Order and dynamics in mixtures of membrane glucolipids from Acholeplasma laidlawii studied by 2H NMR.

The two dominant glucolipids in Acholeplasma laidlawii, viz., 1,2-diacyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerol (MGlcDG) and 1,2-diacyl-3-O-[alpha-D-glucopyranosyl-(1----2)-O-alpha-D-glucopyranosyl ]- sn-glycerol (DGlcDG), have markedly different phase behavior. MGlcDG has an ability to form nonlamellar phases, whereas DGlcDG only forms lamellar phases. For maintenance of a stable lipid bilayer, the polar headgroup composition in A. laidlawii is metabolically regulated in vivo, in response to changes in the growth conditions [Wieslander et al. (1980) Biochemistry 19, 3650; Lindblom et al. (1986) Biochemistry 25, 7502]. To investigate the mechanism behind the lipid regulation, we have here studied bilayers of mixtures of unsaturated MGlcDG and DGlcDG, containing a small fraction of biosynthetically incorporated perdeuterated palmitic acid, with 2H NMR. The order-parameter profile of the acyl chains and an apparent transverse spin relaxation rate (R2) were determined from dePaked quadrupole-echo spectra. The order of the acyl chains in DGlcDG-d31 increases upon addition of protonated MGlcDG, whereas the order of MGlcDG-d31 decreases when DGlcDG is added. The variation of order with lipid composition is rationalized from simple packing constraints. R2 increases linearly with the square of the order parameter (S2) up to S approximately 0.14; then, R2 goes through a maximum and decreases. The increase in R2 with S2, as well as the magnitude of R2, is largest for pure MGlcDG-d31, smallest for DGlcDG-d31, and similar for mixtures with the same molar ratio of MGlcDG/DGlcDG but with the deuterium label on different lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermotropic characterization of the 2-O-acyl,polyprenyl alpha-D-glucopyranoside isolated from palmitate-enriched Acholeplasma laidlawii B membranes.

The thermotropic phase behavior of a monoacylated neutral glucolipid (2-O-acyl,polyprenyl alpha-D-glucopyranoside), isolated from palmitate-enriched Acholeplasma laidlawii B membranes, was studied by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. When equilibrated at low temperatures, aqueous dispersions of this lipid form an ordered, crystal-like lamellar gel phase which transforms to an inverted hexagonal phase at temperatures near 65 degrees C upon heating. However, upon cooling from high temperatures, the inverted hexagonal phase remains stable down to temperatures near 45 degrees C. Further cooling first results in the formation of a metastable lamellar liquid crystalline phase at temperatures near 35 degrees C and then a metastable gel phase at lower temperatures. The metastable gel phase, if immediately reheated at a fast scan rate, undergoes a gel/liquid-crystalline phase transition at temperatures near 33 degrees C. These results indicate that this monoacylated glucolipid exhibits its gel/liquid-crystalline phase transition and its lamellar/non-lamellar phase transition at considerably lower temperatures than does the monoglycosyldiacylglycerol formed under the same conditions. When cultured in media enriched in 'high-melting' fatty acids, Acholeplasma laidlawii B synthesizes large quantities of the 2-O-acyl,polyprenyl alpha-D-glucopyranoside (up to 60 mol%) mainly at the expense of the monoglucosyldiacylglycerol (the only other nonbilayer-forming liquid normally found in the cell membrane of this organism). We thus suggest that the biosynthesis of this novel glucolipid, in response to the biosynthetic incorporation of high-melting exogenous fatty acids, is an adaptive response designed to maintain a predominantly liquid-crystalline membrane lipid bilayer at the growth temperature, while retaining the high proportion of nonbilayer-forming glucolipid species characteristic of A. laidlawii B cells cultured under these conditions.

A cation/proton antiport activity in Acholeplasma laidlawii.

Sealed membrane vesicles of Acholeplasma laidlawii were obtained by controlled lysis of carotenoid-rich intact cells. An imposed delta pH was created by loading membrane vesicles or intact Acholeplasma laidlawii cells with 0.25 M NH4Cl and diluting them into 0.25 M choline chloride. The passive efflux of NH3 from the membrane vesicles or cells resulted in the creation of a delta pH (inside acid) that could be visualized by the quenching of the fluorescence of the weak base acridine orange. Whereas with isolated membrane vesicles, the fluorescence was dequenched by the addition of Na+, with intact cells, K+ in addition to Na+ was required. These results strongly suggest a Na+/H+ exchange activity that in intact Acholeplasma laidlawii cells is K+-dependent. The possible role of the Na+/H+ exchange activity in pH homeostasis at the more alkaline pH range, as well as in the extrusion of excess Na+ from the cells is discussed.

