were observed by atomic force microscopy (AFM). The ability to self-assemble of d(TTAGGG)_n into four-stranded tetraplexes and nanowires depends strongly on the number of repeating units and ionic environment. A model to explain how these structures formed is proposed.     

Similarities and differences in interaction of K+ and Na+ with condensed ordered DNA. A molecular dynamics computer simulation study

Four 20 ns molecular dynamics simulations have been performed with two counterions, K⁺ or Na⁺, at two water contents, 15 or 20 H₂O per nucleotide. A hexagonal simulation cell comprised of three identical DNA decamers [d(5′-ATGCAGTCAG) × d(5′-TGACTGCATC)] with periodic boundary condition along the DNA helix was used. The simulation setup mimics the DNA state in oriented DNA fibers or in crystals of DNA oligomers. Variation of counterion nature and water content do not alter averaged DNA structure. K⁺ and Na⁺ binding to DNA are different. K⁺ binds to the electronegative sites of DNA bases in the major and the minor grooves, while Na⁺ interacts preferentially with the phosphate groups. Increase of water causes a shift of both K⁺ and Na⁺ from the first hydration shell of O1P/O2P and of the DNA bases in the minor groove with lesser influence for the cation binding to the bases in the major groove. Mobility of both water and cations in the K–DNA systems is faster than in the Na–DNA systems: Na⁺ organizes and immobilizes water structure around itself and near DNA while for K⁺ water is less organized and more dynamic.     

Die pH-Abhängigkeit der Aufnahme von H2PO 4 - , SO 4 = , Na+ und K+ und ihre gegenseitige Beeinflussung bei Ankistrodesmus braunii

Summary Ion uptake was studied using ³²P, ³⁵S, ²²Na and ⁴²K as tracers in synchronized cells of Ankistrodesmus, which were slightly starved with respect to the ions to be investigated. In the light and in the dark, phosphate uptake is maximal between pH 5.5 and 6.5. Whereas Na⁺ in comparison to K⁺ enhances phosphate uptake in the light (8 to 9-fold) and in the dark, Ca⁺⁺ exerts only a slightly stimulatory effect. The stimulation of phosphate binding by Na⁺ occurs rapidly, even after less than 5 sec of incubation, and also in the presence of an equimolar concentration of K⁺.The pH-dependence of Na⁺-uptake in the light and in the dark is comparable to a dissociation curve: Na⁺-uptake increases with decreasing extracellular H⁺-concentration and is inversely proportional to phosphate uptake in the absence of Na⁺. The light:dark ratio of Na⁺-uptake at pH 8 amounts to 7:1. Mere adsorption of Na⁺ is similarly dependent on the pH. K⁺ strongly competes with Na⁺-uptake, even at pH 8. K⁺-uptake proceeds in a quite different manner from Na⁺-uptake and has an optimum at pH 7.Sulfate is taken up linearly in a biphasic process as a function of time; the pH-optimum lies between pH 7.5 and 8. K⁺ but not Na⁺ slightly enhances sulfate uptake.The Na⁺-enhancement of phosphate uptake can be related neither to a sodium-potassium exchange pump nor to a photosynthesis-dependent ion-exchange reaction.The results suggest that the uptake of phosphate, Na⁺ and K⁺, and the influence of alkali cations on phosphate uptake, but not sulfate uptake, are strongly dependent on fixed charges of the plasmalemma or even of the cell wall. These fixed charges may even prevent an active ion uptake.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

A Nanosecond Molecular Dynamics Study of Antiparallel d(G)7 Quadruplex Structures: Effect of the Coordinated Cations

Abstract

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G₇) quadruplex structures with different coordinated ions, namely Na⁺ and K⁺ ion, water and Na⁺ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of ～ 1.5 Å from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K⁺ ions are quite immobile. MD studies at 400K indicate that K⁺ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K⁺ ion coordinated structure is more favourable than that for the Na⁺ ion coordinated antiparallel quadruplex structure.     

The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na+,K+)-ATPase

A rat heart plasma membrane preparation isolated in a sucrose medium and some of its enzymatic properties have been investigated.It has been shown that a rat heart plasma membrane fraction contains high creatine phosphokinase activity which can not be diminished by repeated washing with sucrose solution. Creatine phosphokinase extracted from a plasma membrane fraction with potassium chloride and 0.01% deoxycholate solution is electrophoretically identical to MM isoenzyme of creatine phosphokinase. Under the conditions where (Na⁺,K⁺)-ATPase is activated by addition of Na⁺,K⁺ and MgATP, creatine phosphokinase of plasma membrane fraction is able to maintain a low ADP concentration in the medium if creatine phosphate is present. The rate of creatine release is dependent upon MgATP concentration in accordance with the kinetic parameters of the (Na⁺,K⁺)-ATPase and is significantly inhibited by ouabain (0.5 mM). The rate of creatine release is also dependent on creatine phosphate concentration in conformance with the kinetic parameters of MM isozyme of creatine phosphokinase,It is concluded that in intact heart cells the plasma membrane creatine phosphokinase may ensure effective utilization of creatine phosphate for immediate rephosphorylation of ADP produced in the (Na⁺,K⁺)-ATPase reaction.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Na+/H+ exchanger (NHE) in the basolateral membrane is encoded by NHE-1 mRNA in LLC-PK1 clone 4 cells

