Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry.At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 Å with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 Ų to a limiting value of 100 Ų.On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93 000.This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles. (Tanford, C.(1972) J. Phys. Chem. 76, 3020–3024).     

Surface and interlayer structure of vermiculite intercalated with methyl viologen

Molecular modeling using empirical force field revealed the differences between the surface and interlayer arrangement of the dye guest molecules in vermiculite intercalated with the divalent methyl viologen cation (MV²⁺). Conformation and anchoring of MV²⁺ cations on the silicate layer in the interlayer space of vermiculite host structure is different from that on the crystal surface. A preferential position has been found for the anchoring of guests on the silicate layer. Anyway the arrangement of guests in the interlayer space as well as on the crystal surface exhibits a high degree of disorder due to a certain flexibility in guest molecules arrangement and first of all due to the presence of water molecules in the interlayer space. The presence of water disturbs not only the regularity in guest positions and orientations but also in conformation of guest molecules in the interlayer space of the host structure.     

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition

Water dynamics in samples of ceramide tetrasaccharide (G_g4Cer) vesicles and G_M1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (²H-NMR). G_M1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10^?10 s, typical of water molecules near to the polar groups. Pure G_M1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from ?18 to 32°C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to G_M1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into G_M1 micelles substantially increases the mobility of water molecules compared with that found in pure G_M1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid–lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.     

Gelation of chemically cross-linked methylcellulose studied by DSC and AFM

Gelation of methylcellulose (MC) and chemically cross-linked MC via urethane linkage (MCPU) with various molecular weights was investigated in a concentration range from 0.1 to 4.0 wt %. On heating of aqueous solution of MC, three transitions, clear sol to turbid sol, sol to gel and phase separation due to water separation were observed. With increasing molecular weight the transition temperatures decrease. In contrast, no effect of molecular weight on the transition temperatures was observed for MCPU. Structural change of water restrained by MC and MCPU was investigated by DSC. Melting and crystallization of water in both series of sample showed no significant difference, however, a molecular weight dependency of the glass transition was observed for MC. The results obtained in this study indicate that hydrophobic aggregation is restricted by cross-linking. Images of atomic force microscopy (AFM) indicated that from 6 to 16 molecules piled in two layers form a bundle. By chemical cross-linking, molecular chains align in the mono layer, molecular bundles consisting of more than 10 molecules coaggregate and form a large flexible ring.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry. At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 A with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 A2 to a limiting value of 100 A2. On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93,000. This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles.     

Spontaneous formation of detergent micelles around the outer membrane protein OmpX

The structure and flexibility of the outer membrane protein X (OmpX) in a water-detergent solution and in pure water are investigated by molecular dynamics simulations on the 100-ns timescale and compared with NMR data. The simulations allow for an unbiased determination of the structure of detergent micelles and the protein-detergent mixed micelle. The short-chain lipid dihexanoylphosphatidylcholine, as a detergent, aggregates into pure micelles of approximately 18 molecules, or alternatively, it binds to the protein surface. The detergent binds in the form of a monolayer ring around the hydrophobic beta-barrel of OmpX rather than in a micellar-like oblate; approximately 40 dihexanoylphosphatidylcholine lipids are sufficient for an effective suppression of water from the surface of the beta-barrel region. The phospholipids bind also on the extracellular, protruding beta-sheet. Here, polar interactions between charged amino acids and phosphatidylcholine headgroups act as condensation seed for detergent micelle formation. The polar protein surface remains accessible to water molecules. In total, approximately 90-100 detergent molecules associate within the protein-detergent mixed micelle, in agreement with experimental estimates. The simulation results indicate that OmpX is not a water pore and support the proposed role of the protruding beta-sheet as a "fishing rod".     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.     

