Molecular order in acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids

The first application of deuterium magnetic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37°C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.     

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.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.     

Chloride fluxes across Acholeplasma laidlawii membranes.

Chloride fluxes across the cytoplasmic membrane of Acholeplasma laidlawii were studied by using the chloride sensitive fluorescent dye, 6-methoxy-N-(sulfopropyl)quinolinium. Chloride was found to penetrate the membrane passively. Chloride flux was dependent upon the transmembrane electric potential.     

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.     

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii: A 2H-NMR study

We have investigated by ²H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of Acholeplasma laidlawii B. This is the only ²-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristics condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the ²H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.     

Acholeplasma laidlawii membranes: an electron spin resonance study of the influence on molecular order of fatty acid composition and cholesterol.

The effects on membrane structure of including various fatty acids and cholesterol in the growth medium of Acholeplasma laidlawii were investigated by the use of spin-labeled fatty acids. Although the order-mobility parameters varied significantly at some temperatures with the nature of the fatty acid incorporated, the value measured at the growth temperature was only slightly affected by changes in the fatty acid composition of the membranes. The data confirm previous assertions that despite a high level of incorporation of fatty acids of various chain lengths or degree of unsaturation, A. laidlawii regulates its overall membrane fluidity within close limits at the growth temperature. Incorporation of cholesterol increased the degree of order at all temperatures. The coexistence of two lipid phases, one protein-dependent, could be observed in membranes. The order-mobility parameter of spin probes proved less satisfactory for the observation of a gel to liquid crystal transition of the membrane lipid than the partition parameter of a fatty acid spin probe. Order parameters measured by fatty acid spin probes were somewhat higher than those measured by the analogous ²H nmr probes.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

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)     

Proteins of bacterial membranes. Purification of soluble ATPase from Acholeplasma laidlawii

A purified preparation of ATPase (factor F1) from the Acholeplasma laidlawii was obtained. The purification procedure included extraction of the enzyme complex from the isolated membranes by ultrasonication, chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The specific activity of the ATPase was increased 30-fold as compared to the original activity. The Km value for ATP hydrolysis was 7,4 . 10(-4) M. ADP competitively inhibited the enzyme (Ki = 2,0 . 10(-4) M). Ouabain (2,5 . 10(-4) M) and dicyclohexylcarbodiimide (1,0 . 10(-4) M) did not inhibit the ATPase activity. The enzyme was activated by Mg2+, but was inhibited by a combination of Na+ and K+. The enzyme is cold-labile, but can be stabilized by storage in buffer solutions, containing methanol, glycerol or lecithin.     

Proteins of bacterial membranes. H+-adenosinetriphosphatase from Acholeplasma laidlawii cells

The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined.     

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)     

Adenylate energy charge in Acholeplasma laidlawii.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.