Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Layer-by-layer cell membrane assembly.

Quantitative analysis of phospholipids by 31P-NMR.

The phospholipids (PL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were purified from commercial soybean lecithin by silicic acid chromatography and preparative silica gel thin layer chromatography (TLC). Purified phosphatidylinositol (PI) was obtained commercially. Phosphatidic acid (PA) was made from PC by phospholipase D action and purified by preparative TLC. Commercial soybean tocopherols (TOC) were further purified in a florisil column. Combinations of PL and TOC were added to commercially refined, unhydrogenated soybean oil to determine the effects and interaction of PL and TOC on soybean-oil stability. Oil stability was determined by measuring the time in days of oil samples incubated at 110C to reach a peroxide value of 100 meq/kg. Additions of TOC and all PL except PA increased the stability of the oil. PI and PE appear to be more effective than PC in increasing oil stability. The effect of the PL was not simply a matter of pro-oxidant metal inactivation, but rather appeared to extend the effectiveness of the TOC in free-radical termination.

Phospholipids plus tocopherols increase soybean oil stability

Triacylglycerols were synthesized from 1,2-diacylglycerol and fatty acids by lipase entrapped in phosphatidylcholine reverse micelles in n-hexane. In the reaction system without reverse micelles, however, 1,2-diacylglycerol was hydrolyzed into 2-monoacylglycerol and fatty acid, and triacylglycerol was not synthesized. The maximum activity of synthetic reaction was obtained at Wo=10 (Wo=mol water/mol surfactant), which was the water content of this reverse micellar system. Though the optimal pH of theR. delemar lipase reaction is about pH 5.6 in a bulk water system, the enzyme was active for triacylglycerol synthesis at pH’s from 5 to 9 in the reverse micellar system. For the synthesis of triacylglycerols, lauric, myristic, palmitic, stearic, oleic and arachidic acids were effectively used as the fatty acid substrate. 2-Monoacylglycerol was also effective as a substrate of triacylglycerol synthesis. Furthermore, 1,2-diacylglycerol could be replaced by several kinds of aliphatic alcohols as fatty acid acceptors in the reverse micellar system. In this case, those alcohols with chain length more than 4 carbons were effectively used for ester formation.

Synthesis of triacylglycerol by lipase in phosphatidylcholine reverse micellar system

The oxidation of edible oil yields both primary and secondary oxidation products (e.g., hydroperoxides, carbonyls, hydrocarbons, and epoxides), which produce undesirable sensory and biological effects. Consequently, the suppression of lipid oxidation in food matrices is of great importance. The rate and extent of lipid oxidation in many heterogeneous foods are strongly affected by the physicochemical characteristics of water-oil interfaces. This study examined the ability of dioleoylphosphatidylcholine (DOPC) and water to form association colloids within bulk oil, as well as their impact on lipid oxidation kinetics. Attenuation was used to show the DOPC and water concentrations at which association colloids existed without altering the optical properties of the oil. Interfacial tension and fluorescence spectrometry showed the critical micelle concentration (CMC) of DOPC in stripped soybean oil was around 650 μM at room temperature. Small-angle X-ray scattering (SAXS) and fluorescence probes showed that water had a very strong impact on the properties of the association colloids formed by DOPC. Measurement of primary and secondary lipid oxidation products revealed that the association colloids formed by DOPC had a pro-oxidant effect. The characterization of association colloids could provide a better understanding of the mechanisms of lipid oxidation in bulk oils and provide insights into new antioxidant technologies.

Physical structures in soybean oil and their impact on lipid oxidation.

Phospholipid-phospholipid interaction in soybean oil is described. Phosphatidylcholine was effectively removed from soybean oil by degumming (water hydration), whereas phosphatidylethanolamine and phosphatidic acid were hardly hydratable. However, the degree of their hydration increased in the presence of phosphatidylcholine. The spectrophotometric assay based on charge transfer interaction between 7,7,8,8-tetracyanoquinodimethane and phospholipids at 480 nm was used to determine the formation of phospholipid micelles in soybean oil. The critical micelle concentrations were 0.085, 0.84 and 2.6 mM for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, respectively. Phosphatidylcholine interacted with phosphatidylethanolamine or phosphatidic acid to form mixed micelles. The critical micelle concentrations of phosphatidylcholine-phosphatidylethanolamine mixture and phosphatidylcholine-phosphatidic acid mixture were 0.16 and 1.3 mM, respectively. The degree of hydration of phospholipids was related to their critical micelle concentrations. Interaction of phosphatidylcholine with phosphatidylethanolamine or phosphatidic acid was confirmed by determining the changes in the chemical shifts of 31PNMR spectra.

Y. Wada

Papers

Phospholipid- phospholipid interaction in soybean oil