Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ～1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ～1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ～500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

Requirement for sodium in the anaerobic growth of Aerobacter aerogenes on citrate

Anaerobic growth of Aerobacter aerogenes on citrate as a carbon source required the presence of Na(+). The growth rate increased with increasing Na(+) concentration and was optimal at 0.10 m Na(+). The requirement was specific for Na(+), which could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). K(+) was required for growth in the presence of Na(+), the optimal K(+) concentration being 0.15 mm. Enzyme profiles were determined on cells grown in three different media: (i) intermediate Na(+), high K(+) concentration, (ii) high Na(+), high K(+) concentration, and (c) high Na(+), low K(+) concentration. All cells contained the enzymes of the citrate fermentation pathway, namely, citritase and the Na(+)-requiring oxalacetate (OAA) decarboxylase. All of the enzymes of the citric acid cycle were present, except alpha-ketoglutarate dehydrogenase which could not be detected. The incomplete citric acid cycle was, in effect, converted into two biosynthetic pathways leading to glutamate and succinate, respectively. The specific activities of citritase and OAA decarboxylase were lowest in medium (i), and under these conditions the activity of OAA decarboxylase appeared to be limited in vivo by the availability of Na(+). Failure of A. aerogenes to grow anaerobically on citrate in the absence of Na(+) can be explained at the enzymatic level by the Na(+) requirement of the OAA decarboxylase step of the citrate fermentation pathway and by the absence of an alternate pathway of citrate catabolism.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

Synthesis, DNA cleavage, and cytotoxicity of a series of bis(propargylic) sulfone crown ethers

Compounds that couple molecular recognition of specific alkali metal ions with DNA damage may display selective cleavage of DNA under conditions of elevated alkali metal ion levels reported to exist in certain cancer cells. We have prepared a homologous series of compounds in which a DNA reactive moiety, a bis(propargylic) sulfone, is incorporated into an alkali metal ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ethers to bind Li(+), Na(+), and K(+) ions was determined. For the series of crown ethers, the association constants for Li(+) ions are generally low (< 2 x 10(4)M(-1)). Only two of the bis(propargylic) sulfone crown ethers associate with Na(+) or K(+) ions (K(a) 4-8 x 10(4)M(-1)), with little discrimination between Na(+) or K(+) ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of Li(+), Na(+), and K(+) ions was determined. The two crown ethers that bind Na(+) and K(+) display a modest increase in DNA cleavage efficiency in the presence of Na(+) or K(+) ions as compared to Li(+) ions. These two bis(propargylic) sulfone crown ethers are also more cytotoxic against a panel of human cancer cell lines when compared to a non-crown ether macrocyclic bis(propargylic) sulfone.     

Effects of nigericin and monactin on cation permeability of Streptococcus faecalis and metabolic capacities of potassium-depleted cells

At a concentration of 10(-6)m, nigericin and monactin inhibited growth of Streptococcus faecalis, and the inhibition was reversed by addition of excess K(+). In the presence of certain antibiotics, the cells exhibited increased permeability to certain cations; internal Rb(+) was rapidly lost by exchange with external H(+), K(+) Rb(+), and, more slowly, with Na(+) and Li(+). No effect was observed on the penetration of other small molecules. Cation exchanges induced by nigericin and monactin were metabolically passive and apparently did not involve the energy-dependent K(+) pump. When the cells were washed, the cytoplasmic membrane recovered its original impermeability to cations. By use of monactin, we prepared cells whose K(+) content had been completely replaced by other cations, and the metabolic characteristics of K(+)-depleted cells were studied. Cells containing only Na(+) glycolyzed almost as well as did normal ones and, under proper conditions, could accumulate amino acids and orthophosphate. These cells also incorporated (14)C-uracil into ribonucleic acid but incorporation of (14)C-leucine into protein was strictly dependent upon the addition of K(+). When K(+) or Rb(+) was added to sodium-loaded cells undergoing glycolysis, these ions were accumulated by stoichiometric exchange for Na(+). From concurrent measurements of the rate of glycolysis, it was calculated that one mole-pair of cations was exchanged for each mole of adenosine triphosphate produced.     