Purification of membrane proteins in SDS and subsequent renaturation.

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.

Isolation and characterization of a novel monoacylated glucopyranosyl neutral lipid from the plasma membrane of Acholeplasma laidlawii B.

The level of a normally minor component of the membrane polar lipids of Acholeplasma laidlawii B was significantly increased when the glucose supplement in the growth medium was reduced. Under such glucose-limiting conditions the proportion of this component was found to be dependent upon the fatty acid supplement and could approach 55-60% of the total polar lipids when palmitic acid was used to supplement the growth medium. A number of physical measurements, along with specific chemical and enzymic degradation studies followed by a careful analysis of the degradation products, enabled us to tentatively identify this lipid as a polyprenyl-alpha-D-glucoside with a long-chain fatty acid esterified to the 2-hydroxyl group of the sugar moiety. This lipid exhibited some unusual thermotropic phase properties and our observations suggest that it may not be easily miscible with the other membrane lipid components. The structure and physical properties of this unusual glycolipid also suggest that it may be capable of forming non-bilayer phases under physiologically relevant conditions.

Cytotoxic and cytolytic activity of nonadecafluoro-n-decanoic acid on Acholeplasma laidlawii.

We studied the interactions between the perfluorinated fatty acid nonadecafluoro-n-decanoic acid (NDFDA) and the cell wall-less procaryote Acholeplasma laidlawii, which were cultured in an identical medium base but with different serum supplements. When grown in mycoplasma media supplemented with PPLO serum fraction (Difco Laboratories, Detroit, Mich.), A. laidlawii was rapidly killed by low concentrations of toxicant (less than 1.0 mM). At higher concentrations (greater than 10 mM), NDFDA treatment appeared to lyse cells. A. laidlawii cells grown in horse serum-supplemented mycoplasma media were both killed and lysed at the same NDFDA concentration (greater than 10 mM). These data suggest that this perfluorinated fatty acid can be cytotoxic and cytolytic to mycoplasmas. Changes in active concentrations occurred in parallel with changes in growth medium serum supplementation, which is known to alter mycoplasma membrane composition. We propose that NDFDA interacts with the membranes of A. laidlawii cells, resulting in cell death or cell lysis or both.

Calorimetric studies of the thermotropic phase behavior of cells, membranes and lipids from fatty acid-homogeneous Acholeplasma laidlawii B.

We have investigated the comparative lipid thermotropic phase behavior of intact cells, isolated membranes and total membrane lipid dispersions from fatty acid-homogeneous Acholeplasma laidlawii B by high-sensitivity differential scanning calorimetry. We find that the gel to liquid-crystalline phase transition temperature, enthalpy and degree of cooperativity are essentially identical in viable cells and isolated membranes and that these parameters are not affected by the thermal denaturation of the cellular or membrane proteins. In contrast, the extracted lipid dispersions, although having the same phase transition temperature, exhibit a greater enthalpy and a reduced cooperativity than do the lipids in cells or membranes. These results imply that lipid organization is identical in cells and isolated membranes, but different in the derived model membrane, probably due to the influence of membrane proteins and/or the presence of lipid bilayer compositional asymmetry in cells and membranes. We also demonstrate for the first time the existence of gel state polymorphism in a biological membrane.

Cholesterol uptake capacity of Acholeplasma laidlawii is affected by the composition and content of membrane glycolipids.

The composition of the cell membrane of 20 Acholeplasma laidlawii strains grown under identical conditions was studied and correlated with the capacity of these strains to incorporate cholesterol. Membranes of these strains had similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and contained the same lipid species, but the relative amounts of the major polar lipids varied. Statistical analyses revealed that the glycolipids, monoglucosyldiglyceride, and an unidentified glycolipid (glycolipid-X) succeeded in explaining 90% (R2 = 0.90) of the cholesterol uptake variations. The regression coefficients for both glycolipids were negative (P less than 0.001), indicating that the capacity of A. laidlawii strains for cholesterol incorporation is inversely proportional to the relative amounts of these glycolipids. Accordingly, an increased capacity for cholesterol incorporation was detected upon aging of A. laidlawii cells. The aged cells contained significantly smaller amounts of both monoglucosyldiglyceride and glycolipid-X, and a higher amount of diglucosyldiglyceride. The change in cholesterol incorporation as a response to glycolipid composition and content can be explained by the low solubility of cholesterol in glycolipids as well as by the induction by the sterol molecule of a nonlamellar phase state that will destabilize a membrane structure containing monoglucosyldiglyceride and glycolipid-X.

Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy.

The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde-fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction. The cytoplasmic membrane of sectioned Acholeplasma-cells dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature. Glutaraldehyde-fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their chlorophyll content.

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Gel-state disorder in the presence of methyl iso- and anteiso-branched-chain substituents.

The hydrocarbon chain orientational order parameters of membranes of Acholeplasma laidlawii B enriched with large quantities of a linear saturated, a methyl iso-branched, or a methyl anteiso-branched fatty acid plus small quantities of various isomeric monofluoropalmitic acid probes were determined via fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR) over a range of temperatures spanning the gel to liquid-crystalline phase transitions (determined by differential scanning calorimetry). Membrane orientational order profiles in the liquid-crystalline state were generally similar regardless of the particular fatty acyl structure, showing a region of relatively constant order preceding a region of progressive decline in order toward the methyl terminus of the acyl chain. In the gel state, the order profile of the linear saturated fatty acid enriched membranes was characteristically flat, with little head to tail gradation of order. In contrast, the methyl iso-branched and the methyl anteiso-branched enriched membranes exhibited a local disordering in the gel phase reflected in a very pronounced head to tail gradient of order, which remained at temperatures below the lipid phase transition. In addition, the methyl iso- and anteiso-branched fatty acid enriched membranes were overall more disordered than the membrane containing only linear saturated fatty acyl groups. Thus, at a constant value of reduced temperature below the lipid phase transition, overall order decreased in the progression 15:0 greater than 16:0i greater than 16:0ai, suggesting that these methyl-branched substituents lower the lipid phase transition by disrupting the gel phase lipid chain packing.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.

19F nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Orientational order in the presence of a series of positional isomers of cis-octadecenoic acid.

The 19F nuclear magnetic resonance (NMR) spectra of membranes of Acholeplasma laidlawii B enriched with one of a series of positional isomers of cis-octadecenoic acid plus small amounts of one of a number of isomers of monofluoropalmitic acid were interpreted in terms of an orientational order parameter (Smol). The variation of Smol with the position of the fluorine label in the liquid-crystalline state yielded an "order profile" with characteristics similar to those obtained via 2H NMR and which was relatively invariant regardless of the site of cis unsaturation. In the gel state, values of Smol approached the theoretical maximum, and the order profiles in the presence of different isomeric cis-octadecenoic acids displayed distinct dissimilarities. When the cis double bond was located proximal to the methyl terminus of the fatty acyl chain, a gradient of order across the bilayer was still evident in the gel state. When the cis double bond was located near the carbonyl head group, values of Smol were approximately equal at all chain positions. These observations were interpreted as indicating that in the gel state the stringency of packing restrictions is still subject to variation across the width of the bilayer. Relative overall orientational order among all isomers examined (specifically, 18:1c delta 4, delta 5, delta 6, delta 7, delta 8, delta 9, delta 10, delta 11, delta 12, delta 13, delta 14, and delta 15) varied directly as a function of proximity to the lipid gel to liquid-crystalline phase transition (Tm) (determined via differential scanning calorimetry) when compared at a constant temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cholesterol pools in Acholeplasma laidlawii membranes.

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.

Yet another improved silver staining method for the detection of proteins in polyacrylamide gels.

Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands.

The effects of ionizing radiation on the peroxide content of a pure polyunsaturated lipid dispersion and of lipids and membranes derived from Acholeplasma laidlawii.

Dispersions of a pure unsaturated phospholipid, dilinoleoylphosphatidyl choline, formed conjugated diene hydroperoxides when irradiated in air with 7 MeV electrons (150 Gy and 300 Gy). Peroxide formation was optimized when the dispersions were irradiated in air at 37 degrees C at a dose rate of 5 Gy/min. No significant loss of linoleic acid from the irradiated phospholipid dispersions was observed after doses of 150 or 300 Gy. Small amounts of thiobarbituric acid-reactive material were formed in irradiated unsaturated phospholipid dispersions. However, lipids or membranes isolated from 48 hour cultures of Acholeplasma laidlawii grown in media supplemented with either linoleic or linolenic acid did not appear to be peroxidized by irradiation under the same conditions.

