Solution structure of neuromedin B by (1)H nuclear magnetic resonance spectroscopy.
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The solution structure of neuromedin B (NMB) was investigated using two-dimensional nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking environments. NMB adopts a relaxed helical conformation from Trp(4) to Met(10) in 50% aqueous 2,2, 2-trifluoroethanol (TFE) solution and in 150 mM SDS micelles. Sidechain atoms of the three residues, Trp(4), His(8) and Phe(9) orient toward the same direction and these residues might play a key role on interacting with hydrophobic acyl chains of the phospholipids in the membrane. NOESY experiments performed on NMB in non-deuterated SDS micelle show that aromatic ring protons of Trp(4) and Phe(9) residues are in close contact with methylene protons of SDS micelles. In addition, proton longitudinal relaxation data proved that the interactions between NMB with SDS micelle are characterized as extrinsic interaction. Trp(4) and Phe(9) seem to be important in interaction with receptor and this agrees with the previous studies of structure-activity relationship (Howell, D.C. et al. (1996) Int. J. Pept. Protein Res. 48, 522-531). These conformational features might be helpful in understanding the molecular mechanism of the function of NMB and developing the efficient drugs. (+info)
Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.
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Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles. (+info)
Asymmetric distribution of phosphatidylcholine and sphingomyelin between micellar and vesicular phases. Potential implications for canalicular bile formation.
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Both phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids in the outer leaflet of the hepatocyte canalicular membrane. Yet, the phospholipids secreted into bile consist principally (>95%) of PC. In order to understand the physical;-chemical basis for preferential biliary PC secretion, we compared interactions with bile salts (taurocholate) and cholesterol of egg yolk (EY)SM (mainly 16:0 acyl chains, similar to trace SM in bile), buttermilk (BM)SM (mainly saturated long (>20 C-atoms) acyl chains, similar to canalicular membrane SM) and egg yolk (EY)PC (mainly unsaturated acyl chains at sn-2 position, similar to bile PC). Main gel to liquid-crystalline transition temperatures were 33. 6 degrees C for BMSM and 36.6 degrees C for EYSM. There were no significant effects of varying phospholipid species on micellar sizes or intermixed-micellar/vesicular bile salt concentrations in taurocholate-phospholipid mixtures (3 g/dL, 37 degrees C, PL/BS + PL = 0.2 or 0.4). Various phases were separated from model systems containing both EYPC and (EY or BM)SM, taurocholate, and variable amounts of cholesterol, by ultracentrifugation with ultrafiltration and dialysis of the supernatant. At increasing cholesterol content, there was preferential distribution of lipids and enrichment with SM containing long saturated acyl chains in the detergent-insoluble pelletable fraction consisting of aggregated vesicles. In contrast, both micelles and small unilamellar vesicles in the supernatant were progressively enriched in PC. Although SM containing vesicles without cholesterol were very sensitive to micellar solubilization upon taurocholate addition, incorporation of the sterol rendered SM-containing vesicles highly resistant against the detergent effects of the bile salt. These findings may have important implications for canalicular bile formation. (+info)
Cellular internalization of PCL(20)-b-PEO(44) block copolymer micelles.
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The cellular internalization of polycaprolactone-b-poly(ethylene oxide) (PCL(20)-b-PEO(44)) copolymer micelles were investigated in PC12 cells cultures. The micelles were found to be internalized into PC12 cells when followed over the 4-h incubation period. Also, the internalization process was found to fulfill the basic criteria for endocytotic uptake in that it was time, temperature, pH and energy dependent. In addition, the use of other pharmacological manipulations (hypertonic treatment, Brefeldin A) provide further evidence that the mode of cellular internalization is in fact endocytotic. (+info)
A combinatorial approach to producing sterically stabilized (Stealth) immunoliposomal drugs.
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We have developed a method for producing sterically stabilized immunoliposomal drugs (SIL) readily applicable to a 'mix and match' combinatorial approach for the simple manufacture of a variety of ligand-targeted liposomal drugs. Ligands coupled to the terminus of polyethylene glycol (PEG) in micelles formed from PEG-lipid derivatives (mPEG2000-DSPE) could be transferred into preformed, drug-containing liposomes from the micelles in a temperature- and time-dependent manner. Antibody densities up to 100 microg antibody/micromol of phospholipid, and up to 3 mol% of mPEG2000-DSPE, could be simultaneously transferred from the ligand-coupled micelles into the liposomal outer monolayer with negligible drug leakage from liposomes during transfer and good stability in human plasma. Transfer of anti-CD19 into SIL resulted in a three-fold increase in binding of these liposomes to CD19+ human B cell lymphoma cells. (+info)
The recently determined structure of the lipid-binding 'FYVE' domain provides several clues to the mode of interaction for this class of peripheral membrane proteins. However, the application of traditional modes of structural analysis to diffusible membrane-binding proteins exposes some limitations of these techniques. (+info)
Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract.
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BACKGROUND: The extent of fat emulsification affects the activity of digestive lipases in vitro and may govern digestion and absorption of dietary fat. OBJECTIVE: We investigated the effect of the fat globule size of 2 enteral emulsions on fat digestion and assimilation in humans. DESIGN: Healthy subjects received intragastrically a coarse (10 microm) and a fine (0.7 microm) lipid emulsion of identical composition in random order. Gastric and duodenal aspirates were collected throughout digestion to measure changes in fat droplet size, gastric and pancreatic lipase activities, and fat digestion. Blood lipids were measured postprandially for fat assimilation. RESULTS: Despite an increase in droplet size in the stomach (2.75-6.20 microm), the fine emulsion retained droplets of smaller size and its lipolysis was greater than that of the coarse emulsion (36.5% compared with 15.8%; P < 0.05). In the duodenum, lipolysis of the fine emulsion was on the whole higher (73.3% compared with 46.3%). The overall 0-7-h plasma and chylomicron responses given by the areas under the curve were not significantly different between the emulsions, but the triacylglycerol peak was delayed with the fine emulsion (3 h 56 min compared with 2 h 50 min). CONCLUSIONS: Fat emulsions behave differently in the digestive tract depending on their initial physicochemical properties. A lower initial fat droplet size facilitates fat digestion by gastric lipase in the stomach and duodenal lipolysis. Overall fat assimilation in healthy subjects is not affected by differences in initial droplet size because of efficient fat digestion by pancreatic lipase in the small intestine. Nevertheless, these new observations could be of interest in the enteral nutrition of subjects suffering from pancreatic insufficiency. (+info)
Models for enzyme superactivity in aqueous solutions of surfactants.
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Theoretical models are developed here for enzymic activity in the presence of direct micellar aggregates. An approach similar to that of Bru et al. [Bru, Sanchez-Ferrer and Garcia-Carmona (1989) Biochem. J. 259, 355-361] for reverse micelles has been adopted. The system is considered to consist of three pseudo-phases: free water, bound water and surfactant tails. The substrate concentration in each pseudo-phase is related to the total substrate concentration in the reaction medium. In the absence of interactions between the enzyme and the micelles, the model predicts either monotonically increasing or monotonically decreasing trends in the calculated reaction rate as a function of surfactant concentration. With enzyme-micelle interactions included in the formulation (by introducing an equilibrium relation between the enzyme confined in the free water and in the bound water pseudo-phases, and by allowing for different catalytic behaviours for the two forms), the calculated reaction rate can exhibit a bell-shaped dependence on surfactant concentration. The effect of the partition of enzyme and substrate is described, as is that of enzyme efficiency in the various pseudo-phases. (+info)