Microvillus membrane vesicles from pig small intestine Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18:50:29:2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17,17,6,5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either $\text{N-}\text{acetylglucosamine}$ or $\text{N-}\text{acetylgalactosamine}$) in the molar ratio of 1:2:1:1.     

Lipid and lipopolysaccharide composition of Acholeplasma oculi.

The total lipid content of Acholeplasma oculi comprises 13.3% of the dry weight of the organism and is about equally distributed between the neutral lipids plus glycolipids and the phospholipids. The phospholipids were identified as phosphatidyl glycerol and diphosphatidyl glycerol. The glycolipid fraction contained O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol and O-alpha-D-glucopyranosyl-(1 leads to 2)-O-alpha-D-glucopyranosyl-(1 leads to 1)-2,3-diacyl glycerol. The neutral lipid contained pigmented carotenoids. Hot aqueous phenol extraction of lipid-extracted whole cells yielded a polymeric carbohydrate comprising 2.3% of the dry weight of the organism. The A. oculi lipopolysaccharide was found to contain only neutral sugars and no amino sugar, in contrast to other acholeplasmas. The neutral sugars consisted of fucose, galactose, and glucose in a ratio of 2:19:3.     

Chemical Composition and Ultrastructure of Native and Reaggregated Membranes from Protoplasts of Bacillus cereus

Conditions were defined for producing protoplasts with lysozyme and isolating the protoplast membranes from cells of Bacillus cereus T harvested late in the exponential growth phase just before sporogenesis. The membranes contained approximately 60% protein, 30% lipid, 6% carbohydrate, and 1% ribonucleic acid. Seventeen proteins were distinguished by molecular size in the membrane solubilized with sodium dodecyl sulfate, and 12 in that with phenol and acetic acid. The lipid fraction consisted of neutral lipids (28%) and phospholipids (72%). Four phospholipids were identified: diphosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl glycerol, and lysophosphatidyl ethanolamine. Eighteen fatty acids were identified, with a predominance of branched C(15) and C(17) and of normal C(16) acids. The carbohydrate fraction consisted of neutral hexoses. A clear supernatant solution from the solubilized preparation became reaggregated into membrane by dialysis in the presence of MgCl(2). The reaggregated membrane had the same main components as the native membrane, but the amount and ratio of protein and lipid depended on the buffer and the MgCl(2) concentration. By electron microscopy, the reaggregated membranes appeared as vesicles or sheets, depending on the MgCl(2) concentration. Hexagonal lattices were occasionally detected in the negatively stained ultrastructure of both native and reaggregated membrane fragments.     

A simple method of rat renal brush border membrane preparation using polyethylene glycol precipitation

A simple method for preparation of brush border membranes (BBM) from rat kidney using polyethylene glycol (PEG) precipitation has been described. This method avoids the use of cations for the preparation, which might alter membrane lipid composition. These preparations were assessed for enrichment of marker enzymes, contamination by subcellular structures, lipid composition and transport function. An enrichment of 11.8910-fold of alkaline phosphatase, 13.9500-fold of amino peptidase and 13.6500-fold of gamma-glutamyl transpeptidase and an approximate yield of 60% were seen in the final membrane preparation as compared to the homogenate. There was very little contamination of basolateral membranes, peroxisomes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sailic acid as compared to hexoses. Isolated membranes appeared as vesicles as seen by electron microscopy. Lipid analysis indicated the presence of various neutral and phospholipids with a high content of sphingomyelin along with a cholesterol/phospholipid ratio of 0.4850. The isolated membrane vesicles were able to transport glucose. This study has shown a simple method of renal brush border membrane preparation, which is comparatively pure and functionally active.     

Galactose oxidase action on galactose containing glycolipids--a fluorescence method

Features that alter the glycolipid sugar headgroup accessibility at the membrane interface have been studied in bilayer lipid model vesicles using a fluorescence technique with the enzyme galactose oxidase. The effects on oxidation caused by variation in the hydrophobic moiety of galactosylceramide or the membrane environment for galactosylceramide, monogalactosyldiacylglycerol and digalactosyldiacylglycerol were studied. For this study we combined the galactose oxidase method for determining the oxidizability of galactose containing glycolipids, and the fluorescence method for determining enzymatic hydrogen peroxide production. Exposed galactose residues with a free hydroxymethyl group at position 6 in the headgroup of glycolipids were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). Amplex Red reacts with hydrogen peroxide in the presence of horseradish peroxidase with a 1:1 stoichiometry to form resorufin. With this coupled enzyme approach it is also possible to determine the galactolipid transbilayer membrane distribution (inside-outside) in bilayer vesicles.     

Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles.