Combining precision spin-probe partitioning with time-resolved fluorescence quenching to study micelles. Application to micelles of pure lysomyristoylphosphatidylcholine (LMPC) and LMPC mixed with sodium dodecyl sulfate

Micelles of lysomyristoylphosphatidylcholine (LMPC) and mixed micelles of LMPC with anionic detergent sodium dodecyl sulfate (SDS) have been characterized by spin-probe-partitioning electron paramagnetic resonance (SPPEPR) and time-resolved fluorescence quenching (TRFQ) experiments. SPPEPR is a novel new method to study structure and dynamics in lipid assemblies successfully applied here for the first time to micelles. Several improvements to the computer program used to analyze SPPEPR spectra have been incorporated that increase the precision in the extracted parameters considerably from which micelle properties such as effective water concentration and microviscosity may be estimated. In addition, with this increased precision, it is shown that it is feasible to study the rate of transfer of a small spin probe between micelles and the surrounding aqueous phase by SPPEPR. The rate of transfer of the spin probe di-tert-butyl nitroxide (DTBN) and the activation energy of the transfer process in LMPC and LMPC-SDS micelles have been determined with high precision. The rate of transfer increases with temperature and SDS molar fraction in mixed micelles, while it remains constant with LMPC concentration in pure LMPC micelles. The activation energy of DTBN transfer in pure lysophospholipid micelles does not change with LMPC concentration while it decreases with the increasing molar fraction of SDS in mixed LMPC-SDS micelles. Both this decrease in activation energy and the increase in the rate of transfer are rationalized in terms of an increasing micelle surface area per molecule (decreasing compactness) as SDS molecules are added. This decreasing compactness as a function of SDS content is confirmed by TRFQ measurements showing an aggregation number that decreases from 122 molecules for pure LMPC micelles to 80 molecules for pure SDS micelles. The same increase in surface area per molecule is predicted to increase the effective water concentration in the polar shell of the micelles. This increase in hydration with SDS molar fraction is confirmed by measuring the effective water concentration in the polar shell of the micelles from the hyperfine spacing of DTBN. This work demonstrates the potential to design mixed lysophospholipid surfactant micelles with variable physicochemical properties. Well-defined micellar substrates, in terms of their physicochemical properties, may improve the studies of protein structure and enzyme kinetics.     

Structuration in the Interface of Direct and Reversed Micelles of Sucrose Esters,Studied by Fluorescent Techniques

Background

Reactors found in nature can be described as micro-heterogeneous systems, where media involved in each micro-environment can behave in a markedly different way compared with the properties of the bulk solution. The presence of water molecules in micro-organized assemblies is of paramount importance for many chemical processes, ranging from biology to environmental science. Self-organized molecular assembled systems are frequently used to study dynamics of water molecules because are the simplest models mimicking biological membranes. The hydrogen bonds between sucrose and water molecules are described to be stronger (or more extensive) than the ones between water molecules themselves. In this work, we studied the capability of sucrose moiety, attached to alkyl chains of different length, as a surface blocking agent at the water-interface and we compared its properties with those of polyethylenglycol, a well-known agent used for this purposes. Published studies in this topic mainly refer to the micellization process and the stability of mixed surfactant systems using glycosides. We are interested in the effect induced by the presence of sucrose monoesters at the interface (direct and reverse micelles) and at the palisade (mixtures with Triton X-100). We believe that the different functional group (ester), the position of alkyl chain (6-O) and the huge capability of sucrose to interact with water will dramatically change the water structuration at the interface and at the palisade, generating new possibilities for technological applications of these systems.

Results

Our time resolved and steady state fluorescence experiments in pure SEs micelles show that sucrose moieties are able to interact with a high number of water molecules promoting water structuration and increased viscosity. These results also indicate that the barrier formed by sucrose moieties on the surface of pure micelles is more effective than the polyoxyethylene palisade of Triton X-100. The fluorescence quenching experiments of SEs at the palisade of Triton X-100 micelles indicate a blocking effect dependent on the number of methylene units present in the hydrophobic tail of the surfactant. A remarkable blocking effect is observed when there is a match in size between the hydrophobic regions forming the apolar core (lauryl SE/ Triton X-100). This blocking effect disappears when a mismatch in size between hydrophobic tails, exists due to the disturbing effect on the micelle core.     

Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

Model study of interactions of high-molecular dextran sulfate with lipid monolayers and foam films

The interaction of high-molecular dextran sulfate (DS-5000) with dimyristoylphosphatidylcholine (DMPC) monolayers and foam films (FF) at the air–water interface in the presence of Ca²⁺ and Na⁺ ions was studied. DS-5000 was added in monolayer films (MF) and in FF as monomer molecules and in liposomal form. When added in liposomal form in FF, DS-5000 decreased the stability of DMPC common black films (CBF), and no formation of Newton black films (NBF) was observed. However, when included as monomer molecules in FF, DS-5000 caused film thinning, and drastically decreased the expansion rate of the black spots and transition of thick films to NBF, thus avoiding formation of CBF. The above effects were observed in both gel and liquid-crystalline phase states of DMPC in the presence of Ca²⁺ ions only, and not in the presence of Na⁺ ions. We postulate that the interaction of DMPC with DS-5000 in the plane of FF is mediated by Ca²⁺ bridges and results in dehydration of the DMPC polar heads. The interaction between DMPC and DS-5000 in monolayers resulted in slower adsorption and spreading of DMPC molecules at the interface, lower monolayer surface pressure, and penetration of DS-5000 molecules to DMPC monolayers when surface lipid density was higher than 50 Ų per DMPC molecule. The applicability of the FF model for studying the interactions of phospholipids with polysaccharides at interfaces surrounded by bulk solution, and for modeling such interactions in biological systems, e.g. LDL adhesion to the arterial walls, aggregation and fusion of liposomes, etc., is discussed.     

[31P]-Nuclear magnetic resonance spin lattice relaxation in lecithin reverse micelles

[³¹P] -Nuclear magnetic resonance (NMR) spin lattice relaxation times (T₁) have been measured for lecithin-nonpolar solvent-water as a function of added water for three solvents, namely, benzene, carbon tetrachloride and cyclohexane. In benzene and carbon tetrachloride systems, where spherical reverse micelles are formed, [³¹P]-NMR T₁, values increase linearly with added water. However, in cyclohexane, the trends in the [³¹P]-T₁ values indicate very different micellisation processes. Even at the lowest concentration of added water, the [³¹P]-T₁ values in this solvent are substantially larger than the corresponding values in benzene and carbon tetrachloride, which is attributed to the intramolecular chlorinephosphate interaction being the weakest in cyclohexane. At a higher water content of six mols of water per mol of lecithin in cyclohexane solvent, the [³¹P]-T₁ values show a sharp decrease indicating a sudden change in the dynamics of the phosphate group, and this confirms the on set of ‘reverse micelle-to-liquid crystalline’ phase transition observed in this system by other spectroscopic and physical techniques.     

