The isolation and characterization of plasma membranes from calf thymocytes.

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

Fractionation of membrane vesicles: I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose

Affinity chromatography, a separation technique which makes use of biospecific properties, is well established for the separation of molecules in solution. We applied this method to the subfractionation of biomembranes. Using a microsomal fraction mainly consisting of plasma membrane from rabbit or calf thymocytes, 20–40% of the protein adhered specifically to concanavalin A-Sepharose, whereas the majority of the membrane vesicles were recovered from the effluent. The adherence involved the binding of Con A to membranes, as addition of the hapten sugar α-methyl-mannoside completely prevented separation. The fractions which bound to Con A-Sepharose could be eluted by combining mechanical forces with the addition of α-methyl-mannoside. All fractions exhibited the same vesicular appearance and were identical with respect to the phospholipid cholesterol ratio. The method proved to be highly reproducible and it offers a possible way for the subfractionation of membranes according to their biospecific structure.     

Analysis of the proteins in thymocyte plasma membrane and smooth endoplasmic reticulum by sodium dodecylsulfate-gel electrophoresis

We have purified the plasma membranes and membranes of endoplasmic reticulum from calf and rabbit thymocytes and from calf mediastinal lymph node lymphocytes. We disrupted the cells by the “nitrogen cavitation method” and prepared a microsomal isolate by differential centrifugation. We fractionated this by isopycnic ultracentrifugation in dextran gradients into membrane vesicles, PM₁ and PM₂, most likely derived from plasma membrane and a fraction, ER, most likely originating from endoplasmic reticulum. More than 80% of the microsomal 5′-nucleotidase and acid p-nitrophenylphosphatase concentrates in the PM₁ and PM₂ fractions; alkaline p-nitrophenylphosphatase, another presumptive PM marker, is concentrated in the PM₁ fraction. These data are confirmed by the lacroperoxidase radioiodination of intact rabbit thymocytes followed by subcellular fractionation. The specific content of phospholipids (822 nmoles/mg protein) and cholesterol (1032 nmoles/mg protein) is highest in PM₁ and PM₂ plasma membrane fractions. NADH-oxidoreductase, our endoplasmic reticulum marker, is clearly enriched in gradient pellet.The membrane proteins were separated by electrophoretic molecular sieving in sodium dodecylsulfate-polyacrylamide gel electrophoresis, containing dithiothreitol (sodium dodecylsulfate-polyacrylamide gel electrophoresis). We numbered the 10 major protein components of the “microsomal fraction” (apparent molecular weights between 280000 and 15000) from 1–10 according to their decreasing molecular weights. Of these proteins, those with higher molecular weight, predominantly glycoproteins, appear in the PM₁ fraction, while the endoplasmic reticulum fraction contains mainly low molecular weight components.     

Fractionation of membrane vesicles : I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose

Affinity chromatography, a separation technique which makes use of biospecific properties, is well established for the separation of molecules in solution. We applied this method to the subfractionation of biomembranes. Using a microsomal fraction mainly consisting of plasma membrane from rabbit or calf thymocytes, 20–40% of the protein adhered specifically to concanavalin A-Sepharose, whereas the majority of the membrane vesicles were recovered from the effluent. The adherence involved the binding of Con A to membranes, as addition of the hapten sugar α-methyl-mannoside completely prevented separation. The fractions which bound to Con A-Sepharose could be eluted by combining mechanical forces with the addition of α-methyl-mannoside. All fractions exhibited the same vesicular appearance and were identical with respect to the phospholipid cholesterol ratio. The method proved to be highly reproducible and it offers a possible way for the subfractionation of membranes according to their biospecific structure.     

