Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

Conditions for the association of the two clotting proteins of the cockroachRhyparobia (Leucophaea) maderae (Blattaria)

Summary The clotting system ofRhyparobia (Leucophaea) maderae comprises two clotting proteins, plasma coagulogen and hemocyte coagulogen, which during clotting become crosslinked. Cross-linking is thought to be preceded by an association of the two coagulogens. This paper reports an attempt to elucidate the mechanism of association, using an aged hemocyte coagulogen (=hemocyte gel).In a first series of experiments association was studied with a normal, unmodified gel under various conditions (ionic strength, pH, inhibitors). Association is optimal at low ionic strength and a slightly acidic to neutral pH. When the associated proteins are subjected to increased ionic strength or higher pH they dissociate again. Association is not influenced by crosslinking inhibitors such as EDTA, iodoacetamide, hydroxylamine, and hydrazine up to concentrations of 0.01M.In a second series of experiments association was tested with hemocyte gels which had been treated with a variety of chemicals in order to modify the amino acid side chains. Association is inhibited only when carboxyl groups of the gel are modified.The results of both series of experiments suggest that during association the two proteins are held together mainly by electrostatic attractions between negatively charged carboxyl groups of the hemocyte gel and positively charged amino and/or guanidino groups of the plasma coagulogen.     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

The use of a PMR probe for studying the hydrophobic properties of micrococcus lysodeikticus membranes and membrane fractions

The hydrophobic character of the trimethyl group of sodium 2,2-dimethyl-2-silapentane-5-sulfonate, makes it an effective PMR probe for apolar sites on proteins and membranes. By comparing the spin-spin relaxation rates of the free and bound probe the extent and strength of the interaction can be qualitatively compared for bovine serum albumin, membranes from Micrococcus lysodeikticus and for different fractions isolated from this membrane. It is concluded that this membrane has hydrophobic sites on or near its surface and that the number of such sites is sensitive to the ionic composition and to the pH of its aqueous environment. Removal of lipid from the membrane greatly increased the binding of the probe, while vesicular preparations of the lipid fraction itself gave no evidence of an interaction with the probe. The results are discussed in terms of protein-lipid-water interactions.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

The formation of vesicles retaining sodium-dependent transport systems for amino acids from protein-depleted membranes of pigeon erythrocytes

The process of the formation of vesicles from pigeon erythrocyte membranes was studied. Mildly alkaline solutions of low ionic strength, which reduce human erythrocyte membranes to small vesicles depleted of spectrin and other proteins, have no such effect on pigeon erythrocyte ghosts. A distinct phase of removal of membrane proteins, including spectrin, began to occur only when pigeon erythrocyte membranes were exposed to 0.2 mM EDTA adjusted to pH values above 10.2. Vesicles which demonstrated Na⁺-dependent amino acid transport were generated between the pH values 10.8 and 11.4. The results show that peripheral proteins, notably spectrin, maintain the integrity of the pigeon erythrocyte ghost. The interaction of these proteins with the membrane is rather different from that well studied in the human erythrocyte ghost and the possible significance of this for the pigeon erythrocyte is discussed.     

The effects of ionic strength on the protein conformation and the fluidity of porcine intestinal brush border membranes. Fluorometric studies using N-[7-dimethylamino-4-methylcoumarinyl]maleimide and pyrene

The effects of ionic strength on the conformation around the SH groups of the proteins and the lipid fluidity of porcine intestinal brush border membranes were studied using two fluorescent dyes, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM) and pyrene. The extent of DACM labeling to the SH groups of the membrane proteins was accelerated depending on the KCl concentrations in medium. A quenching study of DACM-labeled membranes with acrylamide showed that the proximity of the quencher to the fluorescence-labeled SH groups in the membrane proteins is increased with increasing ionic strength of medium. An implication of the conformational changes around SH groups in the membrane proteins with increase of ionic strength was also obtained from the stimulation of guanidine effect on the fluorescence parameters of DACM-labeled membranes by addition of KCl. On the other hand, the results of the quenching study with KI, excimer fluorescence, and polarization measurements of pyrene-labeled membranes suggested an increase of membrane fluidity on addition of KCl to medium. The temperature dependence of polarization of the complex strongly suggested that the rotational freedom of pyrene molecules embedded into the lipid layers of the membranes is increased by addition of KCl. In fact, the harmonic means of the rotational relaxation times of pyrene molecules in the membranes with and without 100 mM KCl were estimated to be about 2900 and 9000 ns at 25 degrees C, respectively. Based on these results, the salt-induced alterations of the conformation in the vicinity of the bound dyes of the membrane proteins and of the membrane fluidity are discussed.     

