The effect of amine structure on complexation with lasalocid in model membrane systems. II. Ionophore selectivity for amines in lipid bilayers and at oil/water interfaces

The ionophore antibiotic X-537A (lasalocid) transports biogenic amines across biological and artificial membranes. The major portion of amine flux (greater than 99%) occurs as a 1:1 neutral complex. The rank order of ionophore selectivity was determined for lipid bilayer membrane transport of amines based on a comparison of permeability coefficients: p-tyramine ～ β-phenylethylamine ～ amphetamine > methamphetamine > dopamine > phenylephrine ～ metanephrine > norepinephrine > epinephrine. This rank order is in agreement with results obtained from partitioning measurements which were carried out in parallel to the bilayer membrane experiments. A correlation between amine structure and binding characteristics has been developed.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1 %) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K ≈ 10⁴) and the turnover numbers (≈ 3 · 10⁴/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

The Effect of Calcium Ionophores on Fragmented Sarcoplasmic Reticulum

X-537 A and A 23187, two antibiotics which form liphophilic complexes with divalent cations, function as ionophores in vesicular fragments of sarcoplasmic reticulum (SR). Addition of either ionophore to SR preloaded with calcium in the presence of adenosine triphosphate (ATP), causes rapid release of calcium. Furthermore, net calcium accumulation by SR is prevented, when the ionophores are added to the reaction mixture before ATP. On the contrary, ATP-independent calcium binding to SR is not inhibited. This effect is specific for the two antibiotics and could not be reproduced, either by inactive derivatives, or by other known ionophores. Neither ionophore produces alterations of the electron microscopic appearance of SR membranes or inhibition of the calcium-dependent ATPase. In fact, the burst of ATP hydrolysis obtained on addition of calcium, is prolonged in the presence of the ionophores. Lanthanum inhibits ATP-independent calcium binding to SR, ATP-dependent calcium accumulation and calcium-dependent ATPase. However, addition of lanthanum to SR preloaded in the presence of ATP, does not cause calcium release. The reported experiments indicated that: (a) ATP-dependent calcium accumulation by SR results in primary formation of calcium ion gradients across the membrane. (b) Most of the accumulated calcium is not available for displacement by lanthanum on the outer surface of the membrane. (c) Calcium ionophores induce rapid equilibration of the gradients, by facilitating cation diffusion across the membrane.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H+, K+ and Na+ in phospholipid vesicles

Rates of M⁺/H⁺ exchange (M⁺=K⁺, Na⁺) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M⁺ and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M⁺ dissociation constant, K_M, of the M⁺ bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H⁺ bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M⁺ and an anion) by forming 1:2 complexes with valinomycin–M⁺ or nonactin–M⁺. (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K_M increases. The decreases/increases in K_M mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Tyramine and β-phenylethylamine,from fermented food products,as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach

The aromatic amines tyramine and β-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a G_s-coupled receptor in response to tyramine and β-phenylethylamine stimulation. The β-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and β-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.     

Dissipation of the proton electrochemical potential in intact and lysed chloroplasts : I. The electrical potential

Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Trifluoromethyl-modified dipeptides by ZrCl4-promoted aza-Henry reactions

Chiral (R)-1-phenylethylamine was successfully employed in a tandem aza-Henry addition–reduction reaction to give chiral β-nitro α-trifluoromethyl amines. A subsequent coupling reaction with N-Boc-protected amino acids leads to obtain optically pure CF₃-modified dipeptides carrying two different N-protecting groups. These peptidomimetic units are characterized by the presence of the [CH(CF₃)NH] group as mimetic of the natural [CONH] peptidic bond and can be used for the synthesis of more complex CF₃-modified peptides after selective deprotection of one of the two amine functions. 2D NMR spectral analyses were employed to determine the absolute configurations of all newly synthesized chiral compounds.     

Purification and Characterization of Aromatic Amine Dehydrogenase from Alcaligenes xylosoxidans

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on β-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (α₂β₂) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the α-subunit (42.3-kDa subunit) and the β-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the β-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and β-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 °C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, β-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 μM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 °C for one month at least in phosphate buffer (pH 7.0).     

