NMR studies on phospholipid bilayers. Some factors affecting lipid distribution.

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.     

Effects of paramagnetic shift reagents on the 13C nuclear magnetic resonance spectra of egg phosphatidylcholine enriched with 13C in the N-methyl carbons.

Effects of paramagnetic shift reagents on the 13C NMR spectra obtained from single-walled vesicle dispersions of egg phosphatidylcholine enriched with 13C in the N-methyl carbons are investigated. Spectra obtained at 25.1 MHz show that, at Yb3+ to phospholipid molar ratios as low as 0.06, complete resolution of the N-methyl carbon resonances is obtained from molecules on the inner and outer faces of the vesicle bilayer. No precipitation of the vesicles is caused by Yb3+ at these concentrations nor is appreciable line broadening observed. Other paramagnetic shift reagents frequently used in proton NMR investigations of phosphatidylcholine vesicles do not give complete separation of the N-methyl 13C signals from the two bilayer surfaces. K3Fe(CN)b,Eu3+, and Pr3+ cause precipitation of the phosphatidylcholine vesicles at concentrations, which give only incomplete resolution of these signals. T1 measurements of the resonances separated by Yb3+ indicate that the choline groups on the inner bilayer surface are less mobile than are the same groups in the outer surface. Gated proton decoupling measurements, which show that the nuclear Overhauser effect is 2.8 +/- 0.1, indicate that the dominant mode of relaxation is dipolar interaction.     

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henry's law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.     

Salt-induced aggregation and fusion of dioctadecyldimethylammonium chloride and sodium dihexadecylphosphate vesicles.

Small dioctadecyldimethylammonium chloride (DODAC) vesicles prepared by sonication fuse upon addition of NaCl as detected by several methods (electron microscopy, trapped volume determinations, temperature-dependent phase transition curves, and osmometer behavior. In contrast, small sodium dihexadecyl phosphate (DHP) vesicles mainly aggregate upon NaCl addition as shown by electron microscopy and the lack of osmometer behavior. Scatter-derived absorbance changes of small and large DODAC or DHP vesicles as a function of time after salt addition were obtained for a range of NaCl or amphiphile concentration. These changes were interpreted in accordance with a phenomenological model based upon fundamental light-scattering laws and simple geometrical considerations. Short-range hydration repulsion between DODAC (or DHP) vesicles is possibly the main energy barrier for the fusion process.     

Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated phosphatidylcholine liposomes.

1. Phase transitions in sonicated (vesicles) and unsonicated liposomes composed of various synthetic phosphatidylcholines are monitored using differential scanning calorimetry and 31P NMR. 2. The temperature (Tc), heat content and width of the phase transition are comparable in both vesicles and liposomes prepared from 1,2-dipalmitoyl phosphatidylcholine and 1,2-dimyristoyl phosphatidylcholine. In vesicles composed of a (1 : 1) mixture of 1,2-dipalmitoyl phosphatidylcholine and 1,2-dioleoyl phosphatidylcholine phase separation occurs as in the bilayers of the unsonicated liposomes. 3. The linewidth of the 31P resonances in vesicles is not greatly dependent upon the fatty acid composition when the lipids are in the disordered liquid crystalline state (above Tc). When the lipids are in the gel state (below Tc), however, there is a marked increase in linewidth, demonstrating a reduction in motion of the phosphate group. 4. The ratio of the amounts of phosphatidylcholine present in the outside and inside monolayter of the vesicle membrane was determined with 31P NMR using Nd3+ as a non-permeating shift reagent. 5. The outside/inside ratio is dependent upon the hydrocarbon chain length. Increasing chain length gives a lower outside/inside ratio and a larger vesicle. Introduction of cis or trans double bonds in the chain influences the outside/inside ratio slightly. 6. The incorporation of cholesterol decreases the outside/inside ratio and increases the size of 1,2-dimyristoyl phosphatidylcholine vesicles. The cholesterol concentration in the outside and inside monolayer is approximately the same. The size of the 1,2-dioleoyl phosphatidylcholine vesicles is also increased by cholesterol incorporation but the outside/inside distribution is also increased, especially between 30 and 50 mol% cholesterol. In these vesicles cholesterol is asymmetrically distributed and strongly prefers the inside monolayer of the vesicle.     

The effect of chloride on the thermal inactivation of oxygen evolution

The effect of Cl depletion on the sensitivity of the oxygen-evolving complex of Photosystem II (PS II) to heat treatment was examined by a parallel study of the Hill activity (H₂O2,6-dichlorophenolindophenol), Cl^- binding (by ³⁵Cl-NMR) and Mn release (by EPR). The extent of thermal inactivation in spinach thylakoids was found to depend on the degree of Cl^- depletion in the sample. In partially Cl^--depleted thylakoids, mild heating (38°C, 3 min) was found to eliminate inflections in plots of both Hill activity versus [Cl^-] (at low light intensity) and excess ³⁵Cl-NMR linewidth versus [Cl^-] (in the dark). In PS II membranes, the same treatment reduced the differences between the linewidth maxima and minima, particularly in the region of 0.3 mM and 7.0 mM Cl^-, as compared to unheated membranes. These results indicate that mild heating affects the Cl^--binding domains within the oxygen-evolving complex, OEC, EPR measurements of the temperature dependence of Mn release from heated thylakoids show that Mn release begins to correlate with the loss of Hill activity only at higher temperatures, where the OEC is already substantially inactivated. We conclude from these studies that the Cl^--binding domains of the OEC constitute a principal site of damage by heat treatment.     