1. Intact cells, cell fragments (membranes) and matrix vesicles were isolated from the proliferating and calcifying layers of epiphyseal cartilage by sequential hyaluronidase and collagenase digestion and differential centrifugation. Lipids were extracted and analyzed for various lipid classes and their fatty acid composition by column, thin-layer, paper and gas-liquid chromatography. 2. On a protein basis the isolated matrix vesicles had more total lipid than either the membrane or cell fractions, the vesicles and membranes being richer in non-polar lipids and containing smaller quantities of phospholipids than whole cells. Expressed as a percentage of the total lipid, the cells were richer in triacylglycerols and lower in free fatty acids than in the membrane or vesicle fractions. The proportion of free cholesterol and the cholesterol/phospholipid ratio were nearly twice as high in the matrix vesicles as in the other tissue fractions. Choline and ethanolamine phosphoglycerides progressively declined in the membrane and matrix vesicle fractions, whereas serine phosphoglycerides and sphinogomyelin increased. Non-phosphorus-containing polar lipids were present in all fractions, the vesicles being richer in polyhexosyl ceramides, cerebrosides, glycosyldiacylglycerols and certain uncharacterized acidic polar lipids. 3. Fatty acid patterns of the matrix vesicles were distinctive from those of isolated cells, being generally richer in 18 : 0 and 18 : 2, and lower in 16 : 1 and 18 : 1 fatty acids. Monoacyl forms were similarly increased in 16 : 0 and/or 18 : 0, and reduced in 16 : 1, 18 : 1 or 20 : 2 fatty acids, depending on the lipid class. The fatty acid composition of diphosphatidylglycerol from cells and matrix vesicles was markedly different, providing evidence that the cardiolipin in the vesicles was not from mitochondrial components. 4. Based on the fact that the matrix vesicles were significantly enriched in free cholesterol, sphingomyelin, glycolipids and serine-phosphoglycerides, it is concluded that they are derived from the plasma membrane of the cell, supporting earlier conclusions based upon morphological and enzymological evidence.     

Carbohydrate composition of lymphocyte plasma membrane from pig mesenteric lymph node.

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.     

Modification of membrane protein expression and protein secretion in LLC-PK1 cultures grown on different carbohydrates

Membrane glycoproteins and glycolipids play an important role in epithelial organization, transport and function. To study the effects of exogenous carbohydrates on the expression of glycoproteins, cells of the renal epithelial line LLC-PK1 were cultured on different nutritive carbohydrate sources and on uridine, which is, despite striking differences, known to substitute all essential nutritive functions of glucose. LLC-PK1 cultures were long-term adapted to growth in culture medium containing 0.5, 5, 10 and 25 mM glucose, and 5 mM fructose, galactose and uridine, respectively, as the sole carbohydrate source. These growth conditions elicited adaptive changes in the expression of enzyme activities of alkaline phosphatase and gamma-glutamyltranspeptidase, integral membrane glycoproteins exclusively localized in the apical membrane of LLC-PK1 cells. SDS-PAGE of membrane preparations of adapted LLC-PK1 cells revealed a strong induction of several protein bands between 13.5 and 47 kD in fructose-grown cells, while in plasma membranes of cells grown in galactose several protein bands between 62 and 70 kD decreased. Changes in the secretion pattern of proteins into the culture medium were most prominent in uridine-grown cells compared to controls grown on 25 mM glucose.     

Lipid requirement of the branched-chain amino acid transport system of Streptococcus cremoris

The role of the membrane lipid composition on the transport protein of branched-chain amino acids of the homofermentative lactic acid bacterium Streptococcus cremoris has been investigated. The major membrane lipid species identified in S. cremoris were acidic phospholipids (phosphatidylglycerol and cardiolipin), glycolipids, and glycerophosphoglycolipids. Phosphatidylethanolamine (PE) was completely absent. Protonmotive force-driven and counterflow transport of leucine was assayed in fused membranes of S. cremoris membrane vesicles and liposomes composed of different lipids obtained by the freeze/thaw-sonication technique. High transport activities were observed with natural S. cremoris and Escherichia coli lipids, as well as with mixtures of phosphatidylcholine (PC) with PE or phosphatidylserine. High transport activities were also observed with mixtures of PC with monogalactosyl diglyceride, digalactosyl diglyceride, or a neutral glycolipid fraction isolated from S. cremoris. PC or mixtures of PC with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activities. In mixtures of PC and methylated derivatives of PE, both counterflow and protonmotive force-driven transport activities decreased with increasing degree of methylation of PE. The decreased transport activity in membranes containing PC could be restored by refusion with PE-containing liposomes. These results demonstrate that both aminophospholipids and glycolipids can be activators of the leucine transport system from S. cremoris. It is proposed that aminophospholipids in Gram-negative bacteria and glycolipids in Gram-positive bacteria have similar functions with respect to solute transport.     

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.     

Characterization of the bacterial magnetosome membrane.

