Effect of competing self-structure on triplex formation with purine-rich oligodeoxynucleotides containing GA repeats.

Competition between triplex formation with double-stranded DNA and oligonucleotide self-association was investigated in 23mer GA and GT oligonucleotides containing d(GA)5 or d(GT)5 repeats. Whereas triplex formation with GT oligonucleotides was diminished when temperature increased from 4 to 37 degrees C, triplex formation with GA oligonucleotides was enhanced when temperature increased within the same range due to the presence of competing intermolecular GA oligonucleotide self-structure. This self-structure was determined to be a homoduplex stabilized by the internal GA repeats. UV spectroscopy of these homoduplexes demonstrated a single sharp transition with rapid kinetics (Tm = 38.5-43.5 degrees C over strand concentrations of 0.5-4 microM, respectively, with transition enthalpy, delta H = -89 +/- 7 kcal/mol) in 10 mM MgCl2, 100 mM NaCl, pH 7.0. Homoduplex formation was strongly stabilized by multivalent cations (spermine > Mg2+ = Ca2+) and destabilized by low concentrations of monovalent cations (K+ = Li+ = Na+) in the presence of divalent cations. However, unlike GA or GT oligonucleotide-containing triplexes, the homoduplex formed even in the absence of multivalent cations, stabilized by only moderate concentrations of monovalent cations (Li+ > Na+ > K+). Through the development of multiple equilibrium states and the resulting depletion of free oligonucleotide, it was found that the presence of competing self-structure could decrease triplex formation under a variety of experimental conditions.     

A unique binding cavity for divalent cations in the DNA-metal-chromomycin A3 complex

Binding of chromomycin A3 (CRA) to calf thymus DNA was investigated in the presence of divalent cations using visible absorption and 1H-nmr spectroscopies. An apparent equilibrium binding constant (approximately 10(11) M-1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA-M-CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H-nmr spectrum of the above complex using the paramagnetic Co2+ cation, indicating that the metal occupies a unique binding site. Since no induced 1H-nmr chemical shifts were observed for the drug-Co2+ mixture, it was concluded that no metal-drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (less than 0.85 A) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA-M-CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.     

Effect of divalent metal cations on the dimerization of OXA-10 and -14 class D beta-lactamases from Pseudomonas aeruginosa

The factors influencing the oligomerization state of OXA-10 and OXA-14 class D beta-lactamases in solution have been investigated. Both enzymes were found to exist as an equilibrium mixture of a monomer and dimer, with a K(d) close to 40 microM. The dimeric form was stabilized by divalent metal cations. The ability of different metal ions to stabilize the dimer was in the following order: Cd(2+) > Cu(2+) > Zn(2+) > Co(2+) > Ni(2+) > Mn(2+) > Ca(2+) > Mg(2+). The apparent K(d)s describing the binding of Zn(2+) and Cd(2+) cations to the OXA-10 dimer were 7.8 and 5.7 microM, respectively. The metal ions had a profound effect on the thermal stability of the protein complex observed by differential scanning calorimetry. The enzyme showed a sharp transition with a T(m) of 58.7 degrees C in the absence of divalent cations, and an equally sharp transition with a T(m) of 78.4 degrees C in the presence of a saturating concentration of the divalent cation. The thermal transition observed at intermediate concentrations of divalent metal ions was rather broad and lies between these two extremes of temperature. The equilibrium between the monomer and dimer is dependent on pH, and the optimum for the formation of the dimer shifted from pH 6.0 in the absence of divalent cations to pH 7.5 at saturating concentrations. The beta-lactamase activity increased approximately 2-fold in the presence of saturating concentrations of zinc and cadmium ions. Reaction with beta-lactams caused a shift in the equilibrium toward monomer formation, and thus an apparent inactivation, but the divalent cations protected against this effect.     

Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: I. Interaction with δ and μ Sites

In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.     

Cu2+ binding triggers alphaBoPrP assembly into insoluble laminar polymers

Cu(2+) binding is so far the best characterized property of the prion protein. This interaction has been mapped to the N-terminal domain of the prion protein where multiple His residues occur largely embedded within the repetitive PHGGGWGQ sequence known as octarepeats. When Cu(2+) interaction is studied using a solution of full-length bovine prion protein containing six octarepeats at protein concentrations above 25 microM, a drastic increase in solution turbidity is observed due to the formation of insoluble cation-protein complexes that appear as bidimensional polymer meshes. These bidimensional meshes consist of a single layer of protein molecules crosslinked by Cu(2+) cations. Polymer formation is a cooperative process that proceeds by nucleation of protein molecules with a Cu(2+) site occupancy of above 2. These results support the hypothesis that the N-terminal domain of prion protein is a ligand binding module that promotes crosslinked assembly, and suggest the existence of inter-repeat Cu(2+) sites.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

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)     

Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4? and HATP3? in the assay mixture

The effects of Mg(2+) and Ca(2+) on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP(3-) and ATP(4-) on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP(2-), and their ratio to MgATP(2-) is constant if Mg(2+) and H(+) concentrations are unchanged; (ii) Mg(2+) addition decreased the ratio of the minor species to MgATP(2-) and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP(3-) or ATP(4-). On the other hand, kinetic analysis of Mg(2+) effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP(2-)) site, with apparent K(m) values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP(2-) merged into one at high Mg(2+) concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg(2+) as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca(2+) effects showed: (i) competition with Mg(2+) at the metal site (K(i) 20mum for bone and 40mum for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca(2+) binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca(2+) in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.     

