Role of cholesterol in the action of cereolysin on membranes.

The following evidence supports the concept that cholesterol in membranes is the receptor and target site for the cytolytic action of cereolysin. (i) Of the various phospholipids, gangliosides, and steroids tested, only cholesterol and closely related sterols (sitosterol and dihydrocholesterol) significantly inhibited the hemolytic activity of cereolysin. (ii) Acholeplasma laidlawii cells grown in the presence of cholesterol inhibited the hemolytic activity of cereolysin, but A. laidlawii grown in the absence of cholesterol did not. (iii) Incubation of A. laidlawii cells, grown in the absence of cholesterol, with a cholesterol-Tween 80 mixture reestablished the ability of the cells to bind cereolysin. (iv) Treatment of erythrocyte membranes and A. laidlawii cells containing cholesterol with cholesterol oxidase (EC 1.1.3.6, Brevibacterium species) abolished the ability of these membranes to bind cereolysin and inhibit the hemolytic activity of the toxin, (v) Cereolysin could bind to and alter the permeability of both right-side-out ghosts and inside-out vesicles prepared from human erythrocytes, in agreement with other data that cholesterol is present on both sides of the erythrocyte membrane, (vi) Cereolysin caused the release of [¹⁴C]glucose from liposomes containing cholesterol, and this release was dependent on the amount of cholesterol in the liposomes.     

Lipid and protein membrane components associated with cholesterol uptake by mycoplasmas

Membranes of Mycoplasma species take up 2–4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A². Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake.Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.     

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3–5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.     

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.     

Acholeplasma laidlawii PG8 ultramicroforms amplificate selectivelyrrnB nucleotide sequences

Mycoplasmas are frequent contaminants ofin vitro animal cell cultures. Despite a broad spectrum of modern methods, detection of mycoplasmas remains a serious problem. The situation is complicated by the fact that mycoplasmas may be presented in cell cultures or biological samples by viable but unculturable forms (ultramicroforms). We found that the DNA ofAcholeplasma laidlawii PG8 ultramicroforms showed selective amplification of therrnB nucleotide sequences while vegetative cells of the mycoplasma showed amplification both forrrnA andrrnB sequences. The role of enzyme deproteinization in PCR results was also shown. The results presented in this report indicate that the optimisation of primer sequences as well as PCR regime may be crucial steps in detection and differentiation of vegetative forms and ultramicroforms ofA. laidlawii.     

Osmotic behavior of liposomes of phosphatidylcholine and phosphatidylsulfocholine as a function of lipid concentration

A relationship between the initial rate of liposome swelling, d(1/A)/dt and the reciprocal of the lipid concentration of the liposomes has been derived and then utilized to describe the osmotic swelling behavior of serially diluted liposomes and chloroplasts exposed to hypertonic urea solutions. The slopes of plots of d(1/A)/dt vs. the reciprocal of the lipid concentration of liposomes were not affected by differences in the initial absorbance of phosphatidylcholine-sterol bilayers, and were used to assess the ability of sterols to reduce the initial rates of urea permeation through dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline state. Multilamellar liposomes and sonicated vesicles were prepared from dimyristoylphosphatidylsulfocholine (DMPSC), in which the quaternary ammonium group of choline is replaced by -S⁺(CH₃)₂. Cholesterol reduced the initial rate of osmotic urea penetration into liposomes and the rate of 6-carboxyfluorescein efflux from vesicles at 35°C. The effect of cholesterol on bilayers of phosphatidylsulfocholine and phosphatidylcholine was very similar, suggesting that no strict structural requirements need be met in the choline moiety for lecithin-cholesterol interaction. The sulfonium analog could thus functionally replace phosphatidylcholine in natural membranes.     

Heterogeneity in the physical state of the exterior and interior regions of mycoplasma membrane lipids

The electron paramagnetic resonance spectra of spin-labeled fatty acid in intact mycoplasma cells and isolated membrane preparations have been compared. With Mycoplasma hominis and Acholeplasma laidlawii preparations, the freedom of motion of the spin-label was higher in labeled intact cells than in labeled isolated membranes but no differences could be detected between the labeled intact cells and membranes isolated from the labeled intact cells. It is proposed that the higher freedom of motion of the spin-label in the intact cells is due to a higher fluidity of the outer half of the lipid bilayer of mycoplasma membranes rather than to alterations in the structure of the membrane upon isolation.     

Survival of Frozen Mycoplasmas

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.     

Characterization of the mycoplasma membrane proteins: V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50 % of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group.Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [¹²⁵I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core.Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021) has been modified for the separation of Acholeplasma laidlawii proteins.Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein.A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100.Exterior-facing membrane proteins were distinguished from the interiorfacing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinationg intact cells.A. laidlawii was also grown in the presence of NaH₂³²PO₄ and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Mycoplasmatales virus,MV-M1: Discovery inAcholeplasma modicum and preliminary characterization

A new virus, Mycoplasmatales virus-modicum 1 (MV-M1), was recovered from spontaneous plaques in lawns ofAcholeplasma modicum. Strain “mod” produced plaques onA. modicum strains but not on strains ofAcholeplasma laidlawii. Only MV-L3 of the three knownA. laidlawii viruses (MV-L1, MV-L2, and MV-L3) produced plaques onA. modicum. The MV-M1 virus was serologically distinct from the threeA. laidlawii viruses; filterable at 0.1 μm; partially sensitive to heat and Nonidet P-40; and chloroform labile. Spherical particles ranging from 105 to 160 nm were observed in electron micrographs of negatively stained preparations.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.     

Interaction of acidic liposomes with red blood cells: Induction of endocytosis and shedding of particles

Calcium ions induced tight binding of massive amounts of liposomes containing cardiolipin, phosphatidylethanolamine and phosphatidylcholine to erythrocytes. Initially, liposome-liposome fusion occurred and only subsequently the resulting large structures adhered to cells. Large clumps composed of liposomes and cells were formed. Upon prolonged incubation, the clumps were dissipated spontaneously and excess liposomes were released. A constant amount of phospholipid (15–25 nmol/10⁸ cells) were incorporated into the cell membranes. Upon disaggregation, the cells shed erythrocyte particles. The latter were isolated and shown to contain lipids from both cellular and liposomal origin. The particles lacked spectrins and contained variable amounts of band 3 content. Liposomes induced endocytosis in reticulocytes but not in mature erythrocytes. In most cases, the liposomes themselves were not engulfed by the cells and remained attached to their surface. The relevance of this phenomenon to delivery of liposome contents into cells is discussed.     

Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes

The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35 °C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching.Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity.The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.