Intact magnetosomes of Aquaspirillum magnetotacticum were purified from broken cells by a magnetic separation technique. Electron microscopic and chemical analyses revealed the magnetite to be enclosed by a lipid bilayer admixed with proteins. Lipids were recovered in fractions expected to contain (i) neutral lipids and free fatty acids, (ii) glycolipids and sulfolipids, and (iii) phospholipids (in a weight ratio of 1:4:6). Phospholipids included phosphatidylserine and phosphatidylethanolamine. Two of the numerous proteins detected in the magnetosome membrane were not found in other cell membranes or soluble fractions.     

Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation

The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

The isolation and partial characterization of glycolipids of normal human leucocytes

1. The lipids of purified human leucocytes were extracted with chloroform-methanol and the extract was washed with water. Glycolipids, isolated by Florisil chromatography, were subjected to mild alkaline hydrolysis and the alkali-resistant fraction was fractionated on a silicic acid column. 2. Three classes of glycolipid were separated. The less polar, containing 3.6% of the total glycolipid hexose as galactose, was tentatively identified as ceramide monohexoside. The major glycolipid fraction was characterized as ceramide dihexosides. The more polar glycolipids comprised 1.6% of the total glycolipid hexose as galactose and glucose (in the molar ratio 2:1) and were non-acidic. This class was separated as a mixture containing ninhydrin-positive glycolipids. 3. The ceramide dihexosides taken from two leucocyte preparations accounted for 15.2% and 16.4% by weight of the total lipids. 4. The carbohydrate moiety of the ceramide dihexosides contained galactose and glucose in the molar ratio 2:1. Partial acid hydrolysis and paper chromatography indicated that the hexoses are present as disaccharides, lactose being identified as one of them. 5. Palmitic acid (C(16:0)) and nervonic acid (C(24:1)) were the major fatty acids of this glycolipid. Hydroxy fatty acids were not detected.     

Incorporation of MAL, an integral protein element of the machinery for the glycolipid and cholesterol-mediated apical pathway of transport, into artificial membranes requires neither of these lipid species

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Capsular and extracellular polysaccharides of the diazotrophic rhizobacterium <Emphasis Type="Italic">Herbaspirillum seropedicae</Emphasis> Z78

The diazotrophic endophyte Herbaspirillum seropedicae Z78 was shown to possess a capsule containing two high-molecular-weight glycolipids, one of which was of a lipopolysaccharide nature. These glycolipids differed considerably in the fatty acid composition of their lipid components. The polysaccharide moiety of these glycans was composed of glucose, galactose, glucosamine, galactosamine, and a noncarbohydrate component, butanetetraol. In the culture liquid of H. seropedicae Z78, an extracellular polysaccharide and an extracellular form of lipopolysaccharide were revealed. Fatty acid composition of the extracellular lipopolysaccharide differed from that of the capsular glycoconjugates; the polysaccharide moiety of exoglycans contained only neutral sugars (mannose, glucose, and galactose) and a tetraatomic alcohol, butanetetraol. It is assumed that structural diversity of polysaccharide-containing polymers at the surface of H. seropedicae Z78 cells is conditioned by their different roles in plant colonization and formation of efficient symbiosis.     

The lipid composition of plasma membrane from goat mammary gland

Experiments were conducted to examine and characterize the lipid composition of the plasma membrane from the lactating goat mammary gland. The plasma membranes were purified by discontinuous sucrose density centrifugation. Lipids were extracted from these membranes and analyzed by thin-layer and gas-liquid chromatography. The results of these studies demonstrate that (i) the principal phospholipids of mammary-gland plasma membranes are phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; (ii) the principal neutral lipids are triacylglyceride and cholesterol ester; (iii) the major glycolipids are globotetraosylceramide and globotriaosylceramide; and (iv) the major fatty acids are oleic (18:1), palmitic (16:0), stearic (18:0), and myristic (14:0) acids.     

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.     

Charged membrane surfaces impede the protein-mediated transfer of glycosphingolipids between phospholipid bilayers

A lipid transfer protein that facilitates the transfer of glycolipids between donor and acceptor membranes has been investigated using a fluorescence resonance energy transfer assay. The glycolipid transfer protein (23-24 kDa, pI 9.0) catalyzes the high specificity transfer of lipids that have sugars beta-linked to either a ceramide or a diacylglycerol backbone, such as simple glycolipids and gangliosides, but not the transfer of phospholipids, cholesterol, or cholesterol esters. In this study, we examined the effect of different charged lipids on the rate of transfer of anthrylvinyl-labeled galactosylceramide (1 mol %) from a donor to acceptor vesicle population at neutral pH. Compared to neutral donor vesicle membranes, introduction of negatively charged lipid at 5 or 10 mol % into the donor vesicles significantly decreased the transfer rate. Introduction of the same amount of negative charge into the acceptor vesicle membrane did not impede the transfer rate as effectively. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was negative charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (pI congruent with 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that the transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process.