Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

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.     

Membrane lipid metabolism in Acholeplasma laidlawii A EF 22. Influence of cholesterol and temperature shift-down on incorporation of fatty acids and synthesis of membrane lipid species

1. Membrane lipid metabolism in Acholeplasma laidlowii A EF 22 has been studied under different conditions by applying three different techniques for changing membrane viscosity: fatty acid and cholesterol supplementation and temperature changes. 2. The molar relationship between the two dominating membrane lipids, monoglucosyldiglyceride and diglucosyldiglyceride, is to a large extent determined by membrane viscosity properties. This is shown by the varying metabolic responses occurring during incorporation of different fatty acids with and without cholesterol and by temperature shift-down experiments. Higher viscosity in membranes stimulates synthesis of monoglucosyldiglyceride at the expense of diglucosyldiglyceride. Synthesis of phospho and phosphoglucolipids is affected as well. 3. Temperature shift-down from 37 degrees C to 17 degrees C results in an immediate synthesis of monoglucosyldiglyceride accompanied by an increased incorporation of unsaturated fatty acids into this lipid. Synthesis of the other membrane lipid species (containing more unsaturated fatty acids) lags behind temporarily. 4. Incorporation from an equimolar mixture of palmitic and oleic acids together with cholesterol yields greater amounts of oleic acid in membrane lipids than incorporation in the absence of cholesterol, indicating that incorporation is viscosity dependent. 5. Studies of precursor relationships reveal that all main lipids have an active turnover which differs depending on membrane composition and conditions. Furthermore, this turnover proceeds with different intra-lipid pools. 6. Isolated membranes contain no detectable lipolytic enzymes capable of hydrolyzing membrane phospho or glycolipids. It is suggested that lipid turnover is partly mediated by enzymatic interlipid conversions, thus not allowing intermediates to accumulate.     

Metabolic changes of membrane lipid composition in Acholeplasma laidlawii by hydrocarbons, alcohols, and detergents: arguments for effects on lipid packing

The packing of lipids into different aggregates, such as spheres, rods, or bilayers, is dependent on the hydrophobic volume, the hydrocarbon-water interfacial area, and the hydrocarbon chain length of the participating molecules, according to the self-assembly theory [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200]. The origin of the participating molecules should be of no importance with respect to their abilities to affect the above-mentioned parameters. In this investigation, Acholeplasma laidlawii, with a defined acyl chain composition of the membrane lipids, has been grown in the presence of three different classes of foreign molecules, known to partition into model and biological membranes. This results in an extensive metabolic alteration in the lipid polar head group composition, which is expressed as changes in the molar ratio between the lipids monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), forming reversed hexagonal and lamellar phases in excess water, respectively. The formation of nonlamellar phases by A. laidlawii lipids depends critically upon the MGDG concentration [Lindblom, G., Brentel, I., Sj?lund, M., Wikander, G., & Wieslander, A. (1986) Biochemistry (preceding paper in this issue)]. The foreign molecules tested belong to the following groups: nonpolar organic solvents, alcohols, and detergents. Their effects on the gel to liquid crystalline phase transition temperature (Tm), on the order parameter of the acyl chains, and on the phase equilibria between lamellar and nonlamellar liquid crystalline phases in lipid-water model systems are known in several instances.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of variations in growth temperature, fatty acid composition and cholesterol content on the lipid polar head-group composition of Acholeplasma laidlawii B membranes

We have systematically investigated the effect of variations in growth temperature, fatty acid composition and cholesterol content on the membrane lipid polar headgroup composition of Acholeplasma laidlawii B. Two important lipid compositional parameters have been determined from such an analysis. The first parameter studied was the ratio of the two major neutral glycolipids of this organism, monoglucosyldiacylglycerol (MGDG) and diglucosyldiacylglycerol (DGDG). As the former lipid prefers to exist in a reversed hexagonal phase at higher temperatures, with unsaturated fatty acyl chains or in the presence of cholesterol, the ratio of these two lipids reflects the phase state preference of the total A. laidlawii membrane lipids. Although we find that the MGDG/DGDG ratio is reduced in response to an increase in fatty acid unsaturation, increases in growth temperature or cholesterol content reduce this ratio only in cells enriched in a saturated but not an unsaturated fatty acid. The second parameter studied was the ratio of these neutral glycolipids to the only phosphatide in the A. laidlawii membrane, phosphatidylglycerol (PG); this parameter reflects the relative balance of uncharged and charged lipids in the membrane of this organism. We find that the MGDG + DGDG/PG ratio is lowest in cells enriched in the saturated fatty acid even though these cells already have the highest lipid bilayer surface charge density. Moreover, this ratio is not consistently related to growth temperature or changes in cholesterol levels, as expected. We therefore conclude that A. laidlawii strain B, apparently unlike strain A, does not possess coherent regulatory mechanisms for maintaining either the phase preference or the surface charge density of its membrane lipid constant in response to variations in growth temperature, fatty acid composition or cholesterol content.     