Sodium, an Obligate Growth Requirement for Predominant Rumen Bacteria

Sodium is an obligate growth requirement for most currently recognized predominant species of rumen bacteria. The isoosmotic deletion of Na(+) from a nutritionally adequate defined medium completely eliminated growth of most species. Growth yields and rates were both a function of Na(+) concentration for Na(+)-requiring species, and Na(+) could not be replaced by Rb(+), Li(+), or Cs(+) when these ions were substituted for Na(+) at a concentration equivalent to an Na(+) concentration that supported abundant growth. Li(+), Cs(+), or Rb(+) was toxic at an Na(+)-replacing concentration (15 mM) but not at a K(+)-replacing concentration (0.65 mM). K(+) was also an obligate growth requirement for rumen bacteria in media containing Na(+) and K(+) as major monovalent cations, but K(+) could be replaced, for most species, by Rb(+). The quantities of Na(+) that support rapid and abundant growth of Na(+)-requiring rumen bacteria show that these organisms are slight halophiles. A growth requirement for Na(+) appears more frequent among nonmarine bacteria than has been previously believed.     

Kinetics and mechanism of K+- and Na+-induced folding of models of human telomeric DNA into G-quadruplex structures

Cation-induced folding into quadruplex structures for three model human telomeric oligonucleotides, d[AGGG(TTAGGG)(3)], d[TTGGG(TTAGGG)(3)A] and d[TTGGG(TTAGGG)(3)], was characterized by equilibrium titrations with KCl and NaCl and by multiwavelength stopped flow kinetics. Cation binding was cooperative with Hill coefficients of 1.5-2.2 in K(+) and 2.4-2.9 in Na(+) with half-saturation concentrations of 0.5-1 mM for K(+) and 4-13 mM for Na(+) depending on the oligonucleotide sequence. Oligonucleotide folding in 50 mM KCl at 25 degrees C consisted of single exponential processes with relaxation times tau of 20-60 ms depending on the sequence. In contrast, folding in100 mM NaCl consisted of three exponentials with tau-values of 40-85 ms, 250-950 ms and 1.5-10.5 s. The folding rate constants approached limiting values with increasing cation concentration; in addition, the rates of folding decreased with increasing temperature over the range 15-45 degrees C. Taken together, these results suggest that folding of G-rich oligonucleotides into quadruplex structures proceeds via kinetically significant intermediates. These intermediates may consist of antiparallel hairpins in rapid equilibrium with less ordered structures. The hairpins may subsequently form nascent G-quartets stabilized by H-bonding and cation binding followed by relatively slow strand rearrangements to form the final completely folded topologies. Fewer kinetic intermediates were evident with K(+) than Na(+), suggesting a simpler folding pathway in K(+) solutions.     

The effect of sodium, potassium and ammonium ions on the conformation of the dimeric quadruplex formed by the Oxytricha nova telomere repeat oligonucleotide d(G(4)T(4)G(4)).

The DNA sequence d(G(4)T(4)G(4)) [Oxy-1.5] consists of 1.5 units of the repeat in telomeres of Oxytricha nova and has been shown by NMR and X-ray crystallographic analysis to form a dimeric quadruplex structure with four guanine-quartets. However, the structure reported in the X-ray study has a fundamentally different conformation and folding topology compared to the solution structure. In order to elucidate the possible role of different counterions in this discrepancy and to investigate the conformational effects and dynamics of ion binding to G-quadruplex DNA, we compare results from further experiments using a variety of counterions, namely K(+), Na(+)and NH(4)(+). A detailed structure determination of Oxy-1.5 in solution in the presence of K(+)shows the same folding topology as previously reported with the same molecule in the presence of Na(+). Both conformations are symmetric dimeric quadruplexes with T(4)loops which span the diagonal of the end quartets. The stack of quartets shows only small differences in the presence of K(+)versus Na(+)counterions, but the T(4)loops adopt notably distinguishable conformations. Dynamic NMR analysis of the spectra of Oxy-1.5 in mixed Na(+)/K(+)solution reveals that there are at least three K(+)binding sites. Additional experiments in the presence of NH(4)(+)reveal the same topology and loop conformation as in the K(+)form and allow the direct localization of three central ions in the stack of quartets and further show that there are no specific NH(4)(+)binding sites in the T(4)loop. The location of bound NH(4)(+)with respect to the expected coordination sites for Na(+)binding provides a rationale for the difference observed for the structure of the T(4)loop in the Na(+)form, with respect to that observed for the K(+)and NH(4)(+)forms.     

