Synthesis of labeled phospholipids in high yield.
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Carbon Radioisotopes↗
Biomedical subjects
H Eibl
Publications and source records attributed to H Eibl.
Effects of lipids on acetylcholine receptor. Essential need of cholesterol for maintenance of agonist-induced state transitions in lipid vesicles.
The effects of lipids on the maintenance of characteristic functional properties of the acetylcholine receptor during the course of reconstitution into lipid vesicles were studied by following the kinetics of agonist-induced state transitions. The requirements for successful preservation of these properties could be dissected into two components: (a) adequate nature and concentration of lipids during detergent solubilization; (b) correct lipid environment during reincorporation into lipid vesicles by the cholate dialysis procedure. Optimal bulk lipid concentrations and lipid:cholate ratios for preserving state transitions during solubilization were studied by using both crude soybean lipids and pure synthetic phospholipids. The latter class of lipids was found to be unsuitable substitutes for the crude soybean lipids, irrespective of their polar head group and/or fatty acyl chain, even when detergent:lipid ratios as high as 1:1 (w/w) were employed. Addition of cholesteryl hemisuccinate was able to make up this deficiency, attaining preservation of acetylcholine receptor state transitions at cholate:steroid ratios of about 6:1 (w/w). The presence of steroid decreased the amount of protein solubilized. The correct choice of lipid type was also essential to the reincorporation step, and higher concentrations of lipid were required--about 20 mg/mL for soybean lipids. Pure phospholipids at similar concentrations, however, were unable to maintain the state transitions. Again, steroid (40-46% cholesteryl hemisuccinate/mol of total lipid) provided the adequate conditions at the reincorporation stage and enhanced the amount of protein reincorporated into the vesicles. A large (70-90%) percentage of the receptor was reincorporated with the correct vectorial sidedness. No specificity could be detected for the phospholipid polar head or alkyl chain in relation to any of these findings. The effect of the protein on the physical state of the lipids in the reconstituted vesicles was studied by diphenylhexatriene fluorescence depolarization. The results may be interpreted as a disordering of the acyl chains in the gel state and an ordering in the liquid-crystalline state in the presence of protein, accompanied by shifts in the transition temperatures of the pure phospholipids to lower values.
Animals↗
Lipid dependence of the membrane-bound D-lactate dehydrogenase of Escherichia coli.
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Cell Membrane↗
Hexagonal phases in phospholipids with saturated chains: phosphatidylethanolamines and phosphatidic acids.
The structure of phospholipids with saturated chains was investigated by differential scanning calorimetry and by X-ray diffraction. It is shown that both phosphatidylethanolamines and phosphatidic acids can exhibit a hexagonal phase at high temperature. The temperature of the transition to the hexagonal phase is dependent on chain length and sodium salt concentration. Increasing the chain length or the sodium salt concentration results in a decrease in the transition temperature. In addition to this transition at high temperature, a calorimetric transition at low temperature, a calorimetric transition at low temperature is detected in some phospholipids.
Calorimetry, Differential Scanning↗
Calorimetric studies on saturated mixed-chain lecithin-water systems. Nonequivalence of acyl chains in the thermotropic phase transition.
Aqueous dispersions of synthetic lecithins with different fatty acids in positions 1 and 2 of the glycerol molecule were studied by calorimetry. The data show that variation of the acyl chains in different positions of the glycerol backbone either have no influence upon or contribute 0.5 kcal/mol per CH2 segment to the phase transition enthalpy. Different molecular ordering of the mixed acyl chain lecithins in the bilayer is discussed in light of the results.
Calorimetry↗
A calorimetric study of the thermotropic behaviour of 1,2-dipentadecylmethylidene phospholipids.
Differential scanning calorimetry was used to study the thermotropic behaviour of 1,2-dipentadecylmethylidene phospholipids with various head groups. The structural variation in the glycerol backbone region leads to a strong restriction of conformational freedom for the first two methylene segments of the chains, so that dipentadecylmethylidene phospholipids show lower transition temperatures, lower enthalpies and lower cooperativity of the transition from the gel to the liquid crystalline phase. The extreme chemical stability of these lipids in the alkaline pH region enables investigations of phosphatidylethanolamine and phosphatidic acid dispersions at high pH values. Both phospholipids show a decrease in the transition temperature and in the transition enthalpy as they become singly and doubly charged, respectively. A complex behaviour of the transition enthalpy of doubly charged 1,2-dipentadecylmethylidene phosphatidic acid was observed when the NaCl concentration of the dispersion was increased.
Calorimetry, Differential Scanning↗
A new method for membrane reconstitution: fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature.
A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).
Animals↗
Acyl bis(monoacylglycero)phosphate, assumed to be a marker for lysosomes, is a major phospholipid of vaccinia virions.
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Chemical Phenomena↗
Preparation of phospholipids and their analogs by phospholipase D.
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Egg Yolk↗
Agonist-activated ionic channels in acetylcholine receptor reconstituted into planar lipid bilayers.
Planar lipid bilayers were formed with the mixed chain phospholipid 1-stearoyl-3-myristolglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Single-channel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonist-activated ionic channel seems, therefore, to be contained within the receptor monomer.
Acetylcholine↗
Characterization of intracellular and extracellular vaccinia virus variants: N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine interferes with cytoplasmic virus dissemination and release.
