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PubMed1984

Statistical mechanics of lipid membranes. Protein correlation functions and lipid ordering.

An expression is derived for the lipid-mediated intermolecular interaction between protein molecules embedded in a lipid bilayer. It is assumed that protein particles are accommodated by the bilayer, but they distort the lipids in some manner from their equilibrium protein-free configuration. We treat this situation by expanding the free energy density in the plane of the membrane as a Taylor series in some arbitrary parameter and its gradient. Minimization of the total membrane energy for a given particle configuration yields the interparticle interaction energy for that configuration. A test of the model is provided by measurement of the protein-protein pair distribution function from freeze-fracture micrographs of partially aggregated membranes. The measured functions can be simulated by adjustment of two parameters (a) a lipid correlation length that characterizes the distance over which a distortion of the bilayers is transmitted laterally through the bilayer, and (b) a term quantifying the energy of the protein-lipid interaction at the protein-lipid boundary. Correlation lengths obtained by fitting the calculated particle distribution functions to the data are found to be several nanometers. Protein-lipid interaction energies are of the order of a few kT.

Biophysical Phenomena↗
PubMed1998

Effect of extrusion pressure and lipid properties on the size and polydispersity of lipid vesicles.

The production of vesicles, spherical shells formed from lipid bilayers, is an important aspect of their recent application to drug delivery technologies. One popular production method involves pushing a lipid suspension through cylindrical pores in polycarbonate membranes. However, the actual mechanism by which the polydisperse, multilamellar lipid suspension breaks up into a relatively monodisperse population of vesicles is not well understood. To learn about factors influencing this process, we have characterized vesicles produced under different extrusion parameters and from different lipids. We find that extruded vesicles are only produced above a certain threshold extrusion pressure and have sizes that depend on the extrusion pressure. The minimum pressure appears to be associated with the lysis tension of the lipid bilayer rather than any bending modulus of the system. The flow rate of equal concentration lipid solutions through the pores, after being corrected for the viscosity of water, is independent of lipid properties.

Biophysics↗
PubMed2000

The effect of dietary treatment on erythrocyte lipid peroxidation, superoxide dismutase, glutathione peroxidase, and serum lipid peroxidation in patients with type 2 diabetes mellitus.

OBJECTIVES: The aim of the present study was to investigate the effect of dietary treatment on serum and erythrocyte lipid peroxidation and erythrocyte antioxidative enzyme activity of patients with Type 2 diabetes. DESIGN AND METHODS: A total of 30 patients with newly diagnosed as Type 2 diabetes were enrolled to the study. A total of 30 healthy subjects served as controls. Diabetic patients were given standard dietary treatment that was composed of 50% to 55% carbohydrate and 30% fat for 2 months. No diet was applied for controls. For both groups serum and erythrocyte lipid peroxidation and erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were obtained at first and at the end of 2 months. RESULTS: Diabetic patients had higher serum and erythrocyte lipid peroxidation than those of controls before dietary treatment(p < 0.05). However, there was no absolute differences in erythrocyte SOD and GSH-Px (p > 0.05). At the end of 2 months of dietary treatment, while diabetics had still higher glucose and erythrocyte lipid peroxidation than controls (p < 0.05), serum lipid peroxidation, erythrocyte SOD, and GSH-Px levels did not differ significantly from those of controls (p > 0.05). In diabetic patients, after 2 months of dietary treatment, whereas serum and erythrocyte lipid peroxidation decreased, erythrocyte SOD and GSH-Px activities showed significant increase (p < 0.05). CONCLUSIONS: Our results showed significant alteration in serum and erythrocyte lipid peroxidation and erythrocyte antioxidant enzyme status of patients with Type 2 diabetes by dietary treatment. However, whether such alterations have clinical importance for diabetic patients needs further investigation.

Adult↗
PubMed1980

Pulmonary arterial lipid deposit in newborn infants receiving intravenous lipid infusion.