Absorption of surfactants by membranes: erythrocytes versus synthetic vesicles.

Three surfactants (chlorpromazine hydrochloride, thioridazine hydrochloride, and sodium deoxycholate) are found to absorb just as strongly into the protein-containing membranes of erythrocytes as into the phospholipid bilayers of synthetic vesicles. In the concentration region where hemolysis occurs and the Langmuir adsorption isotherm is no longer valid, one may use a phase partition model in which the erythrocyte membrane is one of the phases. The partition coefficients, expressed as the ratio of mole fraction surfactant in the membrane lipid phase to concentration of surfactant in the aqueous phase, have been calculated at the point of saturation in the erythrocyte membrane. These values are Ky = 430 M-1 (chlorpromazine, pH 5.9), 550 M-1 (deoxycholate, pH 7.6), and 640 M-1 (thioridazine, pH 5.9), in isotonic buffer at 27 degrees C. Corresponding values for synthetic vesicles made from dimyristoylphosphatidylcholine are Kx = 230 M-1 (chlorpromazine, 0.12 M buffer/KCl pH 5.9), 440 M-1 (deoxycholate, 0.20 M buffer/NaCl pH 8.0) and 510 M-1 (thioridazine, 0.12 M buffer/KCl pH 5.9), at 27 degrees C. It appears that the surfactants become an integral part of the bilayer in both vesicles and natural membranes and that the absorption is not of a peripheral nature. There is no evidence that the presence of proteins in the natural membrane inhibits the absorption of these surfactants in any way.     

Cation-induced aggregation of acidic phospholipid vesicles: the role of fatty acid unsaturation and cholesterol

Cation-induced aggregation of acidic phospholipid vesicles consisting of dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylserine (DPPS), phosphatidylserine from bovine brain (brPS), and phosphatidylglycerol from egg yolk (eggPG) was studied. Significant differences were evident in the NaCl-induced aggregation of fully saturated and unsaturated acidic phospholipid vesicles. The threshold NaCl concentration of vesicle aggregation ([NaCl]Thr) for DPPS vesicles was 320 mM compared to 610 mM observed for brPS vesicles. For DMPG vesicles the [NaCl]Thr was 430 mM and no aggregation of eggPG vesicles could be observed upon addition of NaCl. The threshold CaCl2 concentrations of aggregation of DMPG and eggPG vesicles were 2.3 and 4.9 mM, respectively. The corresponding threshold CaCl2 concentrations for DPPS and brPS vesicles were 0.85 mM and 1.3 mM, respectively. The inclusion of cholesterol into vesicles attenuated NaCl- and CaCl2-induced aggregation of DMPG and DPPS vesicles. However, enhancement of aggregation by inclusion of cholesterol was observed in the case of NaCl-induced aggregation of brPS vesicles. It is concluded that cation mediated membrane-membrane interactions depend, in addition to polar headgroup structure, on the fatty acid composition of the phospholipids also.     

13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles

1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.     

Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.     

Kinetics of the actions of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of caffeine and procaine on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx in SR vesicles was measured by following the change in light scattering intensity using a stopped flow apparatus. From the kinetic analysis of the rate of choline influx, the following results were obtained. (1) The rate of choline influx was enhanced when Ca2+ bound to the Ca2+-receptor site of the Ca2+-gated cation channel. (2) Caffeine enhanced the choline influx by increasing only the affinity of Ca2+ for the receptor site of the channel and thus regulated the equilibrium between open and closed states of the channel. The affinity increased about 14-fold upon caffeine binding. The dissociation constant of caffeine was 10 mM. (3) In contrast, procaine itself blocked the choline influx mediated by the Ca2+-gated cation channel. The blockade followed a single-site titration curve with a Ca2+-dependent dissociation constant of 0.44 mM at 2 x 10(-6) M Ca2+. The Ca2+-dependence was explained by assuming that procaine would bind to the inhibitory site only when the channel was open. (4) Procaine also inhibited the choline influx enhanced by caffeine. The blockade could be explained on the basis of the above kinetic model.     

Low pH-Induced Pore Formation by the T Domain of Botulinum Toxin Type A is Dependent upon NaCl Concentration

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Nuclear magnetic resonance studies of amphiphile hydration: Effects of cholesterol on phosphatidyl choline hydration

Water which remains unfrozen at ?25 °C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, ~150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, ~300–1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.     

