Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture,by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E₁ (PGE₁) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10^?9 M VIP promotes a rapid and specific activation of the low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ cyclic AMP phosphodiesterase (1.7-fold); at 25°C the effect is maintained for more than 15 min, while at 37°C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 4 · 10}{}^{\mathrm{?10}}\text{M}$) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 · 10^?7 M secretin, 10^?5 M isoproterenol and 10^?5 M PGE₁; PGE₂ and epinephrine are without effect. In HRT 18 cells VIP is less active ($\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 2 · 10}{}^{\mathrm{?9}}\text{M}$) whereas 10^?6 M PGE₁, 10^?6 M PGE₂ and 10^?5 M epinephrine are potent inducers of the phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.     

Cyclic AMP transport in human erythrocyte ghosts

10^?5 M cyclic AMP has high permeability in human erythrocyte ghosts ($\text{p = 0.061 · 10}{}^{\mathrm{?6}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$). Saturation of influx and efflux occurs. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}\text{= 4.43}\text{mM}$. $\text{V}{}_{\mathrm{zt}}{}^{\mathrm{oi}}\text{= 259.6 μ}\text{M · min}{}^{\mathrm{?1}}$. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 0.475 μ}\text{M}$. $\text{V}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 28.3 μ}\text{M · min}{}^{\mathrm{?1}}$ at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 3.9 · 10}{}^{\mathrm{?7}}\text{M}$).Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

The bimolecular decay rates of the flavosemiquinones of riboflavin,FMN and FAD

The bimolecular decay rates (2k) of the flavosemiquinones ($\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}$) of riboflavin, FMN and FAD have been determined using pulse radiolysis. The rates (defined as $\text{d}\text{[}\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}\text{]}\text{d}\text{t}\text{= ?2}\text{k}\text{[}\text{FH}{}^{\mathrm{·}}\text{F}{}^{\mathrm{?}}\text{]}{}^2$) for the neutral flavosemiquinones at zero ionic strength and pH 5.9 are (in units of mol^?1·dm³·s^?1): (1.2 ± 0.1)·10⁹, (5.0 ± 0.2)·10⁸ and (1.4 ± 0.1)·10⁸; and for the anionic flavosemiquinones at pH 11.2 (5.4 ± 0.9)·10⁸, (4.5 ± 0.3)·10⁷ and (8.5 ± 1.3)·10⁶, respectively. The kinetic salt effect has been used to formulate rate equations for each flavin to adjust for ionic strength effects.     

Occurrence of passive furosemide-sensitive transmembrane potassium transport in cultured cells

Furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibits a proportion of the total passive (ouabain-insensitive) K⁺ influx into primary chick heart cell cultures (85%), BC₃H1 cells (75%), MDCK cells (40%) and HeLa cells (57%). This action of furosemide upon K⁺ influx is independent of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{pump}$ inhibition since the furosemide-sensitive component of the K⁺ influx is identical in the presence and absence of ouabain ($\text{1 · 10}{}^{\mathrm{?3}}\text{M}$). For HeLa cells the passive, furosemide-sensitive component of K⁺ influx is markedly dependent upon the external K⁺, Na⁺ and Cl^? content. Acetate, iodide and nitrate are ineffective as substitutes for Cl^?, whereas Br^? is partially effective. Partial Cl^? replacement by NO₃^? gave an apparent affinity of 100 mM [Cl]. Na⁺ replacement by choline⁺ abolishes the furosemide-sensitive component, whereas Li⁺ replacement reduces this component by 48%. Partial Na⁺ replacement by choline⁺ gives an apparent affinity of 25 mM [Na⁺]. Variation in the external K⁺ content gives an affinity for the furosemide-sensitive component of approx. 1.0 mM. Furosemide inhibition of the passive K⁺ inflúx is of high affinity, half-maximal inhibition being observed at $\text{5 · 10}{}^{\mathrm{?6}}\text{M}$ furosemide. Piretanide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) and phloretin ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) inhibit the same component of passive K⁺ influx as furosemide; ethacrynic acid and amiloride ($\text{both}\text{1 · 10}{}^{\mathrm{?4}}\text{M}$) partially so. The stilbene, SITS ($\text{1 · 10}{}^{\mathrm{?6}}\text{M}$), was ineffective as an inhibitor of the furosemide-sensitive component.     

Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Homogenates of adult $\text{Schistosoma mansoni}$ (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E₁, E₂ or B₁. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1×10}{}^{\mathrm{?7}}\text{M}$) over cyclic GMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5×10}{}^{\mathrm{?6}}\text{M}$). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.     

Carnitine uptake into human heart cells in culture

The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carnitine is 3–10-fold higher in heart cells than in fibroblasts ($\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}$). The uptake of carnitine increases with temperature coefficient $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of 1.6 in the interval 10–20° C and with a negligible uptake at 4 and 10° C. The uptake of carnitine follows Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of $\text{4.8 ± 2.2 μ}\text{M}$ and $\text{V = 8.7 ± 3.2}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Carnitine uptake is suppressed 90% by NaF (24 mM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of 5.7–17.3 μM and $\text{V = 8.7–9.3}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary

An $\text{in}$$\text{vitro}$ perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release ($\text{IC}{}_{50}{}^1\text{= 6 × 10}{}^{\mathrm{?8}}\text{M and}\text{10}{}^{\mathrm{?5}}\text{M}$ respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.     