Quantum mechanical calculations of charge effects on gating the KcsA channel

A series of ab initio (density functional) calculations were carried out on side chains of a set of amino acids, plus water, from the (intracellular) gating region of the KcsA K⁺ channel. Their atomic coordinates, except hydrogen, are known from X-ray structures [D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. MacKinnon, The structure of the potassium channel: molecular basis of K⁺ conduction and selectivity, Science 280 (1998) 69-77; R. MacKinnon, S.L. Cohen, A. Kuo, A. Lee, B.T. Chait, Structural conservation in prokaryotic and eukaryotic potassium channels, Science 280 (1998) 106-109; Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait, R. MacKinnon, The open pore conformation of potassium channels. Nature 417 (2001) 523-526], as are the coordinates of some water oxygen atoms. The 1k4c structure is used for the starting coordinates. Quantum mechanical optimization, in spite of the starting configuration, places the atoms in positions much closer to the 1j95, more tightly closed, configuration. This state shows four water molecules forming a “basket” under the Q119 side chains, blocking the channel. When a hydrated K⁺ approaches this “basket”, the optimized system shows a strong set of hydrogen bonds with the K⁺ at defined positions, preventing further approach of the K⁺ to the basket. This optimized structure with hydrated K⁺ added shows an ice-like 12 molecule nanocrystal of water. If the water molecules exchange, unless they do it as a group, the channel will remain blocked. The “basket” itself appears to be very stable, although it is possible that the K⁺ with its hydrating water molecules may be more mobile, capable of withdrawing from the gate. It is also not surprising that water essentially freezes, or forms a kind of glue, in a nanometer space; this agrees with experimental results on a rather different, but similarly sized (nm dimensions) system [K.B. Jinesh, J.W.M. Frenken, Capillary condensation in atomic scale friction: how water acts like a glue, Phys. Rev. Lett. 96 (2006) 166103/1-4]. It also agrees qualitatively with simulations on channels [A. Anishkin, S. Sukharev, Water dynamics and dewetting transitions in the small mechanosensitive channel MscS, Biophys. J. 86 (2004) 2883-2895; O. Beckstein, M.S.P. Sansom, Liquid-vapor oscillations of water in hydrophobic nanopores, Proc. Natl Acad. Sci. U. S. A. 100 (2003) 7063-7068] and on featureless channel-like systems [J. Lu, M.E. Green, Simulation of water in a pore with charges: application to a gating mechanism for ion channels, Prog. Colloid Polym. Sci. 103 (1997) 121-129], in that it forms a boundary on water that is not obvious from the liquid state. The idea that a structure is stable, even if individual molecules exchange, is well known, for example from the hydration shell of ions. We show that when charges are added in the form of protons to the domains (one proton per domain), the optimized structure is open. No stable water hydrogen bonds hold it together; an opening of 11.0 Å appears, measured diagonally between non-neighboring domains as glutamine 119 carbonyl O-O distance. This is comparable to the opening in the MthK potassium channel structure that is generally agreed to be open. The appearance of the opening is in rather good agreement with that found by Perozo and coworkers. In contrast, in the uncharged structure this diagonal distance is 6.5 Å, and the water “basket” constricts the uncharged opening still further, with the ice-like structure that couples the K⁺ ion to the gating region freezing the entrance to the channel. Comparison with our earlier model for voltage gated channels suggests that a similar mechanism may apply in those channels.     

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition

Water dynamics in samples of ceramide tetrasaccharide (Gg4Cer) vesicles and GM1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (2H-NMR). GM1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10(-10) s, typical of water molecules near to the polar groups. Pure GM1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from -18 to 32 degrees C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to GM1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into GM1 micelles substantially increases the mobility of water molecules compared with that found in pure GM1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid-lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.     

Conformational preferences of Leu-enkephalin in reverse micelles as membrane-mimicking environment

Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single-bend, and a double-bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu-enkephalin solubilized in negatively charged reverse micelles of bis (2-ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu-enkephalin in reverse micelles using a synthetic [¹³C, ¹⁵N]-backbone-labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and ¹⁵N-filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double-bend fold of the backbone that is comparable to one of the known x-ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ-receptor-bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody

Summary Previous evidence has established the similarity between (Na⁺+K⁺)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na⁺+ K⁺)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105000 and 51000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na⁺+K⁺)ATPase expressed by various tissues and cells.     

Intra and intermolecular charge effects on the reaction of the superoxide radical anion with semi-oxidized tryptophan in peptides and N-acetyl tryptophan

The reaction of the superoxide radical anion (O^-·₂), with the semi-oxidized tryptophan neutral radical (Trp^·) generated from tryptophan (Trp) by pulse radiolysis has been observed in a variety of functionalized Trp derivatives including peptides. It is found that the reaction proceeds 4–5 times faster in positively charged peptides, such as Lys-Trp-Lys, Lys-Gly-Trp-Lys and Lys-Gly-Trp-Lys-O-tert-butyl, than in solutions of the negatively charged N-acetyl tryptophan (NAT). However, the reactivity of O^-·₂ with the Trp^· radical is totally inhibited upon binding of these peptides to micelles of negatively charged SDS and is reduced upon binding to native DNA. By contrast, no change in reactivity is observed in a medium containing CTAB, where the peptides cannot bind to the positively charged micelles. On the other hand, the reactivity of the Trp^· radical formed from NAT with O^-·₂ is reduced to half that of the free Trp^· in buffer but is markedly increased in CTAB micelles. The models studied here incorporate elements of the complex environment in which Trp^· and O^-·₂ may be concomitantly formed in biological system and demonstrate the magnitude of the influence such elements may have on the kinetics of reactions involving these two species.     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.