Qualitative and quantitative variations of membrane lipid species in Acholeplasma laidlawii A

In Acholeplasma laidlawii A, strain EF 22, the relative amounts of the membrane polar lipids vary as a consequence of different fatty acid supplements to the growth medium. The number of lipid species also varies; a new apolar monoglucolipid containing four fatty acid residues was present only when saturated fatty acids dominated in the growth medium. A new phosphoglucolipid, probably with a glycerophosphoryl-monoglucosyldiglyceride structure, was also found. The most pronounced variations occurred between the two dominating glucolipids, monoglucosyldiglyceride and diglucosyldiglyceride; the former being found in larger amounts when a saturated or a trans-unsaturated fatty acid was present in the medium. The amount of diglucosyldiglyceride decreased accordingly. A qualitative relationship between fatty acid properties and membrane lipid variations was established over a wide fatty acid concentration range. Incorporation of supplied fatty acids reached higher levels than normally found in other acholeplasmas. The ratio between membrane protein and lipids exhibited significant and coherent variations during growth and was to some extent influenced by the fatty acids in the medium. These changes indicate variations in lipid-protein organization in the membranes during growth.     

Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells

Razin, S. (University of Connecticut, Storrs), M. E. Tourtellotte, R. N. McElhaney, and J. D. Pollack. Influence of lipid components of Mycoplasma laidlawii membranes on osmotic fragility of cells. J. Bacteriol. 91:609-616. 1966.-Lipid composition of Mycoplasma laidlawii membranes could be significantly changed by variations in the growth medium. The effect of these changes on the osmotic fragility of the cells was studied. Cholesterol, incorporated into the membrane from the growth medium, had no significant effect on osmotic fragility. Carotenoids, synthesized by the cells from acetate, were likewise without effect. Unsaturated long-chain fatty acids increased markedly the resistance of M. laidlawii to osmotic lysis and promoted growth. The fatty acids of the growth medium were incorporated mainly into membrane phospholipids. The ratio between saturated and unsaturated fatty acids in membrane lipids depended on that of the growth medium.     

Lipid distribution in Acholeplasma laidlawii membrane. A study using the lactoperoxidase-mediated iodination.

The lactoperoxidase-mediated radioiodination has been applied to study the transbilayer distribution of phospho- and glycolipids in Acholeplasma laidlawii membranes. After radioiodination, about 5% of the 125I-iodine was found in membrane lipids. A comparison of the labeling intensities of the various lipid species between iodinated intact cells and isolated membranes revealed that the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride are located almost exclusively in the outer half of the bilayer, whereas the phospholipids phosphatidylglycerol and diphosphatidylglycerol as well as the phosphoglycolipids glycerophosphoryl-diglucosyldiglyceride and glycerophosphoryl-monoglucosyldiglyceride are almost equally distributed in the outer and inner halves of A. laidlawii membranes.     

A rapid,sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol

A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at $\text{m}\text{e}$ 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.     

Biochemical characterization of cholesterol-reducing Eubacterium.

We characterized two isolates of cholesterol-reducing Eubacterium by conducting conventional biochemical tests and by testing various sterols and glycerolipids as potential growth factors. In media containing cholesterol and plasmenylethanolamine, the tests for nitrate reduction, indole production, and gelatin and starch hydrolyses were negative, and no acid was produced from any of 22 carbohydrates. Both isolates hydrolyzed esculin to esculetin, indicating beta-glycosidase activity. In addition to plasmenylethanolamine, five other lipids which contain an alkenyl ether residue supported growth of Eubacterium strain 403 in a lecithin-cholesterol base medium. Of six steroids tested, cholesterol, cholest-4-en-3-one, cholest-4-en-3 beta-ol (allocholesterol), and androst-5-en-3 beta-ol-17-one supported growth of Eubacterium strain 403. All four steroids were reduced to the 3 beta-ol, 5 beta-H products. The delta 5 steroids cholest-5-en-3 alpha-ol (epicholesterol) and 22,23-bisnor-5-cholenic acid-3-beta-ol were not reduced and did not support growth of the Eubacterium strain.     

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.     

