Lateral proton conduction in monolayers of phospholipids from extreme halophiles

Studies have been carried out on the lateral proton conductance properties of monolayers of the major and minor phospholipids of extremely halophilic archaebacteria, 2,3-diphytanyl-sn-glycero-1-phospho-3'-sn-glycerol 1'-phosphate (PGP) and 2,3-diphytanyl-sn-glycero-1-phospho-3'-sn-glycerol (PG), respectively, as well as on their respective deoxy analogues: 2,3-diphytanyl-sn-glycero-1-phospho-1'-propanediol 3'-phosphate (dPGP), 2,3-diphytanyl-sn-glycero-1-phospho-1'-1',3'-propanediol (dPG), and 2,3-diphytanyl-sn-glycero-1-phospho-1'-propanol (ddPG). Lateral proton conduction was found to occur with monolayers of all ether phospholipids examined at reduced surface pressure (pi greater than 25 mN/m) on subphases of low (1 mM) and high (4 M) ionic strength. Proton conduction was also detected in highly condensed monolayers (greater than 35 mN/m) of the naturally occurring phospholipids (PGP, PG) but was abruptly terminated in tightly packed monolayers (greater than 35 mN/m) of the corresponding deoxy compounds (dPGP, dPG, ddPG) on subphases with low ionic strength. conduction did occur, however, along monolayers of the deoxy compounds at high surface pressure when spread on a subphase of high ionic strength (4 M). The abrupt termination of conduction with monolayers of the deoxy compounds at low ionic strength cannot be attributed to a lipid phase transition or to changes in the lateral fluidity of the monolayers, nor was the pK of the fluorescent interfacial proton indicator affected at high surface pressures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lateral interactions among phosphatidylcholine and phosphatidylethanolamine head groups in phospholipid monolayers and bilayers

We develop theory for the lateral interactions among the zwitterionic head groups of phospholipids in monolayers and bilayers, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE). With the P- end of the head group anchored at the water/hydrocarbon interface, a balance of two effects dictates the angle that the P--N+ dipole makes with respect to the plane of the bilayer: N+ is driven toward water due to the (Born) electrostatic free energy, but the hydrophobic effect drives the methyl and methylene groups around the N+ charge toward the hydrocarbon. The only adjustable parameter of the model is the average fluctuation of the oil/water interface or, alternatively, the dielectric constant of the hydrocarbon phase. The model predicts that at 5 degrees C the head group dipole should lie largely in the bilayer plane, in accord with X-ray, neutron diffraction, and NMR studies. The theory makes the novel prediction that the N+ end of the dipole becomes increasingly submerged in hydrocarbon with increasing temperature, leading to strongly enhanced lateral repulsion between PC head groups. This prediction is in good agreement with second and third viral coefficients of monolayer lateral pressures, and with the temperature dependence of the former. The theoretical model is consistent with head group fluctuations measured by neutron diffraction of PC and PE bilayers. Because PE has a smaller hydrophobic cluster near N+, its lateral repulsion should be much smaller and less temperature dependent than for PC, also in agreement with equation-of-state measurements. This suggests why at high density PE monolayers have higher melting temperatures than PC monolayers and more propensity for reversed curvature.     

Proton conduction in phosphatidylethanolamine

The dc conductivity of polycrystalline phosphatidylethanolamine (PE) was measured in the temperature range 60–120°C. Since no conclusive evidence had so far been obtained for the presence of proton conduction in this phospholipid, hydrogen gas was shown in the present experiment to evolve during the electrolysis in its premelted state between 91 and 124°C. In this temperature range molecules assume rotation around the molecular axes and proton conduction of the Grotthus type takes place possibly along two chains of intermolecular hydrogen bonds running in parallel. Zwitter-ions behave cooperatively as proton donors and acceptors in transferring proton from molecule to molecule via the hydrogen bond networks. This efficient push-pull way of proton transferring seems to account for the fact that no polarization was observed in the dc conduction experiments. The amount of evolved gas appears to be not exactly in accordance with Faraday's law and discussions are made on possible causes for this slight deviation.     

Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers.

Lipopolysaccharide and phosphatidylethanolamine are the two major lipid constituents of the membrane of Salmonella typhimurium. Interactions between the purified lipopolysaccharide and phosphatidylethanolamine were studied in molecular monolayers at air-water interfaces. The equilibrium surface pressures of mixed films of lipopolysaccharide and phosphatidylethanolamine were determined as a function of the film composition. The plot of the equilibrium surface pressrue vs. the area occupied by phosphatidylethanolamine molecules exhibited two distinct regions. Below a phosphatidylethanolamine surface concentration at which 55% of the surface was occupied by phosphatidylethanolamine molecules, the equilibrium pressure was invariant and had the value of a pure lipopolysaccharide monolayer at maximum compression. At phosphatidylethanolamine surface concentrations in excess of 55% surface area occupation (phosphatidylethanolamine/lipopolysaccharide (mol/mol) greater than 16), the equilibrium surface pressure was a function of the surface concentration of phosphatidylethanolamine. The results suggest a simple model in which lipopolysaccharide and phosphatidylethanolamine form a complex in which each lipopolysaccharide molecule is surrounded ("lipidated") by a shell of approx. 16 phosphatidylethanolamine molecules.     

Lateral diffusion of cholesterol in monolayers.

