Release of Calcium from Suspension-Cultured Glycine max Cells by Chitosan, Other Polycations, and Polyamines in Relation to Effects on Membrane Permeability

Treatment with chitosan of suspension-cultured Glycine max cells labeled with ⁴⁵Ca²⁺ caused a rapid release of calcium, which was complete much earlier than the chitosan-induced leakage of intracellular electrolytes and probably reflects calcium loss primarily from the cell wall and/or plasma membrane. A linear correlation was found between calcium release from chitosan-treated whole cells or isolated cell walls and the amount of chitosan bound. Other polycations (poly-l-lysine, histone, DEAE-dextran, and protamine sulfate), low molecular weight polyamines (spermine, spermidine, and putrescine) and polyanions (polygalacturonate and poly-l-aspartate, which act as chelating agents) also released calcium from whole cells and isolated cell walls; however, only the polycations increased membrane permeability. Poly-l-lysines of differing molecular weight showed a similar ability to release calcium, but their effect on membrane permeability increased with increasing molecular weight. The results suggest that the effect of polycations on permeability is not the direct result of calcium displacement from the cell surface but is probably due to cross-linking of surface components. The order of effectiveness of inorganic cations in displacing calcium from whole cells and isolated cell walls was Ca²⁺, Ba²⁺, Sr²⁺ > Mg²⁺ > K⁺, Na⁺.     

The enzymic composition of the isolated cell wall and plasma membrane of baker''s yeast

A study was made of the enzyme content of the isolated cell walls and of a plasma-membrane preparation obtained by centrifugation after enzymic digestion of the cell walls of baker's yeast. The isolated cell walls showed no hexokinase, alkaline phosphatase, esterase or NADH oxidase activity. It was concluded that these enzymes exist only in the interior of the cell. Further, only a negligible activity of deamidase was detectable in the cell walls. Noticeable amounts of saccharase, phosphatases hydrolysing p-nitrophenyl phosphate, ATP, ADP, thiamin pyrophosphate and PP(i), with optimum activity at pH3-4, and an activity of Mg(2+)-dependent adenosine triphosphatase at neutral pH, were found in the isolated cell walls. During enzymic digestion, the other activities appearing in the cell walls were mostly released into the medium, but the bulk of the Mg(2+)-dependent adenosine triphosphatase remained in the plasma-membrane preparation. Accordingly, it may be assumed that the enzymes released into the medium during digestion are located in the cell wall outside the plasma membrane, whereas the Mg(2+)-dependent adenosine triphosphatase is an enzyme of the plasma membrane. This enzyme differs from the phosphatases with pH optima in the range pH3-4 with regard to location, pH optimum, substrate specificity and different requirement of activators.     

Copper reduction by yeast cell wall materials and its role on copper uptake in Debaryomyces hansenii

Copper binding reducing activities of cell wall materials (CWM) prepared from cells of the yeast Debaryomyces hamsenii were examined. When CWM was treated with copper sulfate (0.1 mM CuSO₄), the copper was partially reduced from Cu (II) to Cu (I) and bound to CWM (below 10 nmol per mg dry wt.). The bound copper was mostly in the fraction of mannan-protein. Both copper-binding ability and protein content decreased with protease treatments. Mannan-protein prepared from CWM bound more copper than mannan did. This suggests that Cu (II) bound to the protein portion in CWM and was reduced to Cu (I). The optimum pH of copper reduction by CWM was about 5.0. The amount of copper bound to CWM increased with reducing agents and decreased with oxidizing agents. On the other hand, the copper uptake by yeast whole cells and spheroplasts was also stimulated by reducing agents, but inhibited by oxidizing agents. Furthermore, copper uptake by spheroplasts was stimulated in the presence of CWM. The optimum pH of copper uptake coincided with that of copper reducing activity. These results suggest that yeast cell wall not only supplies copper binding but also reduces copper, and the reduced copper is transported into yeast cells. The yeast cells may have copper-reducing proteins in the cell wall.     

Spin label study of erythrocyte deformability. III. Further characterizations of electron spin resonance spectral change in shear flow

It is demonstrated that the change in the spin label ESR spectrum induced by shear flow reflects the whole cell deformation as a function of the cell surface area-to-volume ratio (s/v), the morphology and intracellular viscosity. Since the effect of the change in the membrane mechanical property on the ESR spectrum has been described previously, the spin label ESR spectrum is now shown to contain full information concerning the whole cell deformability which is determined by the major intrinsic and extrinsic properties of the red blood cells. The result of microphotographic observations shows also that the cells in the flow are elongated and aligned approximately along the flow direction to an increasing extent as the cells flow near the surface of the flat channel walls. Thus, the entire observation confirms the view that the ESR spectral difference-shear rate profile is closely related to the elongation ratio shear rate characteristics obtained by other (optical) methods.     

