alpha-Lactalbumin binding and membrane integrity--effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine alpha-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation-leading to looser packing-promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human alpha-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Surface charge, surface dipoles and membrane conductance

The conductance of lipid membranes in the presence of nonactin is changed by the adsorption of small amounts of ionic and zwitterionic surfactants. The conductance changes are, in many instances, not accounted for by the variation in surface charge or diffuse double layer potential as calculated from Gouy-Chapman theory. The changes are, however, all accurately accounted for by the variation in total potential across the membrane interface. This potential includes contributions from surface dipoles and specific adsorption, as well as any diffuse double layer effects not included in the Gouy-Chapman theory.The total potential changes were inferred from Volta or compensational potential changes at bulk oil (and monolayer)/aqueous solution interfaces. Surface charge densities were found by standard thermodynamic methods involving the use of the Gibbs equation. Electrokinetic potentials for the appropriate surfaces were also measured and, in general, agreed well with the diffuse double layer potentials calculated from the Gouy-Chapman theory.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Electrochemistry at DNA-modified surfaces: new probes for charge transport through the double helix

Electrochemistry at DNA-modified surfaces provides an alternative approach to photochemistry or radiation biology for studying charge migration through the double helix. Using short duplexes self-assembled onto gold, electrochemical reduction of redox-active reporter molecules has been observed through DNA films more than 50 A thick, with heterogeneous rate constants as great as approximately 100 s(-1). Though apparently insensitive to base content and sequence, even small distortions in the electronic structure of the pi-stack (caused, for example, by single-base mismatches and other DNA lesions) attenuate the rate of electron transport. Understanding the role of conformational dynamics within the double helix, as well as the cooperative effects of self-assembling individual duplexes into ordered superlattices remain important challenges for theory and experiment.     

Interaction of heparin with cationic molecular probes: Probe charge is a major determinant of binding stoichiometry and affinity

Fluorescent perylenediimide probes modified with 2, 4, 6, or 8 ammonium groups were synthesized and their binding to the antithrombotic drug heparin was studied by fluorescence spectroscopy in solution. The polyanionic polysaccharide strands of heparin bind more probe molecules per sugar unit when the charge of the latter is low, and stability of the probe-heparin complex increases with increasing probe charge.     

Design and characterization of electrochromic membrane probes

Electrochromic membrane probes display a spectroscopic response to membrane poteintial by a direct electronic mechanism. This allows such probes to be designed a priori via quantum-chemical techniques. The detailed behavior of potentiometric optical probes can be elucidated with an apparatus based on phase-sensitive detection from a hemispherical lipid bilayer; several different types of response spectra can be obtained with this apparatus allowing distinction between the electrochromic mechanism and the more common molecular-motion based mechanisms. The development of ‘fast’ potentiometri dyes has now reached a stage where practical and exciting applications are rapidly appearing. It is anticipated that the emergence of a complementary set of electrochromic probes will lead to new applications; in particular, it may be possible to elucidate the molecular events which underlie biological or physiological phenomena.     

A detergent-induced charge shift as a prerequisite for the electrochemical analysis of the adenine nucleotide translocator, a basic membrane protein

The adenine nucleotide translocator is a hydrophobic, basic protein of the inner mitochondrial membrane which is solubilized by the non-ionic detergent Triton X-100. For immunochemical characterization of this membrane-protein by crossed immunoelectrophoresis a charge shift of the protein-Triton X-100 micelle by the introduction of an ionic detergent (deoxycholate) was necessary as a prerequisite to avoid unspecific precipitation of the protein. Beside the charge shift of the protein-detergent micelle, the selection, concentration and ratio of the detergents used and the choice of the agarose with different degrees of electroendosmosis should be carefully considered. The principle derived from these results provides a new methodological possibility for the immunochemical characterization of hydrophobic, basic membrane proteins.     

Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure

Human platelet 12-lipoxygenase (hp-12LOX, 662 residues + iron nonheme cofactor) and its major metabolite 12S-hydroxyeicosatetraenoic acid have been implicated in cardiovascular and renal diseases, many types of cancer and inflammatory responses. However, drug development is slow due to a lack of structural information. The major hurdle in obtaining a high-resolution X-ray structure is growing crystals, a process that requires the preparation of highly homogenous, reproducible and stable protein samples. To understand the properties of hp-12LOX, we have expressed and studied the behavior, function and low-resolution structure of the hp-12LOX His-tagged recombinant enzyme and its mutants in solution. We have found that it is a dimer easily converted into bigger aggregates, which are soluble/covalent-noncovalent/reversible. The heavier oligomers show a higher activity at pH 8, in contrast to dimers with lower activity showing two maxima at pH 7 and pH 8, indicating the existence of two different conformers. In the seven-point C → S mutant, aggregation is diminished, activity has one broad peak at pH 8 and there is no change in specificity. Truncation of the N_t-β-barrel domain (PLAT, residues 1-116) reduces activity to ∼ 20% of that shown by the whole enzyme, does not affect regio- or stereospecificity and lowers membrane binding by a factor of ∼ 2. “NoPLAT” mutants show strong aggregation into oligomers containing six or more catalytic domains regardless of the status of the seven cysteine residues tested. Time-of-flight mass spectrometry suggests two arachidonic acid molecules bound to one molecule of enzyme. Small angle X-ray scattering studies (16 Å resolution, χ∼ 1) suggest that two hp-12LOX monomers are joined by the catalytic domains, with the PLAT domains floating on the flexible linkers away from the main body of the dimer.     

Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles

The binding affinities of polyanions for bovine serum albumin in NaCl solutions from I = 0.01-0.6 M, were evaluated on the basis of the pH at the point of incipient binding, converting each such pH(c) value into a critical protein charge Zc. Analogous values of critical charge for mixed micelles were obtained as the cationic surfactant mole fraction Yc. The data were well fitted as Yc or Zc = KI a, and values of K and a were considered as a function of normalized polymer charge densities (tau), charge mobility, and chain stiffness. Binding increased with chain flexibility and charge mobility, as expected from simulations and theory. Complex effects of tau were related to intrapolyanion repulsions within micelle-bound loops (seen in the simulations) or negative protein domain-polyanion repulsions. The linearity of Zc with radicalI at I < 0.3 M was explained by using protein electrostatic images, showing that Zc at I < 0.3 M depends on a single positive "patch"; the appearance of multiple positive domains I > 0.3 M (lower pH(c)) disrupts this simple behavior.     

Surface charge of plasma lipoproteins from the data of potentiometric studies

The method of potentiometric titration is used to measure a surface very low, low- and high-density charge of the human blood plasma lipoproteins. Density of the surface charges is -0.5 X 10(-2); -0.2 X 10(-2); -3.4 X 10(-2) K/m2, respectively. The surface area of the lipoproteins determined by the method of radiation-free energy transfer between fluorescent probes amounts to 185.9; 241.7 and 426.8 m2 per 1 g of lipoprotein, respectively. Results of the experiments satisfactorily coincide with the data, obtained by other methods.     

S‐perindopril assay using a potentiometric,enantioselective membrane electrode

A potentiometric, enantioselective membrane electrode based on graphite paste (graphite powder and paraffin oil) has been constructed. The graphite paste is impregnated with a 10⁻³ mol/L 2‐hydroxy‐3‐trimethylammoniopropyl‐β‐cyclodextrin (as chloride salt) solution. The potentiometric, enantioselective membrane electrode can be used reliably for enantiopurity tests of S‐perindopril using a chronopotentiometric (zero current) technique, in the 10⁻⁵–10⁻² mol/L concentration range (detection limit 5 × 10⁻⁶ mol/L), with an average recovery of 99.58% (RSD = 0.33%). The enantioselectivity was determined over R‐perindopril and d ‐proline. The response characteristics of the enantioselective, potentiometric membrane electrode were also determined for R‐perindopril. It was shown that l ‐proline is the main interfering compound. The surface of the electrode can be regenerated simply by polishing, obtaining a fresh surface ready to be used in a new assay. Chirality 11:631–634, 1999. © 1999 Wiley‐Liss, Inc.     

Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.     

Perylenoyl- and anthrylvinyl-labeled lipids as membrane probes

The properties of a new family of lipid-specific fluorescent probes, a fatty acid, a phosphatidylcholine and a sphingomyelin, bearing a 3-perylenoyl-labeled hydrophobic chain, are described. Perylenoyl-labeled lipids readily enter the lipid bilayer, the fluorophore being localized in the apolar region of the membrane. The perylenoyl fluorophore is characterized by a high quantum yield, its fluorescence parameters (λ_ex 446 nm, λ_em 479–545 nm) permit to apply it as an acceptor of excitation energy from the 9-anthrylvinyl fluorophore used earlier for phospholipid labeling (Molotkovsky, Jul. G.; Manevich, Y.M., Gerasimova, E.N., Molotkovskaya, I.M., Polessky, V.A. and Bergelson, L.D. (1982) Eur. J. Biochem. 122, 573–579). The anthrylvinyl-labeled lipids were shown to be capable to report phase segregation between the corresponding prototype lipids in model systems. The combined use of anthrylvinyl- and perylenoyl-labeled lipids opens additional possibilities for investigation of lipid-lipid and lipid-protein interactions in artificial and biological membranes. Perylenoyl-labeled lipids appeared also to be useful as fluorescent dyes in cytological studies.     