Ion-exchange properties of cell walls of red seaweed Phyllophora crispa

Research into ion-exchange properties of cell walls isolated from thallus of red seaweed Phyllophora crispa was carried out. Ion-exchange capacity and the swelling coefficient of the red alga cell walls were estimated at various pH values (from 2 to 12) and at constant ionic strength of a solution (10 mM). It was established that behavior of cell walls as ion-exchangers is caused by the presence in their matrix of two types of cation-exchange groups and amino groups. The amount of the functional group of each type was estimated, and the corresponding values of pK(a) were calculated. It can be assumed that ionogenic groups with pK(a) -5 are carboxyl groups of uronic acids, and ionogenic groups with pK(a) -7.5 are carboxyl groups of the proteins. Intervals of pH in which cation-exchange groups are ionized and can take part in exchange reactions with cations in the environment are defined. It was found that protein was a major component of cell wall polymeric matrix because its content was 36%.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

A monolayer (π,ΔV) study of the ionic properties of alanylphosphatidylglycerol: Effects of pH and ions

1,2-Didodecanoyl-sn-glycero-3-phosphoryl-1'-(3'-O-L-alanyl)-sn-glycerol (Ala-PG) has been synthesized. Its ionic properties have been studied at the air-water interface through film compressions and surface potential measurements as a function of subphase pH and ionic content (NaCl, Na₂MoO₄, CaCl₂). The existence of the polar head in a loop conformation allowing for interactions between phosphate and amino groups is suggested. Ionic properties of Ala-PG clearly depended on subphase ionic strength but no specific interactions between either cations or anions in the subphase and phosphate or amino groups in the film could be detected. Results are interpreted in terms of ion-pair interactions at the interface between these two groups and anions and cations from the subphase. Occurrence of charge separation between these two groups, induced by increasing subphase ionic strength, is postulated. Since the molecular packing appeared independent of the subphase ionic content over a large domain of pH (3–8) and surface pressure (π > 5 dyne/cm) and since the lipid can be considered as zwitterionic or slightly positive below pH 5–6, it is suggested that in the parent bacteria, grown under acidic conditions, Ala-PG could play a role in maintaining the membrane intergrity and in preventing the passive diffusion of protons.     

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.     

Ionic permeability of K, Na, and Cl in crayfish nerve. Regulation by membrane fixed charges and pH.

Teorell's fixed charge theory for membrane ion permeability was utilized to calculate specific ionic permeabilities from measurements of membrane potential, conductance, and specific ionic transference numbers. The results were compared with the passive ionic conductances calculated from the branched equivalent circuit membrane model of Hodgkin Huxley. Ionic permeabilities for potassium, sodium, and chloride of crayfish (Procambarus clarkii) medial giant axons were examined over an external pH range from 3.8 to 11.4. Action potentials were obtained over this pH range. Failures occurred below pH 3.8 during protonation of membrane phospholipid phosphate and carboxyl, and above pH 11.4 from calcium precipitation. In general, chloride permeability increases with membrane protonation, while cation permeability decreases. At pH 7.0, PK = 1.33 X 10(-5), PCl = 1.49 X 10(-6), PNa = 1.92 X 10(-8) cm/s. PK: PCl: PNa = 693:78:1. PCl is zero above pH 10.6 and is opened predominately by protonation of epsilon-amino, and partially by tyrosine and sulfhydryl groups from pH 10.6 to 9. PK is activated in part by ionization of phospholipid phosphate and carboxyl around pH 4, then further by imidazole from pH 5 to 7, and then predominately from pH 7 to 9 by most probably phosphatidic acid. PNa permeability parallels that of potassium from pH 5 to 9.4. Below pH 5 and above pH 9.4, PNa increases while PK decreases. Evidence was obtained that these ions possibly share common passive permeable channels. The data best support the theory of Teorell, that membrane fixed charges regulate permiability and that essentially every membrane ionizable group appears involved in various amounts in ionic permeability control.     

