Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.     

Two types of hydrocarbon chain interdigitation in sphingomyelin bilayers

Vibrational Raman spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of synthetic DL-erythro-N-lignoceroylsphingosylphosphocholine [C(24):SPM], a racemic mixture of two highly asymmetric hydrocarbon chain length sphingomyelins. Raman spectral peak-height intensity ratios of vibrational transitions in the C-H stretching-mode region show that the C(24):SPM-H2O system undergoes two thermal phase transitions centered at 48.5 and 54.5 degrees C. Vibrational data for fully hydrated C(24):SPM are compared to those of highly asymmetric phosphatidylcholine dispersions. The Raman data are consistent with the plausible model that the lower temperature transition can be ascribed to the conversion of a mixed interdigitated gel state (gel II) to a partially interdigitated gel state (gel I) and that the higher temperature transition corresponds to a gel I----liquid-crystalline phase transition. The observation of a mixed interdigitated gel state (gel II) at temperatures below 48.5 degrees C implies that biological membranes may have lipid domains in which some of the lipid hydrocarbon chains penetrate completely across the entire hydrocarbon width of the lipid bilayer.     

Tris buffer effects on melanophore aggregating responses

The adverse effects of tris (hydroxymethyl)-aminomethane (Tris) are for the first time reported on melanophore responses to agonists and potassium chloride. Melanophore responses in Tris- or bicarbonate-buffered solutions were compared. In the presence of Tris, the cumulative dose-response curve to norepinephrine was significantly shifted to the left, whereas methoxamine dose-response curves were similar in both buffers. The percentage aggregation in response to synthetic MCH (melanin concentrating hormone) was not affected by Tris in the bathing medium. The cumulative dose-response curve to potassium chloride was leftward shifted (one log case) in Tris-buffered solution. These results suggest that in fish melanophore preparations Tris might exert its actions on the presynaptic membrane and/or on the synaptic cleft enzyme COMT, drawing on a greater availability of neurotransmitters at the melanophore membrane receptors.     

Acyl chain interdigitation in saturated mixed-chain phosphatidylcholine bilayer dispersions

The molecular packing of various fully hydrated mixed-chain phosphatidylcholines was studied by X-ray diffraction and electron microscopy. All of the mixed-chain phosphatidylcholines under study were shown to adopt a lamellar or bilayer form in aqueous media. The bilayer thickness of these mixed-chain phosphatidylcholines was determined from the lamellar repeat distance in the small-anglé X-ray diffraction region by controlled swelling experiments. At T greater than Tm, the bilayer thickness of C(18):C(12)PC and C(18):C-(10)PC is found to be comparable to that of C(14):C(14)PC. In contrast, the bilayer thickness of these highly asymmetric phosphatidylcholines is considerably less than that of the symmetric C(14):C(14)PC at temperatures below Tm. Moreover, the wide-angle X-ray diffraction patterns taken at T less than Tm consist of at least two sharp reflections at 4.2 and 4.6 A. These X-ray diffraction data suggest that these highly asymmetric mixed-chain phospholipids, in excess water, form mixed interdigitated bilayers in the gel state and that the acyl chain packing in the gel-state bilayer is not hexagonal. The freeze-fracture planes of these mixed-chain phosphatidylcholines are discontinuous at T less than Tm, supporting the conclusion drawn from X-ray diffraction data that these highly asymmetric phosphatidylcholines form interdigitated bilayers at temperatures below Tm. The molecular packing of fully hydrated C(18):C(14)PCs in bilayers is distinctively different from that of C(18):C(10)PCs or C(18):C(10)PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of acyl transferase in the biosynthesis of pulmonary microsomal phosphatidylglycerol

Dog lung microsome contain an enzymatic system capable of catalyzing the transfer of radiolabel from [¹⁴C]-acyl CoA, pamitoyl or oleoyl, to 1-acyl-2-lysophosphatidylglycerol thereby forming phosphatidylglycerol. This synthetic activity is consistent with a 1-acyl-2-lyso-phosphatidylglycerol: acyl-CoA acyl transferase. Kinetic analysis suggests the enzyme is preferentially, but not exclusively, orientated to the production of 1-acyl-2-oleoyl-phosphatidylglycerol. When enzymatic activity was examined with palmitoyl-CoA as the donor substrate and either 2-lysophosphatidylglycerol or 2-lysophosphatidylcholine as the acceptor substrates, the perferred product seemed to be phosphatidylglycerol. When the activity was examined with both acceptor substrates present, phosphatidylglycerol formation decreased while phosphatidylcholine formation increased. This observation may represent an effective control mechanism for the differential synthesis of pulmonary phospholipids, according to metabolic or physiological requirements.     

