Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle

Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes.     

Ehrlich ascites tumor cell surface membranes: an abnormality in ether lipid content

Most mammalian neoplasms have a defect in ether lipid content manifested by the presence of abnormally large quantities of 0-alkyl glyceryl ethers, in contrast to normal tissues in which the alk-1-enyl structure predominates. These lipids are for the most part structural. The manner in which tumor cell plasma membranes differ from normal may be important, and it has been hitherto unclear whether or not the 0-alkyl lipid abnormality of neoplasms includes the plasma membrane. The present investigation reveals that 0-alkyl lipids are present in the membranes of Ehrlich ascites tumor cells isolated by several different methods. The amount of 0-alkyl lipid, on a weight basis, represents 1-3 percent of the total phospholipids and 1-4 percent of the total aliphatic lipid. These quantities are the same as or greater than the amount of 0-alkyl lipid found in microsomes, mitochondria, and whole cell homogenate. As is generally the case for intact neoplastic tissues, the quantity of 0-alkyl lipids of Ehrlich ascites tumor plasma membrane is greater than the amount of alk-1-enyl lipids.     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeating labeling experiments indicated that approximately 40% of these proteins were exposed on the extra-cytoplasmic surface of the plasma membranes of these cells. The studies demonstrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.     

Surface peptides on intact Ehrlich ascites tumor cells : Identification by a method not requiring subcellular fractionation

Lactoperoxidase catalysed iodination of tyrosyl residues was used to label the exposed plasma membrane proteins in intact Ehrlich ascites tumor cells. Autoradiography of ¹²⁵I-labeled intact cells revealed that the label was predominantly associated with the plasma membrane. When whole cells were solubilized and subjected to gel electrophoresis, two major labeled peptide classes of 100 000 and 80 000 D along with 4 minor labeled classes were found. An identical labeling pattern was obtained when plasma membranes isolated from labeled cells were solubilized and subjected to gel electrophoresis. These results demonstrate that the number of exposed plasma membrane peptides and their molecular weights can be determined without first isolating the membrane by subcellular fractionation procedures, a standard approach in most studies.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeant labeling experiments indicated that approximately 40% of these proteins were exposed on the extracytoplasmic surface of the plasma membranes of these cells. The studies demostrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.     

Paramagnetic proteoliposomes containing a pure, native, and oriented seven-transmembrane segment protein, CCR5

Seven-transmembrane segment, G protein-coupled receptors play central roles in a wide range of biological processes, but their characterization has been hindered by the difficulty of obtaining homogeneous preparations of native protein. We have created paramagnetic proteoliposomes containing pure and oriented CCR5, a seven-transmembrane segment protein that serves as the principal coreceptor for human immunodeficiency virus (HIV-1). The CCR5 proteoliposomes bind the HIV-1 gp120 envelope glycoprotein and conformation-dependent antibodies against CCR5. The binding of gp120 was enhanced by a soluble form of the other HIV-1 receptor, CD4, but did not require additional cellular proteins. Paramagnetic proteoliposomes are uniform in size, stable in a broad range of salt concentrations and pH, and can be used in FACS and competition assays typically applied to cells. Integral membrane proteins can be inserted in either orientation into the liposomal membrane. The magnetic properties of these proteoliposomes facilitate rapid buffer exchange useful in multiple applications. As an example, the CCR5-proteoliposomes were used to select CCR5-specific antibodies from a recombinant phage display library. Thus, paramagnetic proteoliposomes should be useful tools in the analysis of membrane protein interactions with extracellular and intracellular ligands, particularly in establishing screens for inhibitors.     

Proteoliposome interaction with human erythrocyte membranes. Functional implantation of gamma-glutamyl transpeptidase

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.     