Sodium and Other Inorganic Growth Requirements of Bacteroides amylophilus

Bacteroides amylophilus has growth requirements for Na(+), PO(4) (3-), K(+), and small quantities of Mg(2+). No requirement could be shown for Ca(2+) in media previously found growth-yield-limiting for Bacteroides succinogenes. Deletion of Co(2+), Mn(2+), Cl(-), or SO(4) (2-) did not affect growth. Quantitative studies indicate that Na(+), K(+), and PO(4) (3-) have differing effects on the growth of B. amylophilus. A concentration of sodium and potassium ions affects both growth rate and growth yield, whereas a phosphate concentration markedly affects growth yield, but affects growth rate only slightly, if at all. The sodium requirement of B. amylophilus is absolute. It cannot be replaced by K(+), Li(+), Rb(+), or Cs(+). The latter three monovalent cations are toxic to B. amylophilus if supplied to the organism at Na(+)-replacing concentrations. K(+) is inactive at similar concentrations. The K(+) requirement of B. amylophilus may be satisfied by Rb(+). The concentration of Na(+) required by B. amylophilus for abundant growth suggests that B. amylophilus should be considered a slightly halophilic organism. The results suggest that Na(+) may be a more frequent requirement among terrestial bacteria obtained from relatively low-salt environments than has been previously believed.     

Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase

Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved.     

Sodium binding site of factor Xa: role of sodium in the prothrombinase complex

The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.     

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.     

Potassium ion-stimulated and sodium ion-dependent adenosine diphosphate–adenosine triphosphate exchange activity in a kidney microsomal fraction

1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed.     

Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR.     

An ONIOM investigation on anion recognition of alkali–metal complexes with diurea calix[4]arene receptor

The ONIOM(B3LYP/6-31G(d):AM1) optimized structures of complexes of diurea calix[4]arene receptor (L) with alkali metals Li(+), Na(+) and K(+) and their complexes with halide ions F(-), Cl(-), Br(-), oxygen-containing anions HCO(3)(-), HSO(4)(-) and CH(3)COO(-) ions were obtained. Binding energies and thermodynamic properties of complex receptors LiL(+), NaL(+) and KL(+) with these anions were determined. The binding stabilities according to binding energies of LiL(+), NaL(+) and KL(+) associated with anions computed either at the ZPVE-corrected ONIOM(B3LYP/6-31G(d):AM1) or BSSE-corrected B3LYP/6-31 + G(d,p)//ONIOM(B3LYP/6-31G(d):AM1) are in the same order: F(-) > CH(3)COO(-) ≈ HCO(3)(-) > Br(-) ≈ HSO(4)(-) ≈ Cl(-). All the receptors LiL(+), NaL(+) and KL(+) were found to be selective toward fluoride ion.     

A sodium ion-dependent A1AO ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus

The rotor subunit c of the A(1)A(O) ATP synthase of the hyperthermophilic archaeon Pyrococcus furiosus contains a conserved Na(+)-binding motif, indicating that Na(+) is a coupling ion. To experimentally address the nature of the coupling ion, we isolated the enzyme by detergent solubilization from native membranes followed by chromatographic separation techniques. The entire membrane-embedded motor domain was present in the preparation. The rotor subunit c was found to form an SDS-resistant oligomer. Under the conditions tested, the enzyme had maximal activity at 100 degrees C, had a rather broad pH optimum between pH 5.5 and 8.0, and was inhibited by diethystilbestrol and derivatives thereof. ATP hydrolysis was strictly dependent on Na(+), with a K(m) of 0.6 mM. Li(+), but not K(+), could substitute for Na(+). The Na(+) dependence was less pronounced at higher proton concentrations, indicating competition between Na(+) and H(+) for a common binding site. Moreover, inhibition of the ATPase by N',N'-dicyclohexylcarbodiimide could be relieved by Na(+). Taken together, these data demonstrate the use of Na(+) as coupling ion for the A(1)A(O) ATP synthase of Pyrococcus furiosus, the first Na(+) A(1)A(O) ATP synthase described.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

Kinetic attributes of rat liver microsomal adenosine 5' triphosphate phosphohydrolase (ATPase)

The kinetic properties of the rat liver microsomal ATPase, with respect to Na(+), K(+) and AT P requirements were examined. Presence of Na(+) and K(+), or both hardly caused any stimulation of the enzyme activity. The Km values for Na(+) and K(+) were substantially low (0.32 and 0.05 mM, respectively), compared to those reported for the Na(+), K(+) ATPasesfrom different tissues. Substrate kinetics studies revealed that in the absence of Na(+) and K(+), ATP is an activator of the enzyme. The enzyme displayed increased activity with increase in the energy of activation in the absence of Na(+) and K(+). The activity was partially inhibited by ouabain only in the presence of Na(+) and K(+). The results suggest that the liver microsomal enzyme is not a Na(+), K(+) ATPase, but has requirement of monovalent cations for the regulation of its activity. Also, the beta3 subunit of the enzyme has a Km lowering effect.