Infectious vaccinia virus can be purified from whole cells by experimentally induced lysis (intracellular virus) or from supernatant growth medium (extracellular virus). Extracellular virus and intracellular virus differed by buoyant density (1.237 versus 1.272 g/cm3), phospholipid content and composition, and polypeptide pattern. Differences in structural polypeptides on the virus surface could be detected by lactoperoxidase-catalyzed radioiodination or Brij treatment. Characteristic of extracellular virus was an additional polypeptide, with a molecular weight of 37,000 (37K), which represented 5 to 7% of the total particle protein. Antibodies to the 37K protein detected only some of the cell-associated particles late in normal infection. Upon treatment of infected cultures with N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine, a drug which prevents vaccinia virus release, no particle-associated 37K protein could be detected. In all other properties tested so far, except for a slight difference in phospholipid composition, the virus obtained in the presence of the drug resembled the normal intracellular virus. N1-Isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine prevented vesicularization of intracellular viral particles. Lack of vesicularization was accompanied by the absence of particle-associated 37K viral protein and seemed to correlate with an inhibition of virus dissemination to the cell periphery.
Animals↗
Influence of calcium on phosphatidylethanolamine. An investigation of the structure at high pH.
The influence of calcium on the structure of 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine was investiged at pH 8 and 12 by differential scanning calorimetry and by X-ray diffraction. At pH 12, where the amino group of phosphatidylethanolamine is partly deprotonated, two separate lamellar phases are observed at 60 degrees C in the presence of CaCl2; one phase is metastable and the other is present in a crystalline structure. At high temperature (90 degrees C), the small-angle diffraction lines indicate the existence of a hexagonal phase at pH 8. At pH 12 a hexagonal phase is detected only in the presence of calcium.
Calcium↗
Influence of calcium on phosphatidylglycerol. Two separate lamellar structures.
The influence of calcium on the structure of rac-1,2-ditetradecylglycerol-3-phosphoglycerol is investigated by differential scanning calorimetry and by X-ray diffraction. It is shown that 1 M CaCl2 (pH 4.6) induces two separate lamellar phases in the same sample at 20 degrees C. These two phases can be clearly distinguished by their X-ray diffraction patterns. The type of phase observed depends on the pretreatment of the sample. At high temperature (90 degrees C), when the hydrocarbon chains are in the disordered state, the small angle reflections are in the ratio 1:1/square root 3:1/2 and thus indicate the presence of a hexagonal phase.
Calcium↗
Charge-induced pretransition in phosphatidylethanolamine multilayers. The occurrence of ripple structures.
The influence of pH and ionic strength on the phase transition behavior of 1,2-dihexadecylphosphatidylethanolamine was studied calorimetrically. In the range of ionic strength from 0.75 to 1.5 M NaCl at pH larger than 13, where the amino group of the phosphatidylethanolamine is in the deprotonated state, resulting in one negative charge per lipid molecule, the calorimetric scan shows a pretransition before the main transition. Accompanying freeze-fracture electron microscopic studies on these preparations in the temperature range between the pre- and main transitions show a regular suface, the so-called ripple structure. These are comparable with the structures seen in phosphatidylcholine-water systems at temperatures between the pre- and main transition.
Calorimetry↗
Synthesis of glycerophospholipids.
Recent advances in the synthesis of phosphatidic acids, phosphatidylethanolamines and phosphatidylcholines are described. Methods for the synthesis of some alkylacyl and alk-1-enylacyl analogues of the common diacylglycerophospholipids are also discussed. In addition, synthetic routes are described, that lead to unusual phospholipids such as compounds containing the polar group at position 2 of the glycerol moiety, glycerophospholipids containing alkanolamines of different chain lengths, and glycolphospholipids. All of the common glycerophospholipids can be prepared without the use of protecting groups. Major advances in phospholipid synthesis involve the application of novel phosphorylating agents and the use of cyclic intermediates. Although phosphatidylserines and phosphatidylthreonines as well as phosphatidylglycerols and cardiolipins can be prepared by chemical synthesis, further systematic studies are required to work out procedures that lead to these compounds in high yields.
Cardiolipins↗
Lipid phase transition in planar bilayer membrane and its effect on carrier- and pore-mediated ion transport.
Using mixed-chain lipids, we have recorded cooling and heating curves of planar bilayer membranes while they passed the lipid phase transition range. With unmodified planar bilayers, spontaneous current fluctuations are observed near the lipid phase transition temperature (tc approximately 29 degrees C). This effect coincides with the expected and measured decrease in membrane capacitance. Carrier (valinomycin)-modified planar bilayers exhibit near tc an abrupt change from a high-conducting state above tc to the state of bare membrane conductance below tc. In contrast to this behavior, planar bilayers modified by pore-forming antibiotics (gramicidin A, alamethicin) do not show any peculiar effect at tc. However, at 22--23 degrees C a pronounced maximum in pore-induced conductance is seen. Whereas the gramicidin A pore abruptly stops stepwise fluctuations below approximately 16 degrees C, with alamethicin a few long-lasting pore and pore state fluctuations persist down to 10 degrees C. It is suggested that the carrier may freeze out into the membrane/water interface. The effects observed with pore-forming substances, on the other hand, are interpreted in terms of lateral phase separation into pure lipid and lipid/antibiotic domains.
Alamethicin↗
The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.
The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.
Calorimetry, Differential Scanning↗
Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values.
The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.
Calorimetry, Differential Scanning↗