Spontaneous and detergent-induced vesiculation of thymocyte plasma membranes

An analog of lysophosphatidylcholine (1-dodecyl-propanediol-3-phosphocholine) which does not impair membrane-bound enzymes was used for the induction of shedding of membrane vesicles from intact calf thymocytes. Without liberation of intracellular enzymes such as lactate dehydrogenase (EC 1.1.1.27) the shedded membranes contained 15--25% of the total activity of the plasma membrane enzymes alkaline phosphatase (EC 3.1.3.1), nucleotide pyrophosphatase (EC 3.1.4.1) and gamma-glutamyl transferase (EC 2.3.2.2). Membrane-free supernatants only exhibited trace activities of these enzymes. Without further purification, the specific enzyme activities in shedded membranes were of the same order of magnitude as in purified plasma membranes prepared after nitrogen cavitation of thymocytes. Small amounts of membrane vesicles which showed a different composition could be removed without detergent. These membranes exhibited a 3-fold lower specific activity of the gamma-glutamyl transferase while that of the alkaline phosphatase and nucleotide pyrophosphatase was similar as in detergent induced membrane vesicles. Distinct differences also were found in the protein pattern. The content of total cholesterol and phospholipid in vesicles shed spontaneously or after detergent treatment was nearly identical, however, significant differences were found in the fatty acid composition of the main phospholipids. The content of polyunsaturated fatty acids (linoleic and arachidonic acid) increased in the order: spontaneously shedded membranes, detergent induced vesicles, conventional purified plasma membranes. These results are discussed in terms of the heterogeneous composition of areas of the thymocyte plasma membrane.     

Studies on plasma membranes: XIX. Isolation and characterization of a plasma membrane fraction from calf thymocytes

A plasma membrane fraction was isolated from calf thymocytes by a modification of the method of Wallach and Kamat (Wallach, D. F. H. and Kamat, V. B. (1966) in Methods in Enzymology) (Colowick, S. P. and Kaplan, N. O., eds), Vol. 8, pp. 164–172, Academic Press, New York). Fractions were examined electron microscopically and subjected to chemical and enzymic assays.With respect to the cell homogenate and the final microsomal fraction, respectively, the plasma membrane fraction was enriched by a factor 23 and 5.1 in cholesterol, 11 and 2.4 in phospholipid, 5.1 and 4.2 in sialic acid, 20 and 5.2 in Mg²⁺-ATPase (EC 3.6.1.3), and 8 and 2.6 in 5′-nucleotidase (EC 3.1.3.5). Succinate:cytochrome $\text{c}$ oxidoreductase (EC 1.3.99.1) was lacking, and DNA was hardly if at all present in the plasma membrane fraction. The major part of the RNA found in this fraction (30 μg · mg^?1 protein) was concluded to be an authentic component of the plasma membrane.The concept of membrane “markers” was briefly considered and the conclusion was reached that by current criteria and electron microscopic evidence the plasma membrane fraction obtained from calf thymocytes consisted of reasonably clean plasma membranes.     

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.     

Membrane associated immunoglobulin in pig thymocytes

The polypeptides and glycoproteins in plasma membranes of thymocytes and mesenteric lymph node cells have almost identical patterns in polyacrylamide gels. Thymocyte plasma membranes contain IgG which comprises about 0.3% of the total membrane protein. IgM has not been detected. Previous studies with lymph node lymphocyte membranes have shown 0.6% immunoglobulin, containing both γ and μ determinants. Therefore, in their overall polypeptide composition and the possession of immunoglobulin, the membranes from the two types of cells are strikingly similar to each other.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Phospholipase C-mediated release of low molecular weight follicle-stimulating hormone receptor-binding inhibitor from testis membranes

Low molecular weight inhibitors (FRBI) of FSH binding to receptor have been isolated from a variety of gonadal tissue extracts. Because of similarities noted in the composition of FRBI and that expected for polypeptides anchored to plasma membrane via a glycosyl-phosphatidylinositol linkage, we used bacterial phospholipase C to determine if FRBI could be released from calf testis membranes. FRBI was measured by use of radioligand-receptor assays and by a direct chemical method involving derivatization with dansyl chloride followed by HPLC. Phospholipase C treatment released FRBI from calf testis membranes in a time-dependent fashion. Phospholipase C-mediated release was blocked by O-phenanthroline, a specific inhibitor of phosphoinositol-phospholipase C (PI-PLC) activity. These data suggest that FRBI is anchored to testicular plasma membranes via a phospholipase C cleavable glycosyl-phosphatidylinositol anchor. The quantity of PI-PLC releasable FRBI in the testis and its FSH receptor-binding inhibitory potency suggest the possibility that endogenous regulation of FRBI release from testicular membranes could result in local attenuation of FSH action at the receptor level.     

