B. De Kruyff

The effect of cholesterol and epicholesterol incorporation on the permeability and on the phase transition of intact Acholeplasma laidlawii cell membranes and derived liposomes

1. n1. The effect of incorporated cholesterol and epicholesterol upon the glycerol and erythritol permeability through the membrane of Acholeplasma laidlawii (previously denoted as Mycoplasma laidlawii) is studied. Both sterols, when present in the growth medium, are incorporated to the same extent in the A. laidlawii membrane. Only the cholesterol-containing A. laidlawii membrane shows a reduced permeability towards glycerol and erythritol as compared to the sterol-free cells. The 3α-hydroxy isomer, epicholesterol, does not affect the membrane permeability. n n2. n2. Liposomes prepared from lipids isolated from cells grown on cholesterol-rich media also show a reduced glycerol and erythritol permeability as compared to liposomes prepared from lipids isolated from sterol-free control cells. This permeability-lowering effect can be correlated with a condensing effect of cholesterol upon a monolayer of total A. laidlawii lipids. n n3. n3. The phase transitions occurring in membranes and extracted lipids of A. laidlawii have been studied by differential scanning calorimetry. Incorporated cholesterol causes a considerable reduction of the energy content of this phase transition. This reduction in energy is the same for the intact A. laidlawii cell membrane as for the liposomal bilayer system of the extracted lipids dispersed in water. n n4. n4. Using the synthetic lecithin 1-oleoyl-2-stearoyl-sn-glycero-3-phosphorylcholine the same phenomenon is observed. 32 mole % cholesterol completely eliminated the phase transition of the lecithin. Epicholesterol 5α-androstan-3β-ol and cholest-4,6-dien-3-one are unable to show such an effect, also suggesting the importance of the 3β-OH group of the sterol molecule for the specific sterol-lecithin interaction.

The effect of the polar headgroup on the lipid-cholesterol interaction: a monolayer and differential scanning calorimetry study.

Since the sterol 3β-OH group is essential for the lipid-sterol interaction, the interaction of cholesterol with natural phospholipids and glycolipids and synthetic phospholipids and analogs, differing in the polar moiety was studied in monolayers and liposomes. In monolayers, the interaction was measured as a reduction in the mean molecular area (condensing effect). In liposomes by differential scanning calorimetry as a reduction in the energy content of the crystalline → liquid-crystalline phase transition (liquefying effect). n nThe presence of the oxygen atoms of the acyl ester linkages and of the oxygen atoms connecting phosphorus and carbon are not essential for the lipid-sterol interaction as determined by the above methods. Measurements with monoglucosyldiglyceride and diglucosyldiglyceride even reveal that the phosphorus and choline moiety are not required for the interaction. This indicates that it is unlikely that a specific binding of the sterol-OH group with any polar part of the lipid molecule is essential for the condensing or liquefying effect. The need for a 3β-OH group can neither be explained by a cooperative effect of the C19 methyl group and the 3β-OH group in the lipid-sterol interaction since 19-norcholesterol shows the same effect as cholesterol. n nThe effect of cholesterol upon the crystalline → liquid-crystalline phase transitions in a codispersion of (1,2-dioleoyl)lecithin and (1,2-distearoyl)lecithin was studied by differential scanning calorimetry. At low concentrations (less than 25 mole%) cholesterol preferentially associates with (1,2-dioleoyl)lecithin. At higher concentrations cholesterol interacts with (1,2-distearoyl)lecithin as well. This indicates that when cholesterol is present in membrane with lipids in both the crystalline and liquid-crystalline state, cholesterol preferentially interacts with these lipids which are in the liquid-crystalline state.