Three newborn infants are described with a distinctive pulmonary arterial lipid lesion, characterized by a wide foamy layer of intima partially occluding the lumen of small muscular pulmonary arteries. Lipid stains indicated lipid infiltration of the walls of these arteries, involving not only the intimal layer, but media and adventitia as well. All three infants had received intravenous lipid infusions. All three infants also had histologic evidence of pulmonary hypertension, one on the basis of chronic lung disease (BPD) and two on the basis of congenital heart disease. A retrospective autopsy study of long-term neonatal ICU dwellers receiving such infusions revealed two additional patients with lipid deposits in pulmonary arterial walls. It is speculated that the pulmonary arterial lipid deposits were derived from the lipid infusions and were dependent on antecedent or concurrent vascular damage from pulmonary hypertension.

Chronic Disease↗
PubMed2001

Characterization of oligonucleotide/lipid interactions in submicron cationic emulsions: influence of the cationic lipid structure and the presence of PEG-lipids.

We have recently described how oligonucleotide (ON) stability and release from O/W cationic emulsions are governed by the lipid composition. The aim of the present paper was to investigate the properties of the ON/lipid complexes through fluorescence resonance energy transfer (FRET), size, surface tension measurements and cryomicroscopy. Starting from a typical emulsion containing stearylamine as a cationic lipid, the influence of the lipid structure (monocationic molecules bearing mono or diacyl chains, or polycations) as well as of the presence of PEGylated lipids, were studied. The presence of a positive charge on the droplet surface clearly contributed to enhance the ON interaction with lipid monolayers and to bring the ON molecules closer to the interface. Hydrophobic interactions through the acyl chains were shown to further enhance the anchorage of the ON/lipid complexes. In contrast, the incorporation of PEGylated lipids acted as a barrier against the establishment of electrostatic bindings, the polyethyleneglycol chains acting themselves as interaction sites for the ON leading to hydrophilic complexes. Similar features were observed for the polycationic lipid, and cryomicroscopy revealed the existence of bridges of various intensities between the droplets of the emulsion containing either PEG or the polycation, probably because of the configuration of the ON at the interface.

Algorithms↗
PubMed1989

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers.

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.

Bacteriophages↗
PubMed2006

Microsomal triglyceride transfer protein is required for yolk lipid utilization and absorption of dietary lipids in zebrafish larvae.

Although the absorption, transport, and catabolism of dietary lipids have been studied extensively in great detail in mammals and other vertebrates, a tractable genetic system for identifying novel genes involved in these physiologic processes is not available. To establish such a model, we monitored neutral lipid by staining fixed zebrafish larvae with oil red o (ORO). The head structures, heart, vasculature, and swim bladder stained with ORO until the yolk was consumed 6 days after fertilization (6 dpf). Thereafter, the heart and vasculature no longer had stainable neutral lipids. Following a high-fat meal, ORO stained the intestine and vasculature of 6 dpf larvae, and whole-larval triacylglycerol (TAG) and apolipoprotein B levels increased. Levels of microsomal triglyceride transfer protein (Mtp), the protein responsible for packaging TAG and betalipoproteins into lipoprotein particles, were unchanged by feeding. Since the developing zebrafish embryo expresses mtp in the yolk cell layer, liver, and intestine, we determined the effect of targeted knockdown of Mtp expression using an antisense morpholino oligonucleotide approach (Mtp MO) on the transport of yolk and dietary lipids. Mtp MO injection led to loss of Mtp expression and of lipid staining in the vasculature, heart, and head structures. Mtp MO-injected larvae were smaller than age-matched, uninjected larvae, consumed very little yolk, and did not absorb dietary neutral lipids; however, they absorbed a short chain fatty acid that does not require Mtp for transport. Importantly, the vasculature appeared unaffected in Mtp MO-injected larvae. These studies indicate that zebrafish larvae are suitable for genetic studies of lipid transport and metabolism.

Animals↗
PubMed1999

Organization and dynamics of the proteolipid complexes formed by annexin V and lipids in planar supported lipid bilayers.