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Effects of methyl-branch substitution and of trans unsaturation upon membrane acyl-chain orientational order.

The hydrocarbon-chain orientational order parameters of membranes of Acholeplasma laidlawii B enriched with straight-chain saturated, methyl iso-branched, methyl anteiso-branched, or trans-unsaturated fatty acids have been determined via fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR). A theoretical description of the 19F NMR spectral line shape is presented that permits the determination of the orientational order parameters associated with specifically monofluorinated palmitic acid probes biosynthetically incorporated into membrane glycerolipids. Membrane orientational order profiles determined by 19F NMR in the case of straight-chain saturated fatty acid enrichment were qualitatively similar to profiles obtained by 2H NMR. The methyl iso-branch and methyl anteiso-branch structural substituents induced a local ordering while the trans double bond substituent induced a local disordering evident from alterations to the character of the orientational order profile. These various effects could be understood in terms of an altered probability of the occurrence of rotational isomerization in the presence of particular substituents. At 37 degrees C the overall orientational order decreased in the progression eta-acyl greater than iso branched greater than anteiso branched greater than or equal to trans double bonded. The relative overall order was then a direct function of the relative proximity of the membrane lipids to their respective gel to liquid-crystalline phase transitions. When observed at Tm + 15 degrees C, where the different species of fatty acids could be considered to be in a comparable thermodynamic state, the overall order decreased in the progression anteiso branch greater than trans double bond greater than iso branch greater than eta-acyl.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorine-19 nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. A physical, biochemical, and biological evaluation of monofluoropalmitic acids as membrane probes.

Fluorine-19 nuclear magnetic resonance spectroscopy offers a number of unique advantages for studies of lipid fatty acyl chain order and dynamics in model and biological membranes. However, the germinal difluoromethylene fatty acids commonly employed as 19F membrane probes appear to appreciably perturb the organization of model membranes and biomembranes. We have thus synthesized a series of specifically labeled monofluoropalmitic acids and evaluated these as suitable membrane probes. Differential scanning calorimetric studies of aqueous dispersions of several bis-(monofluoropalmitoyl)phosphatidylcholines reveal that a fluorine substitution near the carbonyl group of palmitic acid has only a modest effect on the thermotropic phase behavior of these model membranes and that substitutions in the center or toward the methyl terminus are relatively nonperturbing. Moreover, all bis(monofluoropalmitoyl)phosphatidylcholines tested exhibit nearly ideal mixing in all proportions with dipalmitoylphosphatidylcholine. The thermotropic phase behavior of membranes of the simple, cell-wall-less prokaryote Acholeplasma laidlawii B is also not detectably altered by the presence of appreciable amounts of biosynthetically incorporated monofluoropalmitic acid. We also find that the biosynthetic incorporation of even large amounts of monofluoropalmitic acids into the membrane lipids of A. laidlawii B has no effect upon the growth and survival of this organism. The presence of exogenous monofluoropalmitic acids in the growth medium does not alter the polar head group composition or lipid/protein ratio of the A. laidlawii B membrane. In addition, all monofluoropalmitic acids tested are biosynthetically incorporated as well as palmitic acid itself and distribute relatively evenly between the various membrane glyco- and phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

19F-nuclear magnetic resonance study of glycerolipid fatty acyl chain order in Acholeplasma laidlawii B membranes.

The technique of 19F-nuclear magnetic resonance (19F-NMR) spectroscopy offers a number of advantages for studies of lipid fatty acyl chain orientation and dynamics in biomembranes. However, the geminal difluoromethylene fatty acid probes usually employed in such studies appreciably perturb the organization of lipid bilayers. We have thus synthesized a series of specifically monofluorinated palmitic acids and carried out biophysical, biochemical, and physiological studies establishing their suitability as relatively non-perturbing probes of lipid hydrocarbon chain organization. These 19F-NMR probes were then used to determine the fatty acyl chain order profiles of Acholeplasma laidlawii B membranes highly enriched in a variety of different exogenous fatty acids, particularly those containing a methyl branch or a trans-double bond.

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid.

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of greater than or equal to 90%. 2H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar 2H spectra. Below 25 degrees C the spectra exhibit a broad gel phase component which at 0 degrees C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30 degrees C) the A. laidlawii B membrane lipids are approximately 85-90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.