Potentiation of TRPM7 inward currents by protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from -100 to -40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an approximately 10-fold increase at pH 4.0 and 1-2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca(2+) and Mg(2+) for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca(2+) and Mg(2+) concentrations were increased. Third, the apparent affinity for Ca(2+) and Mg(2+) was significantly diminished at elevated external H(+) concentrations. Fourth, the anomalous-mole fraction behavior of H(+) permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca(2+) and Mg(2+) bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H(+) concentrations, the affinity of TRPM7 for Ca(2+) and Mg(2+) is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg(2+)-inhibitable cation current (MIC) or Mg nucleotide-regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Characteristics of the binding of tacrine to acidic phospholipids.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrate) is an inhibitor of acetylcholinesterase currently used in the treatment of the symptoms of Alzheimer's disease. The present study demonstrates preferential binding of this drug to acidic phospholipids, as revealed by fluorescence polarization, penetration into lipid monolayers, and effects on the thermal phase behavior of dimyristoyl phosphatidic acid (DMPA). A fivefold enhancement in the polarization of tacrine emission is evident above the main phase transition temperature (T(m)) of DMPA vesicles, whereas below T(m) only a 0.75-fold increase is observed. In contrast, the binding of tacrine to another acidic phospholipid, dimyristoylphosphatidylglycerol, did not exhibit strong dependence on T(m). In accordance with the electrostatic nature of the membrane association of tacrine, the extent of binding was augmented with increasing contents of egg PG in phosphatidylcholine liposomes. Furthermore, [NaCl] > 50 mM dissociates tacrine (albeit incompletely) from the liposomes composed of acidic phospholipids. Inclusion of the cationic amphiphile sphingosine in egg PG vesicles decreased the membrane association of tacrine until at 1:1 sphingosine: egg PG stoichiometry binding was no longer evident. Tacrine also penetrated into egg PG but not into egg PC monolayers. Together with broadening of the main transition and causing a shoulder on its high temperature side, the binding of tacrine to DMPA liposomes results in a concentration-dependent reduction both in the combined enthalpy delta H of the above overlapping endotherms and the main transition temperature T(m). Interestingly, these changes in the thermal phase behavior of DMPA as a function of the content of the drug in vesicles were strongly nonlinear. More specifically, upon increasing [tacrine], T(m) exhibited stepwise decrements. Simultaneously, sharp minima in delta H were observed at drug:lipid stoichiometries of approximately 2:100 and 25:100, whereas a sharp maximum in delta H was evident at 18:100. The above results are in keeping with tacrine causing phase separation processes in the bilayer and may also relate to microscopic drug-induced ordering processes within the membrane.     

Binding effect of Cu(2+) as a trigger on the sol-to-gel and the coil-to-helix transition processes of polysaccharide, gellan gum

The binding effect of divalent cation Cu(2+) on the gelation process with a coil-helix transition in Cu(2+)/gellan aqueous solutions has been successfully elucidated by EPR, CD, and viscoelasticity measurements. Generally, Na-type gellan gum in aqueous solution can make gel when accompanied by an intrinsic coil-helix formation induced by hydrogen bonding between chains without any additional cations at T(ch)(-)(in) ( approximately 29 degrees C) with cooling temperature. An extrinsic coil-helix transition, induced by additional divalent cations in advance of the intrinsic sol-gel transition of gellan gum, is separately detected by CD measurement. The extrinsic coil-helix transition temperatures T(ch)(-)(ex) (>47 degrees C), which increased with the Cu(2+) concentration added, were nearly identical to the sol-gel transition temperature, T(sg), determined by the viscoelasticity measurement. Judging from the molar ellipticity by CD measurement and quantitative analysis of EPR spectra, it was elucidated that the helix forming process via divalent cations is composed of two steps ascribed to the different origins, i.e., a chemical binding effect via Cu(2+) ions in the initial stage and hydrogen bonds subsequently. Finally, we propose the coil-helix and the sol-gel transition mechanism initiated by the binding effect with the divalent cation, in which the partial chelate formation can cause local formation of helices and junction zones in the vicinity of the chelates at the initial stage of the process and stabilize the helices and the junction zones. On the other hand, the stabilized helices and junction zones can induce further formation and further stabilization of the Cu(2+)-gellan chelates. The mutual stabilization promotes the formation of three-dimensional network structure at the higher temperature than the intrinsic temperature for network formation.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

Sodium and potassium ion-dependent adenosine triphosphatase of mammalian brain. Interactions of magnesium ions with the phosphatase site.