Effects of Growth Temperature on Fatty Acid and Alk-1-Enyl Group Compositions of Veillonella parvula and Megasphaera elsdenii Phospholipids

Veillonella parvula ATCC 10790, an anaerobic gram-negative coccus, contains diacyl and alk-1-enyl acyl (plasmalogen) forms of phosphatidylethanolamine and phosphatidylserine. We studied the effect of growth temperature on the lipid composition of this strain. There was a small increase in the phosphatidylethanolamine content but no change in the content of plasmalogens at the lower growth temperatures tested. The total acyl chains and the plasmalogen acyl chains contained between 73 and 80% mono-unsaturated fatty acids at all growth temperatures. The plasmalogen alk-1-enyl chains were significantly more unsaturated in cells grown at 30 and 25°C than in cells grown at 37°C. Differential scanning calorimetry of the hydrated phospholipids showed lower phase transition temperatures for the lipids from the cells grown at the lower temperatures. In Megasphaera elsdenii lipids, which are similar in composition to the lipids of V. parvula, the proportion of phosphatidylethanolamine also increased slightly at lower growth temperatures, with no significant change in the content of plasmalogens. M. elsdenii contained cyclopropane fatty acyl and alk-1-enyl chains in addition to the mono-unsaturated and saturated chains previously reported. As cells entered the stationary phase of growth at 30 and 42.5°C, there was a reciprocal increase in the proportion of cyclopropane acyl chains and decrease in the unsaturated moieties. The total proportion of cyclopropane and unsaturated acyl and alk-1-enyl chains was more than 65% at all growth temperatures studied, and there was no discernible increase in the sum of these moieties at the lower growth temperatures.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

Membrane Lipids of Mycoplasma hominis

Essentially all of the lipids of Mycoplasma hominis (200 mug/mg of cell protein) were found to be located in the cell membrane. Over one-half were neutral lipids incorporated from the growth medium and consisting of 43% free cholesterol, 19% esterified cholesterol, 23% triglycerides, 10% free fatty acids, and small amounts of di- and monoglycerides. The polar lipids accounting for about 40% of the total were synthesized by the organisms. Phosphatidylglycerol was the predominant lipid of this fraction. The minor components, tentatively identified as lysophosphatidylglycerol and phosphatidic acid, seem to represent breakdown products of phosphatidylglycerol. No glycolipids were detected. Being unable to synthesize long-chain fatty acids, M. hominis utilized the fatty acids of the growth medium for polar lipid synthesis, preferentially the saturated ones, so that the polar lipids had highly saturated hydrocarbon chains. It is proposed that the large take up of unsaturated neutral lipids and cholesterol from the medium offsets the marked condensing effect of the saturated polar lipids, although electron paramagnetic resonance spectrometry of spin-labeled fatty acids incorporated into the M. hominis membrane indicated that the lipid region is still more rigid than that of the Acholeplasma laidlawii membrane.     

Sterols of Agarum cribosum: desmosterol in a brown alga

The sterol composition of the cold water brown alga Agarum cribosum was determined by GC—MS. Six of the seven sterols found were identified as stigmata-5,(E)-24(28)-dien-3β-ol (fucosterol), 24-methylenecholest-5-en-3β-ol (24-methylenecholesterol), cholest-5-en-3β-ol (cholesterol), 3β-hydroxycholest-5-en-24-one (24-ketocholesterol), 24ξ-stigmasta-5,28-diene-3β,24-diol (saringosterol) and cholesta-5, 24-dien-3β-ol (desmosterol).     

Conversion of sterols and triterpenes by mycobacteria. II. Transformation of 7-oxygenated sterols into androstane derivatives via a 7-deoxygenation

The bioconversion of 7-oxygenated sterols by Mycobacterium aurum was studied in a preliminary investigation of the microbial conversion of wool wax. 7-Oxocholesterol was found to be transformed mainly into 3,17-dioxygenated androstane derivatives. 7 xi-Hydroxylated sterols were formed in an initial reduction step, and the C-7 hydroxyl group was then eliminated in a dehydration reaction. This was thought to take place during the isomerisation of cholest-4-en-3-one to cholest-5-en-3-one. Deuterium labelling experiments showed that this elimination proceeded faster for the C-7 alpha isomer, although it was not stereospecific. The C-7 alpha and C-7 beta-hydroxy isomers were weakly interconverted via the 7-oxo derivatives. Cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one all lost their side chains following a hydrogenation/dehydrogenation reaction. The resulting 3,17-dioxoandrostene or 3,17-androstadiene derivatives were mainly hydrogenated into 5 alpha-androstane-3,17-dione and 5 alpha-androstane-3 beta-ol-17-one. Elimination of the 3 beta-hydroxyl groups giving cholesta-3,5-dien-7-one, and subsequent microbial degradation of the side chain was not observed to any significant extent. The convergence of the bioconversion pathways of cholesterol and the 7-oxygenated cholesterols enabled crude, partially auto-oxidised cholesterol to be used as a substrate for the production of 3,17-dioxygenated androstane derivatives by M. aurum.     