Purification and reconstitution of the bile acid transport system from hepatocyte sinusoidal plasma membranes

The taurocholic acid transport system from hepatocyte sinusoidal plasma membranes has been studied using proteoliposome reconstitution procedures. Membrane proteins were initially solubilized in Triton X-100. Following detergent removal, the resultant proteins were incorporated into lipid vesicles prepared from soybean phospholipids (asolectin) using sonication and freeze-thaw procedures. The resultant proteoliposomes demonstrated Na+-dependent transport of taurocholic acid which could be inhibited by bile acids. Greatly reduced amounts of taurocholic acid were associated with the phospholipid or membrane proteins alone prior to proteoliposome formation. Membrane proteins were fractionated on an anionic glycocholate-Sepharose 4B affinity column which was prepared by coupling (3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholan-24-oyl)-N alpha-lysine to activated CH-Sepharose 4B via the epsilon-amino group of lysine resulting in the retention of a free carboxyl group. The adsorbed proteins enriched in components in the 54 kDa zone, which were originally identified by photoaffinity labeling to be components of the bile acid transport system, were also incorporated into liposomes. This vesicle system showed almost a 4-fold increase in Na+-dependent taurocholic acid uptake when compared to proteoliposomes formed from total membrane protein, as well as sensitivity to inhibition by bile acids. These results demonstrate that the bile acid carrier system can be reconstituted in proteoliposomes and that utilizing proteins in the 54 kDa zone leads to a significant enhancement in the transport capacity of the reconstituted system, consistent with the role of 54 kDa protein(s) as component(s) of the bile acid carrier system.     

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.     

Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H⁺ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [³H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of d -glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-β-d -glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni²⁺-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.     

Reconstitution of membrane proteins: sequential incorporation of integral membrane proteins into preformed lipid bilayers

Several integral membrane proteins can be inserted sequentially into preformed unilamellar vesicles (ULV's) composed of dimyristoylphosphatidylcholine (DMPC) and cholesterol in a gel phase. Thus, proteoliposomes of DMPC, cholesterol, and bacteriorhodopsin from Halobacterium halobium rapidly incorporate UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase from beef heart mitochondria, and additional bacteriorhodopsin, added sequentially. This process of spontaneous incorporation can be regulated to produce complex artificial membranes that contain phospholipids and proteins at ratios (mol/mol) equivalent to what is found in biological membranes. The ability of the lipid-protein bilayers to incorporate additional integral membrane proteins is not affected by annealing of the proteoliposomes at 37 degrees C nor by the order of addition of the proteins. Bacteriorhodopsin-containing vesicles formed by the sequential addition of integral membrane proteins demonstrate light-driven proton pumping. Therefore, they have retained a vesicular structure. Vesicles containing one or two different proteins will fuse with each other at 21 degrees C or with ULV's devoid of proteins. Incorporation of bacteriorhodopsin or UDPglucuronosyltransferase into proteoliposomes containing DMPC, with or without cholesterol as impurity, also occurs above the phase transition for DMPC. The presence of a protein in a liquid-crystalline bilayer provides the necessary condition for promoting the spontaneous incorporation of other membrane proteins into preformed bilayers.     

Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes

L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y. et al. (1986) J. Biol. Chem. 261, 4549). The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes. The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake. The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH). The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics. These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes.     

Isolation of plasma membrane fragments and vesicles from ascites fluid of lymphoma-bearing mice and their possible role in the escape mechanism of tumors from host immune rejection

Summary Plasma membranes, generated in vivo by actively growing YAC lymphoma cells, were isolated from cell-free ascites fluid of lymphoma-bearing mice. Partial purification of the ascites fluid (AF) by means of ultracentrifugation resulted in the identification of two main fractions: (a) membrane fragments (AFM _s ) and (b) membrane vesicles (AFM _p ). Electron microscopy studies, polyacrylamide gel electrophoresis, marker enzymes, and binding capacity of radioactive lectins, have indicated that these membranes are released from the cell surface of YAC lymphoma cells, presumably by a shedding-off mechanism.In vitro studies have demonstrated that the isolated membranes can specifically inhibit the association of normal macrophages and YAC lymphoma cells. In vivo studies have shown that these membranes can immunize against YAC tumors if injected intramuscularly or subcutaneously into adult mice. The results indicate that the ascites fluid membranes bear tumor-specific antigenic determinants.Our results suggest that in vivo shedding of plasma membrane fragments or of membrane vesicles by actively growing YAC lymphoma cells may induce a self-protection of ascites tumors from host immune rejection.Abbreviations YAC= Moloney-virus-induced lymphoma cells grown in A-strain mice - AF= ascites fluid of YAC lymphoma-bearing mice - AFM_s and AFM_p= membrane fragments and vesicles isolated from AF - PBS= phosphate-buffered saline - Con A= Concanavalin A     

