Acid-bath Phosphorylation in Heptane-extracted Chloroplasts

When spinach (Spinacia oleracea) chloroplast fragments were lyophilized and extracted with n-heptane, acid-bath phosphorylation was significantly reduced. Phosphorylation could be restored to these chloroplasts, if the extracted material (containing quinones, carotenoids, and other lipids) was added back to dry preparation before rehydration. The results reported suggest it was not quinones which were required for restoration of acid-bath phosphorylation but lipids. It is suggested that the lipids which were extracted by n-heptane serves as structural role in the thylakoid membrane.     

Polar lipid and isoprenoid quinone composition in the classification of Staphylococcus

Representatives of 13 species of Staphylococcus were examined using a small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids. Menaquinones were the only isoprenoid quinones found in the 77 test strains which were divided into three groups based upon the predominant isoprenologue detected: (i) S. hyicus subsp. hyicus, S. sciuri subsp. lentus and S. sciuri subsp. sciuri contained unsaturated menaquinones with six isoprene units; (ii) S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus subsp. chromogenes, S. intermedius, S. saprophyticus, S. simulans, S. warneri and S. xylosus contained unsaturated menaquinones with seven isoprene units and (iii) S. aureus contained unsaturated menaquinones with eight isoprene units and varying amounts of the corresponding lower isoprenologue. All of the organisms contained very similar polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol, beta-gentiobiosyl diacylglycerol and a number of glycolipids and phospholipids. One of the glycolipids was chromatographically indistinguishable from beta-gentiotriosyl diacylglycerol. Lysylphosphatidylglycerol was a major component in S. aureus and S. intermedius but was usually present in minor amounts in the coagulase-negative strains. The polar lipid data underline the homogeneity of the genus Staphylococcus and distinguish staphylococci from aerobic, Gram-positive cocci and from the phylogenetically related aerobic, endospore-forming bacteria. Menaquinone composition can also be used to separate staphylococci from other aerobic, Gram-positive cocci.     

Studies in lipid histochemistry

Summary Conditions for a selective extraction of nonpolar lipids from tissue sections with acetone were investigated using methods of lipid chromatography, histochemistry and quantitative determination of lipid phosphorus.Extraction of nonpolar lipids is selective when water (present either in acetone or in tissue sections) is completely excluded from the extraction procedure and the extraction is carried out at 0–4° C for 20–30 minutes. Under these conditions a negligible amount of polar lipids is extracted which cannot be demonstrated histochemically. A very small amount of nonpolar lipids remaining in sections cannot be demonstrated histochemically either.A method for the preparation of anhydrous acetone was recommended and an extraction procedure devised. This is to be applied in cases where nonpolar and polar lipids are to be distinguished and as an integral part of all types of phospholipid stains.When water is present during the extraction procedure (either in acetone or in tissue sections) significant extraction of all polar lipids occurs which is proportional to the content of water, to the length of extraction and to the temperature. Under these conditions the extraction of nonpolar lipids is somewhat slower.The significance of selective extraction with anhydrous acetone in histochemical analysis of lipids is discussed particularly with reference to lipids in atherosclerotic plaques.     

The determination of tocopherols and isoprenoid quinones in the grain and seedlings of wheat (Triticum vulgare)

1. A comparison is made of several procedures for the extraction of tocopherols and isoprenoid quinones from plant tissues. 2. Gradient-elution column chromatography on acid-washed alumina efficiently separates the isoprenoid quinones and tocopherols into groups that can then be assayed spectrophotometrically or, with the tocopherols, separated into their individual components and determined by gas-liquid chromatography. 3. This improved analytical procedure was used to study the distribution of the tocopherols and of ubiquinone in the ungerminated wheat grain.     

Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga,Chlorella

A comparison of three lipid solvent system indicated that they are not equivalent for the analysis of lipid classes in the green alga, Chlorella. Soxhlet extraction (methylene chloride/methanol, 3 h refulx) recovers more neutral lipid than the other methods but is equivalent to the room-temperature Bligh and Dyer (chloroform/methanol/water) extraction modified with phosphate buffer in glycolipid and polar lipid recovery. The Soxhlet method, however, gave a significantly lower recovery of many polyunsaturated fatty acids. The hexane/isopropanol method is selective for algal neutral lipids with poor recovery of membrane lipids (glyco- and polar lipids). Although this selectivity may have some useful applications, for biochemical studies of lipid synthesis in Chlorella, the modified Bligh and Dyer provides the most quantitative and reproducible recovery of all Chlorella lipid classes while minimizing artifacts due to the extraction procedure.     

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction.The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities.Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature.Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both ²H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated ω-d₃-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids.The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration.Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.     

LC-MS method for screening unknown microbial carotenoids and isoprenoid quinones

The structure of secondary metabolites from microorganisms provides a useful tool for microbial characterization and chemotaxonomic classification. Microbial isoprenoid quinones, for example, are well described and used to distinguish among photosynthetic microorganism groups. In addition, isoprenoid quinones can also be found, together with carotenoids, in non-photosynthetic microorganisms. The aim of the present study was to develop a LC-MS/MS method which can analyze and identify these microbial isoprenoids.Positive atmospheric pressure chemical ionization (APCI) together with collisionally induced dissociation was applied for generation of informative fragment spectra by mass spectrometry. Enhanced product ion (EPI) scan in a linear ion trap with information dependent data acquisition (IDA) enabled generation of MS fragment data even from minor isoprenoids. The developed liquid chromatography method enabled separation of isoprenoid patterns from their ester derivatives. Discovery and structural characterization of isoprenoid quinones and carotenoids were carried out by comparing characteristics of fragment spectra from unknown compounds with fragment spectra of a range of isoprenoid standard compounds and using published data. Throughout the study 17 microorganisms (e.g., Acremonium butyri, Arthrobacter spp., Brevibacterium linens, Bullera variabilis, Exophiala dermatitidis, Lecythophora hoffmannii, Panthoea agglomerans, Rhodotorula spp., Xanthophyllomyces dendrorhous) were screened and probable structures of isoprenoid quinones and carotenoids were suggested. The method lays some foundations on the analysis of yet unknown isoprenoids in microorganisms by using LCMS/MS techniques.     

Preparation of lipid-free tissue extracts for chromatographic determination of thyroid hormones and metabolites

We have developed a new method of preparing tissues for analysis of thyroid hormones and metabolites which eliminates troublesome lipids and proteins. Frozen tissue is homogenized by grinding with dry ice in a Waring blender, and the moist powder obtained is extracted with chloroform:methanol (2:1). In a modification of the Folch procedure for the total separation of lipids, the filtered extracts then are partitioned into polar and nonpolar layers by the addition of 0.05% aqueous calcium chloride. The upper phase contains the iodocompounds of interest as well as all salts and small polar molecules. The lipids remain dissolved in the lower phase after it is back-extracted with pure upper phase. The combined upper or aqueous methanol layers are lyophilized and the residue is taken up in methanol to yield a concentrated solution ready for analysis of iodocompounds. The greater clarity of the extract permits application of larger samples for two-dimensional paper chromatography than has been customary. For gradient analysis by reverse-phase ion-pair high-performance liquid chromatography (HPLC), the nitrogen-evaporated methanol extract is dissolved in the initial mobile phase, 0.1% H₃PO₄, for injection onto the column. Using these methods we have achieved the first reported separation of ¹²⁵I-labeled tissue iodothyronine metabolites by HPLC. The new method of extraction is of general applicability to any biological sample which might be analyzed in thyroid hormone metabolism research.     

Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15°C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16°C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15°C for cucumber (Cucumus sativus L.) fruit, at 14°C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15°C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

The influence of carotenoids on the oxidative stability and phase behaviour of plant polar lipids

Carotenoids and total neutral lipids from thylakoids of Nerium oleander were evaluated as antioxidants in liposomes prepared from soybean polar lipids. The extent of lipid oxidation was assessed from the formation of malondialdehyde and conjugated dienes after exposure of the liposomes to free radicals generated by ⁶⁰Co gamma radiolysis. The carotenoids incorporated into the bilayers were isolated from clones of oleander grown at 20° or 45°, growth conditions which are known to result in a difference in the thermal properties of the membrane lipids. The effect of carotenoids on the temperature of the phase transition of thylakoid polar lipids was also examined. The results showed that, in comparison with the effectiveness of a reference antioxidant, α-tocopherol, the carotenoids and total neutral lipids from thylakoids of oleander did not protect the soybean polar lipids from oxidation, nor did they influence the temperature of the phase transition of thylakoid polar lipids.     

Tetraether membrane lipids of <Emphasis Type="Italic">Candidatus “Aciduliprofundum boonei”</Emphasis>, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.     

Application of modified supercritical carbon dioxide extraction to microbial quinone analysis

Supercritical carbon dioxide (scCO₂) was applied to extract microbial quinones from activated sludge. Identification and analysis was then performed using high-performance liquid chromatography (HPLC) equipped with ultraviolet–visible (UV–Vis) detector and photodiode array detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scCO₂ were similar to that of the conventional method (organic solvent extraction).     

Functional diversity of tocochromanols in plants

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. They accumulate in photooxidative organisms, e.g. in some algae and in plants, where they localize to thylakoid membranes and plastoglobules of chloroplasts. Tocochromanols contain a polar chromanol head group with a long isoprenoid side chain. Depending on the nature of the isoprenoid chain, tocopherols (containing a phytyl chain) or tocotrienols (geranylgeranyl chain) can be distinguished in plants. The tocochromanol biosynthetic pathway has been studied in Arabidopsis and Synechocystis in recent years, and the respective mutants and genes were isolated. Mutant characterization revealed that tocopherol protects lipids in photosynthetic membranes and in seeds against oxidative stress. In addition to its antioxidant characteristics, tocopherol was shown be involved in non-antioxidant functions such as primary carbohydrate metabolism. A considerable proportion of tocopherol is synthesized from free phytol suggesting that excess amounts of phytol released from chlorophyll breakdown during stress or senescence might be deposited in the form of tocopherol in chloroplasts.     

Quinones and respiratory activity in Spoendonema epizoum

Conidia, gemmae, submerged mycelium and surface cultures of different ages of Sporendonema epizoum were analyzed for isoprenoid quinones. Respiratory activities of the fungus, metabolizing endogenous substrates and glucose, were determined. Q-9 was the only quinone detected; its concentration, lowest in conidia, increases during germination and then decreases in submerged culture. Q-9 concentrations in surface cultures are much higher than those in submerged cultures and are not related to culture age. Changes in Q-9 concentration and respiratory activity are roughly parallel; a peak is reached during gemma formation.     

Flexibacter elegans and Myxococcus fulvus: Aerobic gram-negative bacteria containing menaquinones as the only isoprenoid quinones

The aerobic Gram-negative gliding bacteria, Flexibacter elegans (Cytophagales) and Myxococcus fulvus (Myxobacterales) contain exclusively menaquinones as isoprenoid quinones. Each strain produces 3 different menaquinones. The menaqinone content of Flexibacter elegans was 1.5 μmol/g dry cells, with MK-7 as the main compound (>99%). MK-7 was identified by mass and n.m.r. spectroscopy. Myxococcus fulvus contained 2.1 μmol menaquinones per g dry mass, with more than 90% being MK-8.     

Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii.

Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins. The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains. Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively. No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids. Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates. The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins. Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114. This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.