Extraction, Characterization, and Cellular Localization of the Lipids of Staphylococcus aureus

Satisfactory extraction and assay procedures have been developed for the lipids of Staphylococcus aureus. The following lipids have been characterized in detail: the vitamin K(2), which is shown to exist as isoprenologues with side chains of 35, 40, and 45 carbon atoms; monoglucosyldiglyceride and diglucosyldiglyceride, which account for all the carbohydrate in the lipid extracts; the lysyl ester of phosphatidyl glycerol, phosphatidyl glycerol, and cardiolipin, which account for 98% of the phosphate in the lipid extract. The extraction procedure removes 98% of the total bacterial fatty acids. Acidification of the medium before harvest and refluxing in isopropanol are critical in the extraction procedure for the maximal recovery of lysyl-phosphatidyl glycerol and the glucolipids. The lipids have been shown to be a part of the same membrane as the respiratory pigments.     

Evidence for the presence of 7-hydroperoxycholest-5-en-3 beta-ol in oxidized human LDL.

Low density lipoprotein (LDL) cholesterol is known to be oxidized both in vitro and in vivo giving rise to oxygenated sterols. Conflicting results, however, have been reported concerning both the nature and the relative concentrations of these compounds in oxidized human LDL. We examined the extracts obtained from Cu(2+)-oxidized LDL. Thin layer chromatography analysis showed that the sterol mixture became more complex with reaction time. Analysis of the components by thin layer chromatography and mass spectrometry allowed to establish that 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha OOH and beta OOH) are largely prevalent among the oxysterols at early times of oxidation. These hydroperoxy derivatives have not been previously identified in oxidized LDL. The concentration of 7-hydroperoxycholest-5-en-3 beta-ol decreased with oxidation time with a concomitant increase of cholest-5-en-3 beta, 7 alpha-diol (7 alpha OH), cholest-5-en-3 beta, 7 beta-diol (7 beta OH), cholesta-3,5-dien-7-one (CD) and cholest-5-en-3 beta-ol-7-one (7CO). After 24 h of oxidation a minor component of the LDL sterols was cholestan-3 beta-ol-5,6-oxide (EP).     

Regulation of fatty acid composition in Escherichia coli: a proposed common mechanism for changes induced by ethanol, chaotropic agents, and a reduction of growth temperature.

Growth of Escherichia coli in the presence of ethanol and chaotropic salts resulted in the synthesis of lipids containing elevated levels of unsaturated fatty acids analogous to the effect of a reduction in growth temperature. Both ethanol and chaotropic agents acted at the level of fatty acid biosynthesis and altered lipid composition by decreasing the proportion of saturated acyl chains available for the synthesis of phospholipids. A reduction in temperature causes similar effects on fatty acid biosynthesis in vivo and in vitro. Ethanol, chaotropic salts, and a decrease in temperature all weaken hydrophobic interactions. Antichaotropic salts antagonized and effects of these treatments on fatty acid synthesis in vitro. These results are consistent with a common mechanism for the effects of chaotropic agents, temperature, and ethanol on fatty acid synthesis. The biosynthesis of saturated and unsaturated acyl chains may be regulated by the strength of hydrophobic interactions. Changes in the strength of hydrophobic interactions could alter enzyme structure, substrate structure, or the equilibrium between the soluble enzymes of fatty acid synthesis and their respective acyl carrier protein substrates.     

Membrane composition and virus susceptibility of Acholeplasma laidlawii.

The membrane composition of 11 strains of Acholeplasma laidlawii, including three strains persistently infected with mycoplasmaviruses MVL51, MVL2, and MVL3, was studied and correlated with mycoplasmavirus sensitivity. Membranes of the strains had similiar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, and all strains were inhibited by an antiserum produced against membranes from one of the strains. The amounts of integral membrane proteins solubilized by the nonionic detergent Tween 20 differed considerably. Therefore, characteristic crossed immunoelectrophoresis patterns were obtained for each strain. Strains persistently infected with MVL2 and MVL3 were notably different from the noninfected host. The ability to propagate any of the viruses was not correlated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis or crossed immunoelectrophoresis patterns. The persistently infected strains had a characteristic lipid composition. MVL51-resistant strains, including a resistant clone selected from a sensitive strain, were characterized by a large monoglucosyldiglyceride/diglucosyldiglyceride ratio and trace amounts of diphosphatidylglyceol (as opposed to the sensitive strains). Differences in lipid composition in A. laidlawii seem to affect the relationship between cells and viruses.