The surface diffusion coefficient of cholesterol in cholesterol monolayers has been measured as a function of cholesterol surface concentration. Two different radiochemical methods, one integral and the other differential, were developed which gave comparable results. In the integral method two cholesterol monolayers, one of which is radioactive, are isolated on inert hydrophilic supports and then brought into contact. After some time the supports are separated and the radioactivity of the supports is measured. The differential method is an autoradiographic experiment. Two cholesterol monolayers, one of which is radioactive, are separated by means of a thin barrier. Upon removal of the barrier and at later times, an autoradiographic plate is brought to within a fraction of a mm from the aqueous surface and exposed. The plates are developed and analysed. The data show that the cholesterol surface diffusion coefficient in the dilute monolayers is approximately 10(-6)cm2/s and is nearly independent of surface concentration up to a concentration corresponding to an area of 40 A2/molecule. As the monolayer becomes compressed beyond this surface concentration, the diffusion coefficient decreases ubruptly with the deeply decreasing surface tension to about 10(-7) cm2/s, when a fully condensed surface layer of 38 A2/molecule is reached. This diffusion coefficient is of the same order of magnitude as the diffusion coefficients measured in lipid bilayers and in membranes.     

Kinetics of proton exchange of phosphatidylethanolamine in phospholipid vesicles.

The rate of proton exchange of the amino protons of phosphatidylethanolamine (PE) in sonicated mixed phospholipid vesicles has been determined by NMR spectroscopy. The rate of exchange increases with increasing pH and phosphate concentration. In the absence of buffer the dominant exchange process is an intrasurface reaction in which NH2 groups react via water with NH3+ groups on the outer surface. Addition of cholesterol reduces the rate constant for intrasurface exchange. The experiments are evidence that such reactions could be dominant in proton transport in and to membrane surfaces.     

Methylation effects on the microdomain structures of phosphatidylethanolamine monolayers.

Increasing methylation of the headgroup in DPPE results in an increase of minimum area per molecule in highly compressed monolayers at the air-water interface. The shape of solid domains, as observed by epifluorescence microscopy, also exhibits marked changes upon increasing headgroup methylation. Branching domains are observed in DPPE and DP(Me)PE, whereas U-shaped or round domains are observed in DP(Me)2PE and DPPC under our experimental conditions. The domain shape is determined more by the headgroup methylatin than by the corresponding shift in critical temperatures, as shown by the study of PCs of different acyl chain moieties. In mixed lipid monolayers, PC (phosphatidylcholine) and PE (phosphatidylethanolamine) do not mix ideally, as indicated by the non-linear variation of the average area per molecule with composition, and by distinct domain shapes in LE/LC (liquid expanded/liquid condensed) coexisting phases representing PE-enriched or PC-enriched domains in those mixed monolayers.     

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.     

Large-scale isolation of plasmid DNA using cetyltrimethylammonium bromide

A rapid procedure for the large-scale isolation of plasmid DNA is described. The method utilizes cetyltrimethylammonium bromide to precipitate the plasmid following extraction of DNA by lysozyme digestion and boiling. The plasmid is then purified by passing through the spin column pZ523. The purity and yield of the plasmid obtained with this method is similar to that isolated by cesium chloride-ethidium bromide gradient centrifugation. The method does not involve any phenol-chloroform extractions and takes five to six hours for completion after growth of the bacterial cells. The plasmid obtained is amenable to digestion with various restriction endonucleases, can be used for cloning with high efficiency and is also suitable as template for dideoxy sequencing.     

Stabilization of cetyltrimethylammonium bromide permeabilized yeast whole cell lactase

Summary Cetyltrimethylammonium bromide-permeabilized cells ofK. fragilis loose -galactosidase activity due to leaking of the enzyme into the medium. This leakage of the enzyme can be prevented by storing the permeabilized cells either in buffer containing 50% glycerol or by treating the permeabilized cells with 0.2% glutaraldehyde at 4°C for 10 min. In repeated batch hydrolysis of lactose in milk, glutaraldehyde treated cells could be repeatedly used very efficiently.     

Circular dichroism and difference spectra of members of the troponin-tropomyosin system in cetyltrimethylammonium bromide.

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The delta epsilonM values at their difference maxima were found to be 1300 at 292 nm for Ac-Trp-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studies by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.     

Properties of bacteriophage T4 modified with cetyltrimethylammonium bromide

Treating of the bacteriophage T4B with cetyltrimethylammonium bromide results in extension of long fibers and contraction of tail sheaths. The unreorganized hexagonal baseplate attachment to the distal end of the tail core remains intact. Such aberrantly contracted phages are shown to retain the ability to absorb on the bacterial surface. The absorption is inhibited by sucrose and does not require the presence of 1-tryptophan. The aberrantly contracted phages lose the infection ability.     

Seperation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis

The gel electrophoresis system presented here allows the separation of proteins with the concomitant retention of detectable native activities. The system, referred to as CAT gel electrophoresis. uses the detergent cetyltrimethylammonium bromide in combination with a discontinuous gel matrix to resolve protein mixtures into discrete bands. Many proteins retain detectable levels of native activity after CAT electrophoresis, and gel bands can be easily identified using assays based on specific enzymatic activities or binding characteristics. The ability to identify protein bands based on BothMnr and activity in a single gel makes the CAT system a powerful adjunct to existing biochemical techniques.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.