The effect of surface curvature on the head-group structure and phase transition properties of phospholipid bilayer vesicles

Proton nuclear magnetic resonance spectra at 360 MHz of small sonicated distearoyl phosphatidylcholine vesicles show easily distinguishable resonances due to choline N-methyl head-group protons located in the inner and outer bilayer halves. A study of the chemical shift of these resonances as a function of temperature reveals that the splitting between them increases below the phase transition. This occurs as a result of an upfield shift of the inner layer resonance at the phase transition. Consideration of the possible causes of this effect results in the conclusion that, at the phase transition, there is a change in the organization of the inner layer head-groups which does not occur for the outer layer head-groups.     

Effect of Phosphatidylcholine on Acrosome Breakdown and Fertilizing Capacity of Amphibian Spermatozoa

The effect of vesicles of purified egg yolk phosphatidylcholine on the fertilizing capacity and acrosome breakdown of amphibian spermatozoa was studied. When Bufo arenarum spermatozoa were incubated with either small unilamellar vesicles (prepared by sonication) or with large unilamellar vesicles (prepared by reverse-phase evaporation) a decrease in the fertilizing capacity of spermatozoa was found. At the same phosphatidylcholine concentration, large unilamellar vesicles were more inhibitory than small unilamellar vesicles. The inhibition was dependent upon the phospholipid concentration and the length of the incubation period. Small unilamellar vesicles did not modify the time course of acrosome breakdown in Leptodactylus chaquensis , while large unilamellar vesicles markedly accelerated the rate of acrosome breakdown. In both biossays, the charge of the vesicles (made either positive or negative by the addition of 5% stearylamine or 5% phosphatidic acid) did not influence their biological effect. Multilamellar vesicles did not alter the fertilizing capacity nor the acrosome breakdown. We conclude that the size and the structure of the vesicles are important parameters in determining the inhibitory capacity of phosphatidyl choline on amphibian fertilization.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

23Na and 1H NMR studies on melittin channels activated by tricyclic tranquilizers.

A dynamic 23Na nuclear magnetic resonance (NMR) technique was applied to the exchange system of Na+ ions present inside and outside large unilamellar vesicles at an equivalent concentration. Addition of melittin to phosphatidylcholine vesicles did not induce any detectable Na+ transport across the membrane but subsequent addition of a trace of chlorpromazine or imipramine did induce Na+ transport. Because the formation of a drug-melittin adduct in a solution was detected by 1H NMR, the activation of melittin channels was assumed to originate from the direct interaction of the drug and melittin.     

Possible involvement of calmodulin in maturation and activation of Chaetopterus eggs

We report the isolation of calmodulin from oocytes of Chaetopterus pergamentaceus. The identification of this protein is based on (1) activation of beef heart cAMP phosphodiesterase, (2) heat stability, (3) sensitivity to chlorpromazine, and (4) electrophoretic mobility identical to that of porcine brain calmodulin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of either Ca2+ or EGTA. We treated oocytes with chlorpromazine and W-7 to investigate the involvement of calmodulin in meiosis initiation and egg activation. Very low concentrations of chlorpromazine inhibited germinal vesicle breakdown (GVBD). This effect was shown to be dependent upon bright indirect light, since the drug was much less effective at GVBD inhibition under conditions of very low illumination. Higher concentrations of chlorpromazine and W-7 (100 microM) inhibited GVBD and activated eggs with intact germinal vesicles as determined by fertilization envelope formation and the onset of ameboid activity. Neither egg activation nor inhibition of calmodulin stimulation of phosphodiesterase activity in vitro was affected by light. These results are consistent with a role for calmodulin in egg activation and GVBD, but suggest that chlorpromazine in bright light may prevent GVBD by some mechanism other than calmodulin inhibition.     

The binding of nitrate to the human anion exchange protein (AE1) studied with 14N nuclear magnetic resonance

The binding of [14N]nitrate to the human erythrocyte anion transport protein, AE1, was studied using 14N nuclear magnetic resonance spectroscopy (14N-NMR). The line-width at half-height of the 14NO3- resonance increased in direct proportion to the concentration of erythrocyte ghost protein. Addition of the AE1 specific inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate markedly reduced this line-broadening, indicating that the broadening was predominantly due to a specific interaction between nitrate and AE1. The dependence of the AE1 specific line-broadening on nitrate concentration had a first-order dissociation constant KD of 6.9 +/- 0.9 mM. In contrast, Cl- interaction with AE1 studied by 35Cl-NMR showed a chloride concentration-dependent line-broadening with a KD of 74 +/- 10 mM, indicating that AE1 has a higher affinity for nitrate than for chloride. Bicarbonate and chloride were found to be competitive inhibitors of the AE1 specific 14NO3- line-broadening (94 +/- 6% and 101 +/- 3% inhibition, respectively). Based on the concentration dependence of inhibition and using a model of competitive inhibition, the KD of bicarbonate binding to AE1 was estimated to be 5.4 +/- 1.3 mM. Nitrate is a structural analog of bicarbonate, making the interaction of nitrate with AE1 a good model for the bicarbonate-AE1 interaction. The 14N-NMR nitrate binding assay, along with the 35Cl-NMR binding assay now in use, will provide a powerful tool for studying the structure of the AE1 binding site for both physiologic substrates, bicarbonate and chloride.