Reconstitution of a partially purified Na+-dependent D-glucose transport system from rat jejunal brush border membranes

The Na+-dependent D-glucose transport system of rat jejunal brush border membranes was partially purified and reconstituted into functional proteoliposomes. Brush border membrane vesciles isolated from villous cells were first extracted with 0.3% cholate to remove extrinsic proteins and the insoluble residual pellet was reextracted with 1.2% cholate. The 1.2% cholate-extracted soluble fraction was then further purified by hydroxylapatite and Concanavalin A affinity chromatography in tandem. When the HLP-unadsorbed-ConA-unadsorbed fraction was reconstituted into proteoliposomes, it showed a characteristic Na+-coupled, phlorizin inhibitable, D-glucose transport activity that was 3 fold higher than that of the reconstituted proteoliposomes of the 1.2% cholate-extracted fraction. This partially purified fraction also displayed the simplest polypeptide composition pattern among all the membrane fractions analysed in SDS-polyacrylamide gels.     

Behavior of vesicular stomatitis virus glycoprotein in mouse LM cells with modified membrane-phospholipids

LM cells in which the membrane phospholipids had been modified with choline analogues were infected with vesicular stomatitis virus. The choline analogues tested were choline, N,N'-dimethylethanolamine, N-monomethylethanolamine and ethanolamine. These modifications per se did not affect the syntheses of individual viral proteins. The viral glycoprotein was detected in the plasma membranes of all the modified cells by pronase digestion in pulse-chase experiments, but the amount of glycoprotein susceptible to proteolysis varied, decreasing in these modified cells in the following order: N,N'-dimethylethanolamine- greater than choline- greater than N-monomethylethanolamine- greater than ethanolamine-treated cells. After a 4-h chase, glycoprotein was mainly distributed in the plasma membranes of cells modified with N,N'-dimethylethanolamine, whereas it was found in both the microsomes and plasma membranes of cells modified with other analogues. Fairly large amounts of glycoprotein were also found in the soluble fraction of ethanolamine-treated cells, but not in that of choline- or N,N'-dimethylethanolamine-treated cells. More precise experiments on the behaviour of glycoprotein with a short period of chase strongly suggested that migration of glycoprotein from the microsomes to the plasma membranes was fastest in cells modified with N,N'-dimethylethanolamine and slowest in cells modified with ethanolamine. Membrane lipid modifications also resulted in release of different numbers of progeny virions from the cells, release of virions from the cells decreasing in the following order: N,N'-dimethylethanolamine- greater than choline- greather N-monomethylethanolamine- greater than ethanolamine-treated cells. These results indicate that modification of membrane phospholipids influences not only the insertion of glycoprotein into the microsomes and its migration to the plasma membranes, but also the production of progeny virions.     

Multiple Mechanosensitive Ion Channels from Escherichia coli,Activated at Different Thresholds of Applied Pressure

Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 m KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system. Received: 23 May 1995/Revised: 31 January 1996     

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.     

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.     

Altered 5'-nucleotidase specific activity and distribution between two plasma membrane domains of ascites tumor cells with modified lipid composition

1. The cholesterol and phospholipid content of the surface membranes of ascites tumor cells cultivated in lipid-depleted medium was reduced to about 60(70)% of the control, but the relative composition of the individual phospholipids was not altered. 2. Differences in lipid composition were also observed between the two plasma membrane domains isolated from the cells cultured in normal and lipid-depleted medium respectively. 3. The fatty acid spectrum of the lipid-depleted membranes showed a greater fraction of saturated vs unsaturated acids. 4. The membrane lipid fluidity measured by fluorescence polarization was decreased in the modified surface membranes. 5. The 5'-nucleotidase specific activity was drastically reduced (46-66%) in the lipid-deleted membranes, and in addition its distribution between the two vesicle fractions was altered.