Non-random distribution of cholesterol in phosphatidyl-choline bilayers

Abstract 1. 1. The effect of cholesterol upon the phase transition occurring in various mixtures of synthetic disaturated phosphatidylcholines with fatty acid constituents of 12, 14, 16, and 18 carbon atoms was measured by differential scanning calorimetry. 2. 2. Mixtures which differ 2 carbon atoms show cocrystallisation of the paraffin chains. In these mixtures cholesterol interacts randomly with the various phosphatidylcholine species. 3. 3. Mixtures which differ 4 or more carbon atoms show monotectic behaviour (phase separation). In these mixtures cholesterol interacts preferentially with the phosphatidylcholine species with the lowest transition temperature. This results in a non-random distribution of cholesterol at temperatures at which phase separation occurs. The implications of these findings for cholesterol-containing biological membranes are discussed.

Studies on the biological properties of polyene antibiotics: Comparison of other polyenes with filipin in their ability to interact specifically with sterol

1. n1. The interaction fo the five polyene antibiotics filipin, etruscomycin, pimaricin, nystatin and amphotericin B with sterol, primarily free, liposomal and membrane bound cholesterol has been examined. Each of these antibiotics has a characteristic ultraviolet absorption spectrum in aqueous or organic solvents with three or four ultraviolet absorption maxima. n nAddition of free cholesterol to aqueous solutions of these antibiotics results in a change of the ratio of the ultraviolet absorbance maxima. The order of effectiveness of interaction with cholesterol as judged by this criterion was filipin, amphotericin B, etruscomycin and pimaricin. n n2. n2. The same alteration in ultraviolet absorption spectra of filipin etruscomycin and amphotericin B observed with addition of free cholesterol to aqueous solutions of these antibiotics also occurs upon addition of these antibiotics to liposomal, erythrocyte or Acholeplasma membrane bound cholesterol. No spectral change was found in membranes devoid of cholesterol. This spectra alteration of the antibiotic was accompanied by a binding of the antibiotic to the membrane. Both nystatin and pimaricin showed little change in spectrum with sterol in these systems, either the artificial or natural membranes which contained cholesterol. n n3. n3. The structural requirements of the sterol for the spectral change with filipin, etruscomycin and amphotericin B include a planar sterol nucleus, and intact side chain at C-17 and 3β-hydroxyl group. The spectral change was not affected by the pH except in the case of amphotericin B. n n4. n4. Measurements by differential scanning calorimetry of the effect of these polyene antibiotics on the phase transition of lecithin and lecithin-cholesterol showed that all polyenes can reduce the ecithin cholesterol interaction.

Cholesterol in mycoplasm membranes. Correlation of enzymic and transport activities with physical state of lipids in membranes of Mycoplasma mycoides var. capri adapted to grow with low cholesterol concentrations

Abstract 1. 1. Membranes of a Mycoplasma mycoides var. capri strain adapted to grow with very low concentrations of cholesterol undergo a reversible phase transition detectable by differential-scanning calorimetry and fluorescence measurements. No phase transition could be detected in membranes of the cholesterol-containing native strain. 2. 2. EPR spectrometry, as well as fluorescence measurements, demonstrated that at temperatures above phase transition the membranes of the adapted strain were more fluid than membranes of the native strain, although the former contained a higher percentage of saturated fatty acids. 3. 3. Arrhenius plots of the ATPase activity of the adapted strain membranes showed breaks at temperatures corresponding to those of the phase transition of membrane lipids. The temperature of the break depended on the fatty acid composition of membrane lipids and on the age of the culture. No break could be detected in Arrhenius plots of the ATPase activity of the native strain. 4. 4. No break could be demonstrated in the Arrhenius plot of α-methylgucoside uptake by the adapted strain. Yet the activation energy of the uptake process by the adapted strain was much higher than that of the native strain. On the other hand, activation energies of α-methylglucoside phosphorylation were the same for membranes of both strains. However, Arrhenius plots of α-methylglucoside efflux from cells of the adapted strain showed breaks at temperatures corresponding to those of the lipid phase transition. 5. 5. It is concluded that cholesterol, by preventing the crystallization of membrane lipids maintains them in a state of fluidity essential for the optimal manifestation of several key activities of the membrane.