Biosorption of cadmium,copper and lead by isolated mother cell walls and whole cells of Chlorella fusca

Using a new method for the isolation of released mother cell walls of Chlorella fusca, the biosorption of cadmium, copper and lead by purified cell wall isolates and whole cell suspensions was comparatively characterized. In all cases whole cells accumulated more metal ions than isolated cell walls. Both the Langmuir and Freundlich isotherm models were suitable for describing the short-term adsorption of cadmium, copper and lead by cell walls and the cadmium and copper adsorption by whole cells. However, neither model could sufficiently explain the lead accumulation by whole cells. The feasibility of a practical use of whole cells or isolated cell walls as biosorbents is discussed.     

Cell wall-bound trehalase in cultured cells of Japanese morning-glory

Occurrence and distribution of trehalase were examined in cytoplasmic and cell wall fractions of cultured cells of morning-glory, soybean and persimmon. Also, some enzymatic properties and solubilization of the enzyme from cell walls were examined. Trehalase was present in both fractions of morning-glory and persimmon cells while trehalase was present only in the cytoplasmic fraction of soybean cells. Morning-glory trehalases in both fractions showed the same optimum pH at 5.5, while persimmon trehalases in both fractions showed the same optimum pH at 6.0. Soybean enzyme in the cytoplasmic fraction showed two optimum activities at 4.0 and 6.5. Morning-glory cell wall bound trehalase was solubilized with various IM salts at about 70 to 75%. Also, the enzyme was solubilized with various buffers and the solubilization ratio increased with increasing in pH of a same series buffer. After multiple extractions with IM NaCl, about 15% of the original trehalase activity still remained in cell walls. On the other hand, Triton X-100 and the substrate, trehalose, at the various concentrations did not release trehalase from cell walls. Invertase and cellobiase solubilized from morning-glory cell walls were re-adsorbed to the cell walls. However, readsorption of trehalase to cell walls has not yet been attained. Based on these results, physiological roles of plant cell wall-bound trehalase were discussed.     

Conformational changes,plasma membrane penetration,and infection by human rhinovirus type 2: role of receptors and low pH

Human rhinovirus type 2 (HRV2) is internalized by members of the low-density lipoprotein (LDL) receptor (LDLR) family. It then progresses into late endosomes, where it undergoes conversion from D- to C-antigenicity at pH < 5.6. Upon uncoating, the viral RNA is transferred into the cytoplasm across the endsosomal membrane. However, C-antigenic particles fail to attach to LDLR; this raised the question of whether the virus remains attached to the receptors and is carried to late compartments or rather falls off at the higher pH in early endosomes. We therefore determined the pH dependence of virus-receptor dissociation and virus conversion to C-antigen under conditions preventing endocytosis. (35)S-HRV2 was attached to HeLa cells at 4 degrees C and incubated in buffers of pH 7.4 to 5.0; levels of native virus and C-antigenic particles remaining cell associated or having been released into the medium were determined by immunoprecipitation. At pH 6.0, HRV2 was readily released from plasma membrane receptors in its native form, whereas at pH < or = 5.4, it was entirely converted to C-antigen, which, however, only dissociated from the surface upon prolonged incubation. The antigenic conversion occurred at the same pH regardless of whether HRV2 was free in solution or bound to its receptors. These data suggest that, in vivo, the virus is no longer bound to its receptors when the antigenic conversion and uncoating occur in more acidic late endosomes. When virus was bound to HeLa cells at 4 degrees C, converted into C-antigen by exposure to pH 5.3, and subsequently warmed to 34 degrees C in the presence of bafilomycin (to prevent endosomal uncoating), viral de novo synthesis was detected. This study demonstrates for the first time that a nonenveloped virus such as HRV2 can infect from the plasma membrane when artificially exposed to low pH. This implies that the viral RNA can gain access to the cytoplasm from the plasma membrane.     

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.     

Fusion of Semliki forest virus with the plasma membrane can be induced by low pH

When BHK-21 cells with Semliki Forest virus (SFV) bound at the plasma membrane are briefly treated with low pH medium (pH 5-6), fusion between the viral membrane and the plasma membrane occurs, releasing the viral nucleocapsid into the cytoplasm. The fusion reaction resembles that described previously for Sendai virus but with one fundamental difference; it is strictly dependent on low pH. The fusion reaction is highly efficient. Up to 86% of bound viruses fuse, and 6 X 10(6) virus spike proteins can be inserted into the plasma membrane of each cell. The process is very rapid (full activity is observed after 5 s) and it occurs over a wide temperature range and equally well with all five cell lines tested (BHK-21, HeLa B, HeLa suspension, Raji, and 3T3). Low pH-induced fusion of the virus at the plasma membrane can lead to infection of susceptible cells. The artificial nature of this infection pathway is, however, demonstrated by the facts that infection through the plasma membrane occurs only at subphysiological pH and that it is insensitive to inhibitors of the normal entry route. Nevertheless, these results indicate that low pH membrane fusion introduces the viral genome into the cytoplasm in a form suitable for replication.     