Spectrophotometric,potentiometric, and density gradient ultracentrifugation studies of the binding of silver ion by DNA

The equilibrium and the stoichiometry for the reversible complexing of silver ion by DNA have been studied by potentiometric titrations, proton release pH-stat titrations, and by spectrophotometry. The complexing reactions involve primarily the purine and pyrimidine residues, not the phosphate groups. There are at least three types of binding (types I, II, and III), of which the first two have been intensively studied in this work. The sum of type I and type II binding saturates at one silver atom per two nucleotide residues. In the type I and type II reactions, zero and one proton, respectively, are displaced per silver ion bound. At pH 5.6, the reactions occur stepwise, type I being first, while at pH 8.0, they occur simultaneously. The silver ion binding curve is very sharp at pH 8, indicating a cooperative reaction. The strength of the binding increases with increasing GC content. Type I binding is more important for GC-rich DNA's than for GC-poor ones. Denatured DNA binds more strongly than does native DNA. The silver ion complexing reaction is chemically and biologically reversible. We propose that type II binding essentially involves the conversion of an \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm N} - {\rm H} \cdots {\rm N} $\end{document} hydrogen bond of a complementary base pair to an N—Ag—N bond. The nature of type I binding is less clear, but it may involve a π interaction with stacked bases. The buoyant density (ρ₀) of DNA in a Cs₂SO₄ density gradient increases when the DNA reacts with silver ion. The buoyant density change is about 0.15 g./ml. for 0.5 silver bound per nucleotide. The large buoyant density changes and the selective nature of the complexing reaction make it possible to perform good separations between native and denatured DNA or between GC-rich and GC-poor native DNA's by density gradient centrifugation.     

Covalent binding of urease on ammonium-selective potentiometric membranes.

As part of the development of disposable urea bioselective probes, the covalent binding of urease on ammonium-selective potentiometric membranes has been assessed. Nonactin/bis(1-butylpentyl)adipate/poly(vinylchloride) (PVC) membranes, directly applied to an internal solid contact (conductive epoxy-graphite composite), has been used as a support for covalent immobilization of urease. Two types of all-solid-state construction process have been assayed: thin layers of cellulose acetate (CA) were coated on the PVC ammonium-selective membranes (type 1) and blends of PVC and CA at various ratios were used as ammonium-selective membrane matrices (type 2). Urease was covalently attached to CA via aldehyde groups. These groups were created on the polysaccharide with sodium periodate to which the enzyme was immobilized through a spacer (hexamethylenediamine). The viability of both types of probe for the determination of ammonium ions was assessed after each step of the activation process. Results indicated that type 2 potentiometric probes are altered after the treatment with sodium periodate. Good results were obtained with type 1 probes. Their dynamic concentration range of response to urea was from 2 x 10(-5) to 0.01 M with a sensibility of 50 mV/decade.     

Profiling the membrane proteome of Shewanella oneidensis MR-1 with new affinity labeling probes

The membrane proteome plays a critical role in electron transport processes in Shewanella oneidensis MR-1, a bacterial organism that has great potential for bioremediation. Biotinylation of intact cells with subsequent affinity-enrichment has become a useful tool for characterization of the membrane proteome. As opposed to these commonly used, water-soluble commercial reagents, we here introduce a family of hydrophobic, cell-permeable affinity probes for extensive labeling and detection of membrane proteins. When applied to S. oneidensis cells, all three new chemical probes allowed identification of a substantial proportion of membrane proteins from total cell lysate without the use of specific membrane isolation method. From a total of 410 unique proteins identified, approximately 42% are cell envelope proteins that include outer membrane, periplasmic, and inner membrane proteins. This report demonstrates the first application of this intact cell biotinylation method to S. oneidensis and presents the results of many identified proteins that are involved in metal reduction processes. As a general labeling method, all chemical probes we introduced in this study can be extended to other organisms or cell types and will help expedite the characterization of membrane proteomes.     

Dopamine-selective potentiometric responses by new ditopic sensory elements based on a hexahomotrioxacalix[3]arene

New ditopic sensory elements 2 and 3 for catecholamines based on a hexahomotrioxacalix[3]arene, with a boronic acid substituent appended, were designed and synthesized. As an interesting mode of molecular recognition at membrane surfaces, the host, when incorporated into poly(vinyl chloride) (PVC) liquid membranes, displayed excellent potentiometric selectivity for dopamine over other catecholamines (noradrenaline and adrenaline) and inorganic cations (Na+, K+, and NH4+).