Hydrostatic pressure induces hydrocarbon chain interdigitation in single-component phospholipid bilayers

By use of neutron diffraction for structural analysis, the temperature-pressure phase diagrams of several fully hydrated single-component phospholipid bilayers have been explored up to hydrostatic pressures of 2 kbars. The gel to liquid-crystalline phase transition temperature Tm increases linearly with pressure over a 10(-3)-2 kbar range in accordance with the Clausius-Clapeyron relationship giving dTm/dP values of 23.0 degrees C/kbar for 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 28.0 degrees C/kbar for 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The so-called pretransition was not observed in the isothermal pressure experiments, suggesting that no appreciable volume change occurs at this transition. These results are in good agreement with those reported using other techniques. In addition, at pressures higher than the isothermal liquid-crystalline to gel transition pressure, a new pressure-induced phase transition was observed for DPPC and DSPC in which the hydrocarbon chains from apposing monolayers become interdigitated with the chains occupying a cross-sectional area approximately equal to 5% less than in the gel phase. The temperature-pressure phase diagrams show the gel-interdigitated phase boundaries to be highly curved and the minimum pressure at which interdigitation occurs to decrease with increasing hydrocarbon chain length.     

Further assessment of a pharmacological effect of Tris buffer

1. Data are presented confirming that Tris buffer can potentiate teleost melanophore responses to noradrenalin, as recently reported for the first time in Poecilia reticulata (Visconti and Castrucci, 1985). 2. In vitro melanophore responses of Pseudopleuronectes americanus were compared in Tris- and bicarbonate-buffered physiological saline solutions. 3. Dose-response curves demonstrate that Tris significantly potentiates melanophore responses to noradrenalin doses between 10(-6) and 10(-5)M in this species. 4. Time-response curves further characterized this effect of Tris on melanophore activity, quantifying the enhancement of the melanosome aggregation rate as well as the dose-related equilibrium levels.     

Tris is a non-innocent buffer during intein-mediated protein cleavage

Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.     

Membrane thickness and acyl chain length

It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.     

Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer

1. Photophosphorylation was measured with spinach grana sampleswashed by 0.8 M Tris buffer at pH 8.0, which no longer catalyzedthe ferricyanide and NADP HILL reactions with water as the electrondonor. The photophosphorylation with the reaction mixture containing2 10^–4 M 2,6-dichlorophenol indophenol (DCPI) plus above2 10^–3 M ascorbate as the electron donor system insteadof water under anaerobic conditions was, in the most part, dependenton the addition of both PPNR (a nonheme iron protein requisitefor photosynthetic pyridine nucleotide reduction ; spinach ferredoxin)and NADP as the electron acceptor system. However, when ascorbateconcentration only was lowered to 2 10^–4 M, the entirephotophosphorylation proceeded, even in the absence of the electronacceptor system. 2. When the NADP added in the reaction mixture had been reducedby glucose-6-phosphate and glucose-6-phosphate dehydrogenasebefore illumination, the photophosphorylation with 2 10^–4M DCPI plus 6.7 10^–3 M ascorbate decreased to aboutthe same rate as that obtained without NADP. 3. The time course for photophosphorylation in the presenceof NADP was consistent with the time course for the photoreductionof NADP: On the complete reduction of NADP, the photophosphorylationstopped. 4. In the presence of 6.7 10^–3 M dichloropheny 1.1,1-dimethylureaor 3 10^–4 M o-phenanthroline, non-cyclic photophosphorylationwith 2 10^–4 M DCPI plus 6.7 10^–3 M ascorbateas the electron donor system decreased to about half that ofthe control, and the remaining activities were hardly affectedeven at higher concentrations of both inhibitors. The P/2e^–ratios of non-cyclic photophosphorylation in the absence andpresence of ophenanthroline were 0.74 and 0.48, respectively. ¹Present address: Department of Biology, University of California,San Diego, La Jolla, California 92037, U. S. A.     