The effect of different fatty acid and sterol composition on the erythritol flux through the cell membrane of Acholeplasma laidlawii

Abstract 1. 1. A technique is described whereby the [ 14 C]erythritol equilibrium flux through the Acheloplasma laidlawii cell membrane is measured. Cells are separated from the surrounding medium by filtration through membrane filters. it is essential that the A. laidlawii cells are suspended in isotonic NaCl. Cells suspended in sucrose media are fragile and easily damaged by applying a mechanical force such as ultrafiltration under negative pressure. 2. 2. The [ 14 C]erythritol equilibrium flux for cells grown on different fatty acids is in the order of cells grown on linoleic acid > oleic acid > elaidic acid steric acid. 3. 3. It is indicated that the permeation of glycerol through the A. laidlawii cell membrane at temperatures in the transition of the membrane lipids predominantly occurs via those parts of the membrane where the fatty acid chains of the membrane lipids are still in the liquid crystalline state. 4. 4. The a. laidlawii cells remain intact upon cooling below the temperature of the phase transition of the membrane lipids. However, at temperatures below the phase transition of the membrane lipids A. laidlawii cells are damaged when mechanical forces such as ultrafiltration or osmotic swelling are applied. 5. 5. The incorporation of cholesterol into the A. laidlawii cell membrane decreases the [ 14 C]erythritol flux for cells grown on linoleic, oleic, elaidic and stearic acid. 6. 6. Cholesterol, epicholesterol, cholestanol, epicholestanol, ergosterol, stigmasterol and coprostanol, when present in the growth medium, are all incorporated in the A. laidlawii cell membrane to about the same extent. 7. 7. The 3α-hydroxy isomers epicholesterol and epicholestanol and the sterol with a cis -structured steroid nucleus, coprostanol, do not significantly alter the [ 14 C]-erythritol flux through the membrane. 8. 8. The 3β-hydroxy sterols with a flat steroid nucleus cholesterol, cholestanol and ergosterol all decrease the [ 14 C]erythritol flux. Stigmasterol does not influence the [ 14 C]erythritol flux to a marked extend. 9. 9. For the reduction in permeability of the A. laidlawii cell membrane a 3β-hydroxyl group and a planar configuration of the sterol molecule are essential requirements. This is in good agreement with similar studies using liposomes and monolayers. 10. 10. It is demonstrated that the liquefying effect of cholesterol on the membrane lipids of A. laidlawii can be very important in maintaining proper membrane functioning in growing cells. A 3β-hydroxyl group on the sterol molecule is an absolute requirement for this liquefying effect.

The intramitochondrial distribution of some enzymes involved in the biosynthesis of rat-liver phospholipids

Abstract 1. 1. The conversions of 1- and 2-acyl-sn-glycero-3-phosphorylcholine into 3-sw-phosphatidylcholine, of i-acyl-sw-glycero-3-phosphate into i,2-diacyl-sn-glycero-3-phosphate, and of 1,2 diacyl-sw-glycerols into 3-sw-phosphatidylcholine and triacyl-glycerols were stud microsomes, mitochondria and submitochondrial fractions from rat liver and compared with the distribution of glucose-6-phosphatase, rotenone-insensitive NADH-cytochrome c reductase, cytochrome oxidase and succinate dehydrogenase. 2. 2. The information obtained strongly indicates that mitochondria do contain the acyltransferases involved in the conversion of both isomeric mono-acyl-sn-glycero-3-phosphorylcholines into 3-sw-phosphatidylcholine and of acyl-sn-glycero-3-phosphate into 1,2-diacyl-sn-glycero-3-phosphate. These processes were found to occur predominantly in the outer membrane fraction. 3. 3. Mitochondria and mitochondrial subfractions did not catalyze significantly the conversion of 1,2-diacyl-sn-glycerols with [14C]CDP-choline into 3-sn-phosphatidylcholine or that of 1,2-diacyl-sn-glycerols with [14C]fatty acids into triacylglycerols, this in strong contrast with microsomes. The specific activities of these enzymes are actually much lower than that of glucose-6-phosphatase in the submitochondrial fractions. 4. 4. Simultaneous incubation of sn-[2-3H]glycero-3-phosphate and [14C]fatty acids with submitochondrial fractions demonstrated that sn-glycero-3-phosphate was converted, in the presence of outer membranes, into 1,2-diacyl-sn-glycero-3-phosphate. However, the small3H/14C ratios observed in 3-sn-phosphatidylcholine and phosphatidylethanolamine allow the conclusion that, at least under these conditions, the uptake of fatty acids into these phospholipids proceeds mainly via acylation of their mono-acyl derivatives.