The consequences of the binding of annexin V on its lateral mobility and that of lipids were investigated by means of experimental and simulated FRAP experiments. Experiments were carried out on planar supported bilayers (PC/PS 9:1 mol/mol mixtures) in the presence of 1 mM CaCl2 in the subphase. The probes C12-NBD-PS and fluorescein-labeled annexin V were used and the data compared with that previously obtained for C12-NBD-PC [Saurel, O., Cézanne, L., Milon, A., Tocanne, J. F., & Demange, P. (1998) Biochemistry 37, 1403-1410]. At complete coverage of the lipid bilayer by the protein (Cannexin = 80 nM), the lateral mobility of C12-NBD-PC was reduced by 40% while C12-NBD-PS and bound annexin V molecules were nearly immobilized (D < 10(-)11 cm2/s). At moderate protein concentration (20 nM < Cannexin < 80 nM), best fitting of the lipid and protein probe recoveries was achieved with one single diffusion coefficient and a mobile fraction close to 100%, indicating homogeneous lipid and protein populations. In contrast, at low protein concentration (Cannexin < 20 nM), C12-NBD-PS showed a two-component diffusion. The slow PS population at Cannexin < 20 nM and the single PS population at Cannexin > 20 nM moved at the same rate that bound annexin V (mobile fraction close to 100%), indicating strong PS/protein interactions. With the aid of computer simulations of the lateral motion of PC molecules, based on the 2-D crystalline networks formed by annexin V in contact with the lipid bilayer, these FRAP results may be accounted for by considering a rather simple model of a proteolipidic complex consisting of an extended 2-D crystalline protein network facing the lipid bilayer and stabilized by strong interactions between annexin V and PS molecules. In this model, immobilization of annexin V and PS molecules originates from their mutual interactions. The slowing down of PC molecules is due to various obstacles to their lateral diffusion which can be described as: the four PS molecules bound to the protein, the tryptophan 187 which presumably interacts with the lipids at the level of their polar headgroups and probably the three other hydrophobic amino acid residues located on the AB calcium-binding loops of the protein.

Animals↗
PubMed2005

Rhodopsin reconstituted into a planar-supported lipid bilayer retains photoactivity after cross-linking polymerization of lipid monomers.

Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function.

Biosensing Techniques↗
PubMed1991

C-glycosidic analogues of lipid A and lipid X: synthesis and biological activities.

The synthesis of a series of novel analogues of lipid A, the lipophilic terminal of lipopolysaccharides (LPS), and lipid X, the reducing monosaccharide unit in lipid A, is reported. In these compounds, the native 1-O-phosphate group has been replaced by a "bioisosteric" CH2COOH substituent. The new N,O-acylated monosaccharide C-glycosides were obtained by Wittig reaction of suitably protected glucosamine derivatives. These lipid X analogues were recognized as substrates by the enzyme lipid A synthase and could be coupled with UDP-lipid X to afford the corresponding disaccharide analogues of the lipid A precursor on preparative scale. All compounds were characterized by NMR, MS, and elemental analysis, and were tested for their ability to enhance nonspecific resistance to infection in mice and also for endotoxicity. The results clearly show that the new compounds express biological activities similar to those of their O-phosphorylated natural counterparts. Furthermore, these compounds exhibit a better therapeutic index in mouse models than the standard LPS obtained from Salmonella abortus equi.

Animals↗
PubMed2002

Position of residues in transmembrane peptides with respect to the lipid bilayer: a combined lipid Noes and water chemical exchange approach in phospholipid bicelles.

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH2) was incorporated into small unaligned phospholipid bicelles, which provide a 'native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the 'bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively 15N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.

Amides↗
PubMed1999

Human skin surface lipid film: an ultrastructural study and interaction with corneocytes and intercellular lipid lamellae of the stratum corneum.

Sebum is a complex mixture of lipids, which is secreted by mammalian sebaceous glands, and forms a fluid film over the skin surface. After sebum is secreted, it becomes mixed with lipid from the keratinizing epithelium and forms the skin surface lipid film (SSLF). Until now, direct fine structural observation of the SSLF has been lacking. In the present work, we viewed the detailed structures of the human SSLF by ruthenium tetroxide staining. The results showed that the SSLF formed an amorphous sheet of variable thickness on the skin surface instead of forming lipid droplets, as had been the usual assumption. In general, its thickness was < 0.5 microm or even negligible in sebum-poor extremities. However, in the sebum-rich face, its thickness was > 4 microm in focal areas. Consistent with the thickness of SSLF, the sebum quantity showed great regional variation. It varied from 1 microg/cm2 (leg) to 189 +/- 42.7 microg/cm2 (mean +/- SD: face). The SSLF was composed of numerous fine granules of about 4-5 nm in a random orientation. Within the SSLF, variable amounts of deranged lipid lamellae derived from corneocytes were mixed with sebum. As well as on the skin surface, a similar amount of sebum was also found between the desquamating corneocytes in the uppermost several layers of the stratum corneum (SC). We also observed the presence of intercellular lipid lamellae in the outer layers of the SC: their lipid envelope remained intact even in desquamated corneocytes. Our results provide some new insights concerning the structure of the SSLF and its relationship with the SC.

Adult↗
PubMed2003

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum.

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Effects of family history of heart disease, apolipoprotein E phenotype, and lipoprotein(a) on the response of children's plasma lipids to change in dietary lipids.

We examined the effects of family history of coronary artery disease (CAD), apolipoprotein E (apo E) phenotype, and lipoprotein(a) [Lp(a)] on the response of plasma lipids to change in dietary lipid intake after 3 mo of nutrition education in 125 children aged 4-10 y. The subjects were healthy children with elevated low-density-lipoprotein (LDL)-cholesterol concentrations who participated in the Children's Health Project, a nutrition-education program designed to lower plasma cholesterol by means of dietary modifications in accordance with recommendations of the National Cholesterol Education Program. Dietary and plasma lipids were measured by three 24-h recalls and assessments of two fasting plasma samples collected before and 3 mo after the start of intervention. Family history of CAD was determined by questionnaires administered to parents at baseline. Apo E phenotyping was done with isoelectric focusing followed by immunostaining; Lp(a) was measured with two-site immunoradiometric assays of frozen aliquots of plasma samples collected at baseline and 3 mo. After adjustment for intervention group, age, sex, and body mass index, analysis of covariance showed that baseline plasma lipid concentrations were the strongest independent predictors of change in plasma lipids after 3 mo. Plasma total and LDL-cholesterol concentrations in children with less family history of CAD were significantly more responsive to change in dietary cholesterol than concentrations in children with a stronger family history of CAD. Neither apo E phenotype nor Lp(a) significantly influenced change in plasma lipids independently or interactively with change in dietary lipids.

Apolipoproteins E↗
PubMedDate not supplied

Lipid phase structure in the regulation of lipid composition in Acholeplasma laidlawii membranes.

In Acholeplasma laidlawii membranes the ratio between the dominating lipids, monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), depends on temperature, configuration of incorporated fatty acids, and membrane cholesterol content, which affect the molecular geometry of the lipids. MGDG and DGDG have wedge- and rod-like molecular shapes, respectively, that are modifiable. The packing constraints of lipids in amphiphilic aggregates, i.e., the area of the hydrocarbon-water interface and the volume and length of the hydrocarbon chains, are important in determining the aggregate structure. Pure MGDG forms a reversed hexagonal- (HII) phase structure with different acyl chain contents, while DGDG forms a lamellar phase. Depending on the unsaturated acyl chain content in the lipids, an in vitro mixture of MGDG and DGDG forms lamellar or cubic phases at physiologic temperatures. A high degree of cis-unsaturation, large amounts of MGDG and high temperatures favor formation of the cubic phase. Addition of cholesterol corresponding to the maximal amount incorporable into A. laidlawii induces a transition from a lamellar or a cubic phase to a reversed hexagonal phase. Lipid mixtures containing only unsaturated acyl chains are more sensitive to the bilayer-destabilizing effect of cholesterol than are mixtures with equal amounts of saturated and unsaturated acyl chains. The lamellar phase is the only one compatible with a functional biological membrane. Consequently, the balance between lipids that form lamellar and other mesophase structures must keep within certain limits. The cubic and reversed hexagonal structures were discovered under conditions not existing in the living Acholeplasma cell. Thus, the response of A. laidlawii lipid metabolism to external and internal stimuli can be predicted on the basis of molecular shapes and is necessary to the maintenance of optimal membranes stability. The reduced capacity of Acholeplasma membranes to incorporate cholesterol is a consequence of this regulation.

Acholeplasma laidlawii↗
PubMed1988

Inositol lipids: receptor-stimulated hydrolysis and cellular lipid pools.

Our current knowledge of the process by which receptors stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has its origin in the discovery by Hokin & Hokin (J. biol. Chem. 263, 967 (1953] that some pancreatic secretagogues not only elicit exocrine secretion but also stimulate the metabolism of membrane phospholipids. Despite the recent elucidation of many aspects of this widespread signalling system, there is still little information on the control of the supply of its substrate, PtdIns(4,5)P2. In particular, some studies have suggested that inositol-lipid-mediated signalling involves much or all of the inositol lipid complement of the stimulated cells, whereas other observations have equally clearly implicated the receptor-activated hydrolysis of an inositol phospholipid pool that comprises only a small fraction of the total cellular complement of these lipids. These studies, which have largely employed radiochemical analyses using single isotopes, are briefly reviewed. In addition, we report the first information obtained by a new procedure for analysing the metabolic characteristics of the inositol lipids that are broken down during stimulation. This technique employs cells that are doubly labelled in the inositol moiety of their lipids (to isotopic equilibrium with 14C and only briefly with 3H) to search for functional metabolic heterogeneity among the inositol lipids of stimulated cells. Using this method, we have found that the inositol phosphates liberated in stimulated cells during brief stimulation of V1a-vasopressin receptors or prostaglandin F2 alpha receptors come from phospholipid that has a turnover rate typical of the bulk of the cellular inositol lipids.

Animals↗
PubMed1993

Lipid supplemented medium induces lamellar bodies and precursors of barrier lipids in cultured analogues of human skin.

Barrier function of cultured skin substitutes (CSS) is required for their effective use in clinical treatment of skin wounds, and for percutaneous absorption in vitro. Arachidonic, palmitic, oleic, and linoleic free fatty acids, in conjunction with the antioxidant alpha-tocopherol acetate (lipid supplements, "LS"), were added to nutrient media of CSS to provide precursors of epidermal barrier lipids. CSS were composed of human keratinocytes (HK), fibroblasts (HF), and collagen-glycosaminoglycan substrates, and were incubated for 14 d submerged or lifted to the air-liquid interface in media based on MCDB 153 +/- LS. Duplicate samples (30 cm2) were harvested and the epidermal analogue was analyzed for total protein, total DNA, total lipid, lipid fractions including acylglucosylceramide (AGC), and presence of lamellar bodies. Significant increases (p < 0.05) were detected between CSS incubated in +LS medium for total lipid, total DNA, ceramide, glucosylceramide, triglycerides, and diglycerides. AGC and lamellar bodies were detected only in epithelia of CSS incubated in +LS medium. These data show that free fatty acids, vitamin E, and lifting of CSS promote increased epithelial morphogenesis compared to CSS cultured submerged without lipid supplements. Presence of lamellar bodies and AGC suggests enhanced production in vitro of barrier-associated epidermal lipids.

Cell Cycle↗
PubMed1984

Solubilization of mannosyltransferase activities for the biosynthesis of mammary glycoproteins. Elongation of tetrasaccharide-lipid to heptasaccharide-lipid by a solubilized enzyme preparation.

The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP-40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147000 X g for 120 min, the supernatant fraction was incubated with labeled sugar nucleotides, GDP-Man and UDP-GlcNAc. It was found to synthesize a series of lipid-linked saccharides up to (Man)5-(GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after two weeks of storage at 4 degrees C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1). A labeled lipid-linked tetrasaccharide of the structure Man alpha 1----3 Man beta----GlcNAc beta----GlcNAc was isolated by labeling baby hamster kidney cells with [2-3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by high-performance liquid chromatography. When this lipid-linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP-Man, it was elongated to a lipid-linked heptasaccharide having the structure Man alpha 1----2Man alpha 1----2Man alpha 1----3(Man alpha 1----6)Man beta----GlcNAc beta----GlcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid-linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP-Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as mannosyl donor for the elongation reaction.

Animals↗