Interference by Tris buffer in the estimation of protein by the Lowry procedure

Tris buffers were found to distort the measurement of protein by the Lowry method both by decreasing chromophore development with protein and by contributing blank color. Tris at an assay concentration of 0.37 mm markedly affects measured results. Similar Tris effects were observed at all wavelengths between 450 and 800 nm and with diverse protein samples. The distortion due to Tris is not correctable by simple blank correction, but it can be overcome by incorporating the same amount of Tris in the standards used. The distortion at Tris concentrations <0.15 mm appears to be within tolerable limits. No interference or distortion was observed with sodium phosphate buffer to an assay concentration of 40 mm. An automated Lowry procedure is also presented which gives excellent correlation with the manual method and an average coefficient of variation of <4%.     

Ethylene glycol causes acyl chain disordering in liquid-crystalline, unsaturated phospholipid model membranes, as measured by 2H NMR

2H NMR has been used to probe the effects of ethylene glycol at the level of the acyl chains in liposomes prepared from dioleoylphosphatidic acid or dioleoylphosphatidylcholine, labeled with 2H at the 11-position of both oleic acid chains. Increasing concentrations of ethylene glycol lead to a proportional and substantial decrease in the quadrupolar splittings, measured from the 2H NMR spectra of both liposomal systems, indicative of acyl chain disordering.     

Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Evidence for the participation of detergent-resistant phospholipase A and heat-labile membrane-bound factor(s).

The conversion of phosphatidylglycerol to acyl phosphatidylglycerol by extracts of Escherichia coli K-12 strains was examined under various conditions. The maximum rate of conversion was observed at pH 7.2 in the presence of 50% (v/v) diethyl ether and 10 mM CaCl2. This conversion was found to involve two sequential reactions: (1) The formation of 2-acyl glycerophosphoglycerol and 2-acyl glycerophosphoethanolamine from phosphatidylglycerol and phosphatidylethanolamine, respectively, by detergent-resistant phospholipase A in the presence of Ca2+ and (2) transfer of the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol by a heat-labile factor(s) in the presence of diethyl ether. Neither fatty, acid acyl-CoA nor 1-acyl lysophospholipid could act as an acyl donor for phosphatidylglycerol. The heat-labile factor(s) was found in both the inner membrane and supernatant fractions.     

Alcohol induction of interdigitation in distearoylphosphatidylcholine: fluorescence studies of alcohol chain length requirements.

Although it is now well established that the fully interdigitated phase is induced in saturated like-chain phosphatidylcholines (PCs) by a variety of amphipathic molecules including alcohols, no systematic study of the properties of the inducing molecules has been reported. To elucidate the stereochemical features that lead to the alcohol induction of interdigitation in PCs, we have investigated the induction of interdigitation in distearoylphosphatidylcholine (DSPC) by a series of alcohols. Our previously established DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence intensity method has been expanded (P. Nambi, E. S. Rowe, and T. M. McIntosh (1988), Biochemistry 27:9175-9182) and used to determine which of the alcohols induce interdigitation and to determine the threshold concentrations for each. We have found that each of the n-alcohols up to heptanol and several branched alcohols are capable of inducing interdigitation in DSPC; octanol and nonanol do not appear to induce interdigitation by these criteria. The threshold concentrations for interdigitation for each of these alcohols up to heptanol were found to be correlated with the membrane: buffer partition coefficients. The mole fraction of bound alcohol at the threshold concentration was similar for each of the alcohols up to pentanol. These results are discussed in terms of a general mechanism of the formation of the interdigitated phase.     

Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases

Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.     

AXanthomonas maltophilia isolate tolerating up to 1% sodium azide in Tris/HCl buffer

A bacterium tolerating up to 1% NaN₃ found as a contaminant of Sephadex colums being run with Tris/HCl buffer, was identified asXanthomonas maltophilia. It had low nutrient requirements, was strongly proteolytic and interfered with Sephadex columns run with Tris/HCl buffers.The authors are with the Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044, S-750 07 Uppsala, Sweden;     

Phospholipid turnover and acyl chain remodeling in the yeast ER

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.     

Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin

Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work. Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid.