Evidence for the presence of a glycosphingolipid-transfer protein in rat brain cytosol

Proteins in the postmicrosomal supernatant fraction of rat brain catalyzed the transfer of bovine brain galactocerebroside, sulfatide, and ganglioside GM1 from unilamellar liposomes to the rat erythrocytes or ghosts. The vesicles were made with egg yolk lecithin, cholesterol, 3H-labelled glycolipid, and a trace of [14C]triolein as a nonexchangeable marker. The routine assay of the glycosphingolipid transfer consisted of incubation of the donor liposomes with erythrocytes in the presence or absence of supernatant protein in physiological buffer at 37 degrees C for various time intervals. After the incubation, the erythrocytes were separated from the vesicles by centrifugation and the extent of protein-catalyzed transfer of labelled glycolipid in the membrane-bound total lipid fraction was determined by scintillation spectrometry. The fraction of [3H]glycosphingolipid transferred is represented by a change in the 3H/14C ratios at initial and subsequent time intervals. The glycosphingolipid transfer catalyzed by the supernatant protein was found to be logarithmic, whereas the protein-independent transfer was linear over a period of 3-4 h. The rate constant (K) and half time (t1/2) of the protein-catalyzed transfer reaction of cerebrosides and sulfatides were almost the same, while the transfer of ganglioside GM1 occurred at a slightly faster rate, probably owing to the greater aqueous solubility of this lipid. The transfer activity was also increased in a manner dependent on the amount of supernatant protein added up to 10 mg. The catalytic activity of the protein was lost when heated at 70 degrees C for 5 min. The pH optimum of the activity was around 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partition coefficients of fluorescent probes with phospholipid membranes

A method for determination of membrane partition coefficients of five fluorescent membrane probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic acid), 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH propionic acid), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide (4-di-10-ASP), was developed utilizing the fluorescence enhancement of a constant probe concentration by titration with excess phospholipid liposomes. The partition coefficients of DPH, DPH carboxylic acid, DPH propionic acid, TMA-DPH and 4-di-10-ASP into dipalmitoylphosphatidylcholine membranes were determined to be 1.3.10(6), 1.0.10(6), 6.5.10(5), 2.4.10(5) and 2.8.10(6) respectively. Knowledge of the partition coefficients may help select a lipid concentration for membrane studies that necessitate a probe's dominant incorporation into membranes.     

Structure and activity of lipid membrane biosensor surfaces studied with atomic force microscopy and a resonant mirror.

Three variants of the liposome fusion (coalescence) method to produce supported lipid bilayers, containing the ganglioside GM1 on silicon nitride surfaces, were studied. The first procedure involved attachment and fusion of liposomes containing DMPC, GM1 and a small amount of biotinylated lipid (Biotin-LC-DPPE) to a streptavidin coated surface. Direct fusion of liposomes composed of a mixture of DPPC, DPPG, DPPE, GM1 and cholesterol to the surface were the second variant. The final method utilised the second type of liposomes, fused onto a streptavidin layer with a small amount of exposed hydrophobic tails. The methods produced similar lipid layers, but with different ways of attachment to the surface. The binding of cholera toxin B-subunit (CTB) towards these sensor surfaces was measured in a resonant mirror biosensor instrument and the activity and longer-term stability of the layers were examined. The prepared surfaces were also imaged by atomic force microscopy (AFM) in liquid to characterise the topography of the lipid layers. The binding efficiency of CTB towards these surfaces was discussed in terms of lipid fluidity and surface roughness.     

A novel method for the rational construction of well-defined immunogens: the use of oximation to conjugate cholera toxin B subunit to a peptide-polyoxime complex

Cholera toxin B subunit (CTB), capable of binding to all mucous membranes in its pentameric form, is a potential carrier of mucosal vaccines. In our previous work we reported that the N-terminus of CTB, a threonine, could in principle undergo oxidation and oximation to form conjugates with a cascade of immunogenic peptides. In this study, we set up a model by chemically coupling CTB to a polyoxime that possessed five copies of influenza virus-derived peptides displayed in comblike form. The construct was reconstituted into pentameric form when eluted from a Superdex column after conjugation, and the pentameric nature of this CTB-viral peptide complex was confirmed by SDS-PAGE. GM(1)-ELISA assay showed that the binding properties of CTB-viral peptide complex were increased 4-5-fold over native CTB.     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

Novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits

The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid B-subunit, but not CTB or LTB, to slowly migrating non-acid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAcalpha3(Fucalpha2)Galbeta4(Fucalpha3)Glc NAcbeta3Galbeta4Glc NAcbeta3Galbeta4Glcbeta1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Galalpha3(Fucalpha2)Galbeta4(Fucalpha3)GlcNAc beta. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Modification of TSH-stimulated adenylate cyclase activity of bovine thyroid by manipulation of membrane phospholipid composition with a nonspecific lipid transfer protein

The lipid composition of bovine thyroid plasma membranes was modified using the nonspecific lipid transfer protein from bovine liver. Incubation of plasma membranes with transfer protein and phosphatidylinositol-containing liposomes caused a strong, concentration dependent, inhibition of TSH-stimulated adenylate cyclase activity. Other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid were two to four times less effective as inhibitors of TSH-stimulation. The phosphatidylinositol-induced inhibition was not reversed when more than 80% of phosphatidylinositol incorporated was removed using phosphatidylinositol-specific phospholipase C. Incorporation of phosphatidylinositol in plasma membranes provoked no significant change in the fluorescence anisotropies of the fluorophores 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(14-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), indicating that the inhibition was not due to changes in membrane fluidity. At phosphatidylinositol concentrations causing a 66% reduction in TSH-stimulated adenylate cyclase activity cholera toxin- and forskolin-stimulated activity as well as basal activity were decreased by maximally 10%. Since TSH binding to bovine thyroid plasma membranes was not affected it is suggested that phosphatidylinositol can act as a negative modulator of the TSH activation of adenylate cyclase and this probably by interfering with the coupling between the occupied TSH receptor and the stimulatory GTP-binding regulatory protein of the adenylate cyclase complex.     

Synthesis of novel NBD-GM1 and NBD-GM2 for the transfer activity of GM2-activator protein by a FRET-based assay system

The ganglioside-activator protein is an essential cofactor for the lysosomal degradation of ganglioside GM2 (GM2) by beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-embedded glycolipid substrate at the lipid-water interphase. Mutations in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. In order to efficiently and sensitively probe the glycolipid binding and membrane activity of this cofactor, we synthesized two new fluorescent glycosphingolipid (GSL) probes, 2-NBD-GM1 and 2-NBD-GM2. Both compounds were synthesized in a convergent and multistep synthesis starting from the respective gangliosides isolated from natural sources. The added functionality of 2-aminogangliosides allowed us to introduce the chromophore into the region between the polar head group and the hydrophobic anchor of the lipid. Both fluorescent glycolipids exhibited an extremely low off-rate in model membranes and displayed very efficient resonance energy transfer to rhodamine-dioleoyl phosphoglycerol ethanolamine (rhodamine-PE) as acceptor. The binding to GM2-activator protein (GM2AP) and the degrading enzyme was shown to be unaltered compared to their natural analogues. A novel fluorescence-resonance energy transfer (FRET) assay was developed to monitor in real time the protein-mediated intervesicular transfer of these lipids from donor to acceptor liposomes. The data obtained indicate that this rapid and robust system presented here should serve as a valuable tool to probe quantitatively and comprehensively the membrane activity of GM2AP and other sphingolipid activator proteins and facilitate further structure-function studies aimed at delineating independently the lipid- and the enzyme-binding mode of these essential cofactors.     

Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.     

Effect of gangliosides on activation of the alternative pathway of human complement

Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.     

The interaction of albumin and fatty-acid-binding protein with membranes: oleic acid dissociation

Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.     

Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents

Sperm cell activation is a critical step in fertilization. To directly investigate the cell signaling events leading to sperm activation it is necessary to deliver membrane impermeant agents into the cytoplasm. In this study, the use of liposomes as possible agent-loading vectors was examined using (1) the octadecylrhodamine B (R18) and NBD phosphatidylethanolamine (NBD DHPE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk samples, (2) membrane transfer of fluorescence from liposome membranes labeled with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cells, a process that stimulated sperm cell activation. Intermediate-sized unilamellar liposomes (98.17+/-15.34 nm) were prepared by the detergent-removal technique using sodium cholate as the detergent and a phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid composition. In the R18 fusion assays, self-quenching increased logarithmically with increasing concentrations of R18 in the liposome membranes; addition of unlabeled sperm to R18-labeled liposomes lead to a rapid release of self-quenching. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assay, RET was rapidly reduced under similar conditions. In addition, individual sperm became fluorescent when TRITC DHPE-labeled liposomes were incubated with unlabeled sperm cells. Incubation of sperm cells with empty liposomes did not significantly affect sperm cell activation and did not alter cell morphology. However, incubation with Ca (10 mM)-loaded liposomes resulted in a time-dependent increase in sperm cell activation (7.5-fold over controls after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and that delivery occurs via fusion and content intermixing.     

Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.     

Involvement of ethylene in the regulation of growth and development of the fungus Botrytis cinerea Pers. ex. Fr.

Application of ethephon slightly increased the growth of hyphae of Botrytis cinerea. A competitive inhibitor of ethylene binding, 2,5-norbornadiene (NBD), inhibited growth of hyphae and mycelium and retarded the development of Botrytis cinerea. Transfer of the mycelium from an atmosphere containing NBD to air relieved the inhibition, indicating that the NBD effects were non-toxic and reversible. Addition of exogenous ethylene to an atmosphere containing NBD (20 ml 1^-1) effectively reduced the inhibition. Inhibition due to 40 ml 1^–1 NBD was not relieved by ethylene at any of the concentrations tested; however, a positive effect of ethylene appeared following transfer of the mycelia to air. The results suggest that ethylene may be required for the growth and development of Botrytis cinerea.Abbreviations NBD 2,5-norbornadiene - ethephon 2-chloroethyl-phosphonic acid - PDA potato dextrose agar     

Glycosphingolipid specificity of the human sulfatide activator protein

The interaction of the sulfatide activator protein with different glycosphingolipids have been studied in detail. The following findings were made. 1. The sulfatide activator protein forms water-soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe-Seyler's Z. Physiol. Chem. 356, 6588-6591] and various other glycospingolipids. 2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules. 3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein. 4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by beta-galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long-chain fatty acids in their hydrophobic ceramide anchor.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.