THE SUBCELLULAR DISTRIBUTION OF β-PHENYLETHYLAMINE, p-TYRAMINE AND TRYPTAMINE IN RAT BRAIN

—The quantitative subcellular distribution of β-phenylethylamine, p-tyramine and tryptamine in rat brain was investigated using the mass spectrometric integrated ion current technique. More of the total cellular tryptamine was found to be associated with paniculate fractions than was the case for phenyiethylamine and p-tyramine but a significant amount of this tryptamine was found to be labile. Analysis of the particulate fractions indicated that each of the amines was localized predominantly in the crude P₂ pellet and that the bulk of this was associated with the synaptosomal (P₂B) fraction. Inhibition of monoamine oxidase systems with pargyline caused an increase in the level of all three amines in all fractions, but the increase was greater in the supernatant than in the combined particulate fractions. This treatment produced changes in the distribution of β-phenylethylamine and p-tyramine between the various particulate subcellular fractions but did not markedly alter the distribution of tryptamine between the same fractions.     

Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li⁺ among alkali cations, was found to be capable of mediating the transport of NH₄⁺ and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li⁺ (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH₄⁺ (0.05). The molecular factors involved in determining these selectivities are assessed.     

Movement of calcium through artificial lipid membranes and the effects of ionophores

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1 : 1 phosphatidylcholine/phosphatidylserine mixture. The percentages of ⁴⁵Ca capture by these three types of liposomes are 10, 1 and 4% respectively.The efflux rates are 2.5 · 10^?6, 2 · 10^?6 and 4 · 10^?5 s^?1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity.The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1 : 2Ca · ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1 : 1 complex, but A23187 as a 2 : 1 complex.     

Effects of type of ionophore and carrier on in vitro ruminal dry matter disappearance,gas production,and fermentation end products of a concentrate substrate

Effects of ionophore type and carrier on in vitro ruminal digestion and fermentation patterns of a concentrate substrate were evaluated at various incubation times. Treatments were: control (no ionophore); lasalocid sodium commercial premix (Bov); lasalocid sodium mycelium cake (LasBio); laidlomycin sodium salt (LaidNa); laidlomycin propionate commercial premix (LaidPro); monensin sodium salt (Mon); and monensin sodium commercial premix (Rum). The Bov, LasBio, Mon, and Rum treatments supplied 4 μg of ionophore/mL of culture volume, whereas the LaidNa and LaidPro treatments supplied 1.33 μg of ionophore/mL. Total gas and methane production did not differ among treatments at any of the incubation times (P>0.09). Similarly, in vitro dry matter disappearance (IVDMD) was not affected by treatment (P>0.28) at 6, 18, and 24 h of incubation; however, IVDMD (P=0.03) was greater for ionophores than for the control at 12 h of incubation. Molar proportions of acetate (P<0.01), acetate:propionate (P<0.01), and total volatile fatty acid (VFA) concentrations (P<0.01) were decreased and propionate was increased (P<0.001) for the average of all ionophore-containing substrates compared with the control. Total VFA were decreased by Bov, LaidNa, and Rum, contrasted with their specific counterparts (LasBio, LaidPro, and Mon, respectively; P<0.05). Differences were detected among ionophore types for acetate (lasalocid vs. laidlomycin; P<0.05), propionate (lasalocid vs. monensin; P<0.05), and butyrate (monensin vs. lasalocid or laidlomycin; P<0.05). Capture of metabolic hydrogen in end products of fermentation was greater for ionophore-containing treatments (P<0.01) than for the control. These data suggest limited unique effects of ionophore type or carrier on IVDMD, total gas production, and methane; however, VFA proportions varied among ionophore types and carriers, which deserves further study.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Structure-activity relationships among noncyclic dicarboxamide Li(+)-selective carriers studied in lipid bilayer membranes.

The structure-activity relationships among three noncyclic diimide ionophores, designed to be Li+ carriers, were studied in lipid bilayer membranes. These ionophores (ETH1644, ETH1810, and ETH1811) vary in their N-imide substituents, going from two isobutyls to one isobutyl and one cyclohexyl to two cyclohexyls, respectively. ETH1811 was found to form two types of complexes with 1:1 and 1:2 ion-carrier stoichiometries, the former type dominant over most of the ionophore (0.1-10 microM) and salt (0.01-1.0 M) concentration ranges studied. In contrast, ETH1644 and ETH1810 were previously found to form a single type of complex with the 1:2 stoichiometry. The alkali cations selectivity sequence induced by ETH1811 is Li+ (1) greater than Na+ (0.08) greater than K+ (0.02) greater than Cs+ (0.008). These ETH1811-induced ionic selectivites as well as its relative potency in ion transport were found to be inferior to those of ETH1644 and ETH1810 (the latter being the best in this series). The conductance-voltage relationships reported here, for all three ionophores transporting alkali cations, were found to fit with a transport mechanism in which the diffusion of the ion-ionophore complex across the membrane is the single rate-limiting step, with the following exceptions: The addition of the dissociation of the ion-carrier complex as a second rate limiting step, for the Li(+)-ETH1810 and the Li(+)-ETH1811 systems. For ETH1810 the kinetics of the dissociation step is a minor, whereas for ETH1811 case it is a significant, addition (the ratio of the diffusion to dissociation rate constants being 0.08 and 0.2, respectively). The implications of the effects of the continuous structural change of these ionophores on their performance as ion carriers and on the design and synthesis of improved Li(+)-selective ionophores are discussed.     