Catecholamine-stimulation of Cl− secretion in MDCK cell epithelium

Cultured epithelial monolayers of MDCK cells grown upon Millipore filter supports and mounted in Ussing chambers for transport studies respond to addition of $\text{5 · 10}{}^{\mathrm{?7}}\text{M}$ adrenalin from only the basal bathing solution by an increased short-circuit current, due both to an increased transmonolayer potential difference (basal solution electropositive) and an increased transmonolayer conductance. Measurement of tracer Na⁺, K⁺ and Cl^? fluxes demonstrate that the adrenalin-stimulated short-circuit current results primarily from basal to apical net Cl^? secretion. Half-maximal stimulation of the short-circuit current was observed at ($\text{3.1 ± 0.3) · 10}{}^{\mathrm{?8}}\text{M adrenalin}$; the order of potency of adrenergic agonists for short-circuit current stimulation was $\text{isoprenalin}\text{>}\text{adrenalin}\text{>}\text{noradrenalin}$, consistent with adrenalin action being mediated by a β-adrenergic receptor. The adrenalin-stimulated short-circuit current was sensitive to inhibition (75%) by basal additions of furosemide ($\text{1 · 10}{}^{\mathrm{?4}}\text{M}$); phloretin inhibition (54%, 57%) was observed from both epithelial surfaces. Amiloride (10^?4 M) and 4-acetamido-4-isothiocyanostilbene-2, 2′-disulphonic acid (SITS) (10 μM) were ineffective as inhibitors of the adrenalin response. The increased short-circuit current was sensitive to replacement of medium Na⁺ by choline (87%) and Tris (93%). Li⁺ was a partially effective substitute cation for Na⁺ · NO₃^?, and isethionate were ineffective substitutes for Cl^? whereas Br^? was partially effective. Partial replacement of medium Na⁺ by choline gave an upward-curving non-saturable dependence of the adrenalin-stimulated short-circuit current upon [Na]; partial replacement of Cl^? by NO₃^? in contrast gave a saturable increase with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approx. 65 mM Cl^?.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

Effect of angiotensin II on artificial lipid membranes

(5-Isoleucine)-angiotensin II applied to black lipid membranes produced current fluctuations varying between $\text{Δ>G = 5 · 10}{}^{\mathrm{?11}}\text{Ω}{}^{\mathrm{?}}$ and 3.5 · 10^?10 Ω^?1. These fluctuations depend on the voltage and the hydrostatic pressure. The membrane resistance is lowered by $\text{Δ>R = 6.1 · 10}{}^7\text{Ω · cm}{}^2$. With (5-isoleucine, 8-leucine)-angiotensin II the jumps are of a single amplitude ($\text{Δ>G = 2 · 10}{}^{\mathrm{?10}}\text{Ω}{}^{\mathrm{?1}}$). In both cases water and ions are transported across the membrane.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

Binding of uranyl to phosphatidylcholine liposomes. Liposomes aggregation effect on surface area

The binding of uranyl ion, UO₂²⁺, to egg phosphatidylcholine liposome was studied as a potential method for the determination of liposome surface areas. Unbound uranyl was determined spectrophotometrically as the Arsenazo III complex with centrifuge supernatant. There is an apparent positive cooperativity in uranyl binding capacity per mol increases upon liposome dilution. The data are consistent with liposomes existing in a highly aggregated state. The binding constant in the limit of low concentration of bound uranyl was $\text{(9 ± 3) · 10}{}^6\text{M}{}^{\mathrm{?1}}\text{in}\text{0.1}\text{M NaCl, pH}\text{4.1}$. At saturation about four uranyl ions are bound per 100 phosphatidylcholine molecules. Relative surface areas of different dispersions may be calculated from intercepts of extrapolated binding isotherms, and absolute surface areas may be calculated if a value for the uranyl-phosphatidylcholine stoichiometry is assumed.     

The interaction of ions with phosphatidylcholine bilayers

The interaction of lanthanides and other cations with phosphatidylcholine bilayers present as single bilayer vesicles in ²H₂O has been investigated in terms of stoichiometry, apparent binding constants and environmental conditions.Lanthanides are shown to form 2 : 1 (molar ratio) phosphatidylcholine to metal ion complexes.The apparent binding constant $\text{K}{}_{\mathrm{<mtext>b</mtext>}}$ varies as a function of the quantity of metal ion bound and as a function of the Cl^? concentration. The apparent binding constant at “zero loading” is $\text{K}{}_0\text{= 1.25 · 10}{}^4\text{L}{}^2\text{·}\text{M}{}^{\mathrm{?}}\text{at}\text{0.15}\text{M KCl}$. It decreases exponentially with increased “loading” expressed as the molar ratio of metal ion bound to effective phosphatidylcholine concentration and increases exponential with Cl^? concentration.The interaction of lanthanides and divalent cations such as Ca²⁺ and Mg²⁺ is independent of pH in the pH range 3–7⁺ and 3–10 respectively, but is sensitive to the nature of the anion. The presence of anions enhances the interaction with polyvalent cations, the chaotropic anions showing the largest effect. The order of enhancement is $\text{Cl}{}^{\mathrm{?}}\text{< Br}{}^{\mathrm{?}}\text{< NO}{}_3{}^{\mathrm{?}}\text{< SCN}{}^{\mathrm{?}}\text{< I}{}^{\mathrm{?}}\text{< ClO}{}_4{}^{\mathrm{?}}$. The nature of the monovalent counterion (cation) has little effect on the enhanced binding of lanthanides in the presence of the above anions.The affinity of other polyvalent cations for phosphatidylcholine bilayers has been determined by competition with lanthanides. The physiologically important divalent cations Ca²⁺ and Mg²⁺ both bind less strongly (by about an order of magnitude) to the lipid surface. The order of binding of cations reflects direct binding to the phosphodiester group, with UO₂²⁺ showing the highest affinity.