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the ¹²⁵I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains.Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

Shedding of gangliosides from calf thymocytes

In the course of our work on calf thymus gangliosides [Dyatlovitskaya, Zablotskaya, Azizov & Bergelson (1980) Eur. J. Biochem. 110, 475-483] we studied the gangliosides exfoliated from the cell surface of thymocytes. It was shown that calf thymocytes shed gangliosides both in vivo and in vitro. Various gangliosides were found to be present in high amounts both in extracellular plasma membrane vesicles and in the 64000 X g supernatant. The extracellular membrane fragments were comparatively higher in disialosyllactosylceramide and the 64000 X g supernatant was higher in sialosyllactosylceramide than the cells. Comparison of the ganglioside composition of extracellular membrane fragments, thymocytes and lymphocytes led us to suggest that the shedding of gangliosides from the surface of thymocytes may be involved in the transformation of immunologically incompetent cortical thymocytes into immunocompetent virgin T-cells.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes

ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.     

Studies on the association of NAD glycohydrolase with membranes in calf spleen

The subcellular distribution of NAD glycohydrolase was studied by fractionation of calf spleen homogenates using differential and discontinuous density gradient centrifugations. The highest amount of NAD glycohydrolase activity was associated with microsomes, which in this tissue were found to contain, in addition to endoplasmic reticulum, a large proportion of vesicles derived from plasma membranes. The distribution pattern of NAD glycohydrolase was found to parallel that of plasma membrane markers. When microsomal vesicles were treated with digitonin, NAD glycohydrolase activity and plasma membranes specifically increased in density. We conclude that in calf spleen the bulk of NAD glycohydrolase is associated with plasma membranes. Microsomal NAD glycohydrolase was associated with sealed vesicles; its activity could not be increased by disruption of the sidedness of the vesicles. This result and further observations based on the known restricted permeability of biological membranes to charged substances, and on the activity of the enzyme with non-penetrating substrates and inhibitors, indicate that the NAD glycohydrolase active site is located on the exterior side of the vesicles. It is proposed that calf spleen NAD glycohydrolase is an ecto-enzyme.     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Extracellular ATP induces a nonspecific permeability of thymocyte plasma membranes

We have previously demonstrated that extracellular ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis, without mobilization of intracellular calcium. We describe here the effects of extracellular nucleotides on membrane permeability to monovalent and divalent cations in mouse thymocytes. Among all nucleotides tested, under physiological conditions, only ATP and, to a lesser extent, 2-methylthio-ATP, adenosine 5'-O-(3-thio-triphosphate), and ADP were able to depolarize thymocyte plasma membranes and to induce Na+ and Ca2+ influxes into thymocytes; other nonhydrolysable ATP analogs were only effective in the absence of Mg2+. The ATP-induced effects were inhibited in a dose-dependent manner by Mg2+ and greatly potentiated in its absence, which suggests that the tetrabasic ATP4 is probably the active species and that a phosphotransferase activity is not involved in its effects. There ATP-mediated changes in ion fluxes result from an increase in nonspecific permeability of thymocyte membranes, probably by pore formation. These ion flux changes might be responsible for the mitogenic induction of phorbol 12-myristate 13-acetate treated medullary thymocytes. The potency order for the adenine derivatives to affect these fluxes (ATP greater than ADP much greater than AMP greater than adenosine) suggests the presence of ATP specific receptors (P2 purinergic receptors) on thymocyte plasma membranes.