The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme

1. The reaction of exo-cis-3,6-endoxo-Delta(4)-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the in-amino group of N(alpha)-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4-5h at pH3.0 and 25 degrees C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.     

Membranes of bacteria and mechanism of action of the antibiotic gramicidin S]

The cyclopeptide antibiotic gramicidin S taken at a concentration of 100--200 mkg/mg membrane protein rapidly increases the permeability of M. lysodeikticus protoplast membranes for substrates of respiratory chain and exogenous cytochromes c. Prolonged incubation of gramicidin S with protoplasts results in their lysis which is more fast at low temperatures. In contrast to natural gramicidin, a derivative of gramicidin S with acetylated amino groups does not inhibit either the micrococcus membrane dehydrogenase or the whole of respiratory chain and does not affect the osmotic barrier of protoplasts. Aliphatic diamines (at concentrations up to 0.1 M) and Ca2+ ions (10(-2) M) do not affect the functioning of the respiratory chain in isolated micrococcus membranes. Another derivative of the antibiotic with an increased distance of loaded amino groups from the cyclopeptide framework (diglycyl gramicidin S) affects the membrane in a way similar to that of natural gramicidin. Washing of gramicidin-treated membranes with NaCl enhances the inhibitory effect of the antibiotic on membrane enzymes. The data obtained suggest that in addition to ionic interactions some hydrophobic interactions also occur during gramicidin S binding to the bacterial membrane, probably at the expense of a hydrophobic peptide ring. It is assumed that gramicidin S, similar to Ca2+ and some other membranotropic agents provides for phase separation of negatively charged phospholipids from other groups of phospholipids, manifesting itself in an appearance of "frozen" sites on the membrane which destroys its barrier properties. This is due to the formation of ionic bonds of negatively charged phospholipids. Simultaneously, unlike Ca2+, gramicidin S, when interacting with membrane proteins, prevents their redistribution in more liquid parts of the membrane, which results in a situation when the respiratory enzymes become surrounded by alkyl chains with restricted motion.     

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Membrane properties of Thermoplasma acidophila.

Plasma membranes were isolated from Thermoplasma acidophila, a mycoplasma-like organism which grows optimally at pH 2 and 59 degrees. Cells in concentrated suspensions were lysed by titrating to pH 9.3. The membranes were purified by washing at pH 10 and centrifuging in a discontinuous sucrose gradient. Membrane purity was assessed by electron microscopy, determination of deoxyribonucleic acid content, polyacrylamide gel electrophoretic behavior. Gel patterns and amino acid composition of cells and membranes were found to differ significantly. The lipid contained small amounts of fatty acid esters and larger amounts of branched long-chain alkyl ethers.     

The polyelectrolyte properties of elastin.

The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.     

Fourier-transform infrared studies on cation binding to native and modified purple membranes

Fourier-transform infrared spectroscopy has been used to examine the structural differences in the protein moiety between the native purple and the deionized blue membranes, both at pH 5.0. The spectra demonstrate that deionization of purple membrane decreases the content of the distorted alpha II-helices in favor of the more common alpha I-helices. Changes in the signals from beta-turns are also observed. The changes corresponding to the carboxyl groups suggest that deionization leads to a decrease in the strength of the hydrogen bonds involving carboxyl groups. Most of these effects are reversed progressively upon binding of one to five Mn2+ per bacteriorhodopsin to the deionized membrane. Binding of Hg2+ to the deionized membranes does not restore the purple color but induces global changes similar to, but less intense than, those brought about by Mn2+ binding. However, the effects attributed to the carboxyl groups are opposite to those found for Mn2+. Schiff base reduction or bleaching induces a decrease of the content of the alpha II-helix in favor of the alpha I-helix and a decrease in the strength of hydrogen bonds to carboxyl groups. Deionization of these modified membranes leads to a further loss in the alpha II content. These results indicate a conformational rearrangement of the protein structure between the native purple membrane and the deionized membrane, which could arise from surface potential changes elicited by bound cations. The changes observed in the carboxyl groups suggest that some of them are located structurally close to the retinal environment and may be involved in cation binding.