Localization of sialidase-positive cells expressing Mac-1 and immunoglobulin in the mouse thymus

We have sought an endogenous membrane bound sialidase acting at neutral pH in immune system, because the removal of sialic acid from cell surfaces will affect the cell-cell interaction directly or indirectly. The levels of activity of unique membrane-bound sialidase at neutral pH and also soluble sialidase are high in the thymus but low in the spleen and lymph nodes. These are thought to be plasma membrane and cytosolic types based on the behavior of inhibition by Cu(2+) and 2-deoxy-2, 3-dehydro-N-acetylneuraminic acid. Newly synthesized 5-bromo-4-chloro-3-indolyl-N-acetylnueraminic acid was used for histochemical staining of sialidase-positive thymic cells, and the results showed positive cells sparsely distributed in the corticomedullar region or medullary region of the thymus. They expressed immunoglobulin and Mac-1 antigen on their surfaces. These cells must therefore be of a B cell lineage, not a T cell lineage. We also found that some vessels in the thymus were sialidase-positive.     

Lipids of whole cells and plasma membrane fractions from Balb/c3T3, SV3T3, and concanavalin A-selected revertant cells.

The lipid composition of Balb/c3T3, SV3T3, and the concanavalin A-selected SV3T3 revertant cells has been analyzed at the whole cell and plasma membrane levels. In comparison to untransformed 3T3 whole cells, SV3T3 cells showed an unchanged content of triacylglycerols, free fatty acids, and glycerylether diesters but a lower concentration of total phospholipids, while no significant difference was found in the phospholipid composition. Whole SV3T3 revertant cells exhibited a lipid composition similar to that in untransformed 3T3 cells with the exception of a higher proportion of sphingomyelin. Analysis of isolated plasma membranes did not reveal any significant differences in the cholesterol to phospholipid molar ratio between 3T3 and SV3T3 or SV3T3 revertant cells. The major changes in the acyl chain pattern SV3T3 compared with whole 3T3 cells consisted of an increase of oleic and palmitoleic acids coupled with a decrease of C20 and C22 polyunsaturated acids in phosphatidylethanolamine and phosphatidylcholine; an increase of oleic acid was also evident in SV3T3 phosphatidylinositol plus phosphatidylserine. An increase of palmitoleic and oleic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine of SV3T3 plasma membranes; the only change in SV3T3 plasma membrane phosphatidylcholine was an increase of oleic acid. An increase of monoenoic acids together with a decrease of arachidonic acid was also found in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine of SV3T3 revertant cells at the level of both whole cells and plasma membranes.     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Trehalase activation in yeasts is mediated by an internal acidification

It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m-chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.     

Biochemical characterization of the human copper transporter Ctr1.

The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated (64)Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.     

Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Isolation of frog urinary bladder plasma membranes with polycation coated beads

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.     

The relationship between plasma membrane lipid composition and physical-chemical properties. III. Detailed physical and biochemical analysis of fatty acid-substituted EL4 plasma membranes

Murine leukemia EL4 cells were modified by supplementation of culture media with fatty acids for 24 h. A plasma membrane-enriched fraction was prepared from substituted and normal cells. Analyses were performed to determine fatty acyl composition, phospholipid headgroup composition and cholesterol content. The two major membrane phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were isolated by thin-layer chromatography and ESR measurements were done on liposomes prepared from these lipids as well as on the intact plasma membrane preparations. Slight perturbations in overall plasma membrane lipid composition were observed when EL4 cells were supplemented with a single exogenous fatty acid. This may be consistent with the idea that the incorporation of exogenous fatty acid induces compensatory changes in membrane lipid composition. On the other hand, we observed no significant difference in two ESR motional parameters between the unsubstituted control and various fatty acid-substituted plasma membranes. ESR measurements carried out on PE and PC liposomes derived from 17:0- and 18:2c-substituted membranes also failed to detect major differences between these liposomes and those made from normal EL4 phospholipids. In the case of liposomes prepared from 18:2t,-substituted membranes, the order parameter was significantly changed from the normal. However, the change was in opposite directions in PE and PC, perhaps accounting for the fact that no change parameter is seen in intact 18:2t-substituted plasma membrane. Measurements of order parameter (S) in mixed lipid vesicles showed that at up to 50 mol% mixture of a synthetic PC with plasma membrane PC, the value of S was only marginally different from that of the plasma membrane PC vesicles. We interpret these data as an indication that the two ESR parameters used are not sufficiently sensitive to detect changes due to modifications of the acyl chain composition of a complex biological membrane.