Several isoforms of Na⁺/H⁺ exchanger (NHE-1–5) have been identified. LLC-PK1 clone 4 (CL4) expresses the amiloride-sensitive type of NHE predominantly in the basolateral membrane, which is believed to be NHE-1. It is not clear whether CL4 expresses NHE in the apical membrane and which side of NHE is encoded by the NHE-1 mRNA. Using acidified CL4 cells on the filter membrane, we examined Na⁺-dependent pH recovery of the apical and basolateral membranes separately. Na⁺ applied to the apical membrane recovered cell pH. Na⁺-dependent pH recovery in the apical membrane was not inhibited by SITS, DIDS, or contralateral amiloride. Li⁺ but not K⁺, chol⁺, or NMG⁺ could replace Na⁺. These data are consistent with the presence of NHE in the apical membrane. Transfection with an antisense oligonucleotide corresponding to the 5′ terminal site of NHE-1 cDNA of CL4 decreased NHE activity in the basolateral membrane but not in the apical membrane. We conclude that CL4 expresses NHE activities in both apical and basolateralmembranes and that NHE-1 mRNA encodes NHE only in the basolateral membrane. J. Cell. Physiol. 171:318–324, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

Structural polymorphism exhibited by a homopurine.homopyrimidine sequence found at the right end of human c-jun protooncogene

Homopurine·homopyrimidine (Pu·Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu·Py DNA segment within human c-jun protooncogene 3′-region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na⁺/K⁺ with Mg²⁺ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na⁺ in acidic as well as neutral pH facilitated the duplex formation, while K⁺ favored the formation of self-associated structures. In Na⁺ and Mg²⁺, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.     

Properties of (Na+ + K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na⁺ + K⁺)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na⁺ + K⁺)-activated ATPase activity was documented by demonstrating specific cation requirements for Na⁺ and K⁺, while the divalent cation, Ca²⁺, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na⁺ + K⁺)-activated ATPase activity averaged 10.07 ± 2.80 μmol P_i/mg protei per h compared to 50.03 ± 11.41 for Mg²⁺-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na⁺ + K⁺)-activated ATPase without any effect on Mg²⁺-activated ATPase. Both (Na⁺ + K⁺)-activated ATPase and Mg²⁺-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na⁺ + K⁺)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na⁺ + K⁺)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.     

Metabolic and ionoregulatory responses of the Amazonian cichlid, <Emphasis Type="Italic">Astronotus ocellatus</Emphasis>, to severe hypoxia

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 ± 0.19 mg O₂/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD⁺]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP_free], redox state, or [pyruvate]). Hypoxia exposure caused a ∼65% decrease in gill Na⁺/K⁺ ATPase activity, which was not matched by changes in Na⁺/K⁺ ATPase α-subunit protein abundance indicating post-translational modification of Na⁺/K⁺ ATPase was responsible for the decrease in activity. Despite decreases in gill Na⁺/K⁺ ATPase activity, plasma [Na⁺] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na⁺/K⁺ ATPase α-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na⁺/K⁺ ATPase α-subunit relative to other genes.     

Examination of the substrate stoichiometry of the intestinal Na+/phosphate cotransporter

Summary The substrate stoichiometry of the intestinal Na⁺/phosphate cotransporter was examined using two measures of Na⁺-dependent phosphate uptake: initial rates of uptake with [³²P] phosphate and phosphate-induced membrane depolarization using the potential-sensitive dye diSC₃(5). Isotopic phosphate measures electrogenic and electroneutral Na⁺-dependent phosphate uptake, while phosphate-induced membrane depolarization measures electrogenic phosphate uptake. Using these measures of Na-dependent phosphate uptake, three parameters were compared: substrate affinity; phenylglyoxal sensitivity and labeling; and inhibiton by mono- and di-fluorophosphates. Na⁺/phosphate cotransport was found to have similar Na⁺ activations (apparentK _0.5's of 28 and 25mm), apparentK _m 's for phosphate (100 and 410 m), andK _0.5's for inhibition by phenylglyoxal (70 and 90 m) using isotopic phosphate, uptake and membrane depolarization, respectively. Only difluorophosphate inhibited Na⁺-dependent phosphate uptake below 1mm at pH 7.4.Difluorophosphate also protected a 130-kDa polypeptide from FITC-PG labeling in the presence of Na⁺ with apparentK _0.5 for phosphate of 200 m; similar to the apparentK _m for phosphate uptake, andK _0.5 for phosphate protection against FITC-PG inhibition of Na⁺-dependent phosphate uptake and FITC-PG labeling of the 130-kDa polypeptide. These results indicate that the intestinal Na⁺/phosphate cotransporter is electrogenic at pH 7.4, that H₂PO ₄ ^– is the transport-competent species, and that the 130-kDa polypeptide is an excellent candidate for the intestinal Na⁺/phosphate cotransporter.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.