Calorimetric and freeze-etch study of the influence of Mg2+ on the thermotropic behaviour of phosphatidylglycerol

In the presence of Mg2+ ions phosphatidylglycerol shows supercooling which leads to the formation of a metastable gel phase. This contrasts with the behaviour of this negatively charged phospholipid in the presence of Ca2+ ions (Biochim. Biophys. Acta 39. (1974)432)9 It is demonstrated that the heat content of this phospholipid is dependent on the ionic environment.

Outside-inside distribution and translocation of lysophosphatidylcholine in phosphatidylcholine vesicles as determined by 13C-NMR using (N-13CH3)-enriched lipids

Abstract 1. 1. The outside-inside distribution of palmitoyl lysophosphatidylcholine and dioleoyl phosphatidylcholine in mixed sonicated vesicles is measured with (N- 13 CH 3 )-labelled lipids using 13 C NMR and Dy 3+ as an impermeable shift reagent. 2. 2. Palmitoyl lysophosphatidylcholine is preferentially localised in the outside layer of the vesicle membrane. Incorporation of cholesterol in the vesicle diminishes the extent of lysophosphatidylcholine asymmetry. 3. 3. Palmitoyl lysophosphatidylcholine added to dioleoyl phosphatidylcholine vesicles is incorporated in the outer monolayer of the vesicle. Even after 40 h less than 2% of the lysophosphatidylcholine could be detected in the inner monolayer. Since in the cosonicated vesicles 17% of the lysophosphatidylcholine is present in the inner monolayer it can be concluded that the transmembrane movement of lysophosphatidylcholine across the lipid bilayer of these vesicles is an extremely slow process.

Utilization of diacylglycerol species by cholinephospho-transferase, ethanolaminephosphotransferase and diacylglycerol acyltransferase in rat liver microsomes

Abstract 1. 1. Several mixtures of 3H- and 14C-labelled diacylglycerols at varying degrees of unsaturation were incubated in the simultaneous presence of CDP-choline and CDP-ethanolamine with rat-liver microsomes. The results obtained allowed the conclusion that both choline- and ethanolaminephosphotransferase show no specificity with respect to the nature of the diacylglycerol molecule, at least for the species tested so far. However, it was found that, per mg microsomal protein, the diacylglycerol molecules were incorporated to a greater extent into phosphatidylcholine than into phosphatidylethanolamine. 2. 2. The synthesis of triacylglycerols by rat-liver microsomes was measured in the presence of various mixtures of labelled 1,2-diacyl-sn and sn-glycerols, containing four, two and one double bond, and several nonlabelled acyl-CoA esters. The rate of synthesis of triacylglycerols was the same regardless of the fatty acid composition of the diacylglycerol. 3. 3. Nonlabelled i -palmitoyl-2-oleoyl-sn-diacylglycerol was incubated in the presence of rat-liver microsomes with various mixtures of 3H- and 14C-labelled acyl-CoA esters. The diacylglycerol acyltransferase exhibited some specificity with respect to the acyl-CoA ester. Linoleic acid appeared to be a somewhat superior substrate compared to palmitic and oleic acids. 4. 4. Acyl-CoA esters, exogenous or synthesized in situ, were found to inhibit drastically the conversion of i -palmitoyl-2-[3H]oleoyl-sn-glycerol with [14C]CDP-choline into phosphatidylcholine in the presence of microsomes, whereas the triacylglycerol synthesis proceeded at a normal level. Acyl-CoA esters did not affect the rate of phosphatidylethanolamine synthesis.