Kinetic parameters are reported for Mg2+, Na+ and K+ as activators of the p-nitrophenylphosphatase activity associated with (Na+ + K+)-ATPase (ATP-phosphohydrolase, EC 3.6.1.3) of beef brain. In each case the phosphatase reaction is activated at low concentrations of the cation and inhibited by higher concentrations. The concentrations of cation that produced half-maximal activation and half-maximal inhibition are increased as the concentration of either of the other two cations is increased. These second ligand effects are all saturable functions. The apparent binding constant that characterizes the effect on activation is closely similar to that acting upon the inhibitory phase in each case.     

Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis

Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Weakly bound calcium ions involved in the thermostability of aqualysin I, a heat-stable subtilisin-type protease of Thermus aquaticus YT-1.

Aqualysin I is a heat-stable protease; in the presence of 1 mM Ca(2+), the enzyme is stable at 80 degrees C and shows the highest activity at the same temperature. After gel filtration to remove free Ca(2+) from the purified enzyme sample, the enzyme (holo-aqualysin I) still bound Ca(2+) (1 mol/mol of the enzyme), but was no longer stable at 80 degrees C. On treatment of the holo-enzyme with EDTA, bound Ca(2+) decreased to about 0.3 mol/mol of the enzyme. The thermostability of holo-aqualysin I was dependent on the concentration of added Ca(2+), and 1 mM added Ca(2+) stabilized the enzyme completely, suggesting that aqualysin I has at least two Ca(2+) binding sites, i.e. stronger and weaker binding ones. Titration calorimetry showed single binding of Ca(2+) to the holo-enzyme with an association constant of 3.1 x 10(3) M(-1), and DeltaH and TDeltaS were calculated to be 2.3 and 6.9 kcal/mol, respectively, at 13 degrees C. La(3+), Sr(2+), Nd(3+), and Tb(3+) stabilized the holo-enzyme at 80 degrees C, as Ca(2+) did. These results suggest that the weaker binding site exhibits structural flexibility to bind several metal cations different in size and valency, and that the metal binding to the weaker binding site is essential for the thermostability of aqualysin I.     

Brain (Na+,K+)-ATPase. Opposite effects of ethanol and dimethyl sulfoxide on temperature dependence of enzyme conformation and univalent cation binding

We examined effects of ethanol and dimethyl sulfoxide on the regulation and apparent thermodynamic properties of moderate affinity Na+ and K+ binding that regulates the K+-dependent phosphatase activity of (Na+,K+)-ATPase. Ethanol and other alcohols reduced the apparent affinity for Na+ and K+ at their moderate affinity sites and increased the negative delta H and delta S of cation binding. Dimethyl sulfoxide had the opposite effects. Inhibition by ethanol was favored by high temperature or low K+. Ethanol potentiated inhibition of K+ binding by ATP or Mg2+. Ethanol also shifted the equilibrium between K+-sensitive and -insensitive forms of (Na+,K+)-ATPase toward the K+-sensitive form; in this case, it reduced the negative delta H and delta S for the transition to K+-sensitive enzyme. Again, dimethyl sulfoxide had the opposite effects. These data indicate that ethanol and other agents considered to affect membrane fluidity act by a combination of membrane (on cation binding) and solvent (on conformation) effects. The most important effect of ethanol and similar agents on the enzyme is to prevent the formation of K+-sensitive enzyme by cation binding and to destabilize K+-sensitive enzyme in the presence of ATP. These results also add further evidence that the sites by which Na+ and K+ produce K+-sensitive enzyme are similar or identical.     

Adsorption of the cationic antitumoral drug celiptium to phosphatidylglycerol in membrane model systems. Effect on membrane electrical properties

The binding of the cationic antitumoral drug Celiptium to the anionic phospholipid phosphatidylglycerol was studied by measuring surface potentials and surface pressures in monolayers, and by determination of electrophoretic mobility on liposomes. Surface potential and zeta potential data were interpreted in terms of the Gouy-Chapman-Stern theory of the diffuse electrical double layer. A unique drug-to-lipid adsorption constant KaD, could not be calculated. KaD was observed to increase rapidly from 10(4) M-1 to 10(6) M-1 with an increase in drug concentration from 5 x 10(-7) M to 7 x 10(-6) M. This was accompanied by a marked decrease (in absolute value) in the corresponding electrophoretic mobilities which, from negative at low drug concentrations, became positive at drug concentrations of 10(-5) M and above. This indicates that the drug-to-lipid binding cannot be accounted for by a simple Langmuir adsorption isotherm, but corresponds to a more complex process, probably of a cooperative nature. Comparison of delta V and zeta potential data shows that adsorption of Celiptium to phosphatidylglycerol not only lowers the electrical surface potential, psi 0 (in absolute value) but also markedly reduces the polarization potential, delta Vp. These observations suggest that Celiptium destabilizes the electrical properties of cell plasma membranes.     

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.