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H+/M+ exchange across phospholipid vesicular membranes

From a study of the decay of the pH difference across vesicular membranes (delta pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M+ = K+, the rate of delta pH decay by H+/M+ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M+ = Na+) depending on whether the exchange ionophore chosen is monensin or nigericin. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anaesthetic (i) translocation rate constants increase, (ii) the concentrations of the M+ bound ionophores increase at the expense of H+ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigericin-H (Nig-H) for M+ = K+ whereas they are monensin-H (Mon-H) and nigericin-Na (Nig-Na) for M+ = Na+. Possible anaesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B), an associated increase in the membrane hydration (and membrane dielectric constant). An analysis of the consequent changes in the various transport step shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation of the observed changes in the delta pH decay rates. (b), Changes in the rates of fast equilibria at the interface contribute to changes in KH and KM. (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the delta pH decay data confirms the validity of the transport scheme used. In our experiments delta pH across the vesicular membrane was created by pH jump coming from a temperature jump.     

A fluorometric assay for beta-phenylethylamine in rat brain.

β-Phenylethylamine was found to react with ninhydrin in the presence of l-leucyl-l-alanine to yeild a highly fluorescent compound which seems to be specific for β-phenylethylamine, phenylalanine, and 5-hydroxytryptamine. The amount of β-phenylethylamine in rat brain could be determined by this reaction after its separation from phenylalanine and 5-hydroxytryptamine by n-heptane extraction. The content of endogenous β-phenylethylamine in rat brain was 5 ng/g of wet tissue. Administration of pargyline caused more than a 10-fold increase in brain β-phenylethylamine. After the injection of both pargyline and phenylalanine, β-phenylethylamine was increased about 100-fold. This increase was reduced by prior injection of reserpine, while enhanced by that of calcium fusarate.     

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Current-voltage studies on the thylakoid membrane in the presence of ionophores.

The reversibility of the binding of ionophores to the thylakoid membrane is studied. While gramicidin binds practically irreversibly, valinomycin and nonactin bind reversibly, however, only a small fraction (about 1%) of the membrane-bound valinomycin or nonactin is active in ion transport. The current-voltage relationship is evaluated under these circumstances. We have found that it is practically linear. This together with the relationship between current and ion concentration agrees qualitatively with the results reported for bimolecular lipid membranes, which contain a large fraction of negatively charged lipids. For the ionophores, valinomycin and nonactin, the binding equilibria (K approximately equal to 10-4) and the turnover numbers (approximately equal to 3-10-4/s) are evaluated for their action on the thylakoid membrane. Possible reasons for the inactivity of the majority of membrane-bound ionophore molecules are discussed.     

Comparative enzymologic study of catalytical properties of liver monoamine oxidases of sturgeon fish

We carried out the comparative study of the substrate and inhibitory specificity of liver monoamine oxidases (MAO) of the giant sturgeon Huso huso, the starred sturgeon Acipenser stellatus, the Persian sturgeon Acipenser persicus, and the Russian sturgeon Acipenser gueldenstaedtii. Results of the substrate-inhibitor analysis with use of inhibitors chlorgilin and deprenil, as well as five specific substrates indicate homogeneity of these enzymes. All studied MAO have the several orders higher sensitivity to chlorgilin than to deprenil, with essential interspecies differences being observed. There are determined kinetic parameters of enzymatic deamination (K _M and V) of tyramine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, and N-methylhistamine. All studied enzymes have been established to have the higher activity toward serotonin and noradrenalin-substrates of the MAO A form as compared with benzylamine, β-phenylethylamine, and N-methylhistamine-substrate of the mammalian MAO B form, the maximal activity being characteristic of the giant sturgeon.