Phospholipid vesicles measuring vesicle concentrations

The increase in Ca is initiated rapidly and begins to recover after 1 min. The order of potency correlates fairly well with the solubilities of these compounds in organic solvents (37) and their abilities to accumulate in phospholipid vesicles (38), i.e., 6>y>a>p, but not with their insecticidal activity (y 6>a p 39). At these concentrations, crystals of p-, a-, and y-HCH were evident in the cell suspensions when we made simultaneous measurements of the right-angle light scatter, indicating that the order of aqueous solubilities is 6>y>a>p. However, stimulation by 6-HCH at concentrations below its aqueous solubility limit shows a typical dose dependency of the response (Figure 10). 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

The transfer of radiolabeled phospholipids between vesicles and erythrocyte membranes could be used to assay lipid transfer activity. Intact erythrocytes are not an ideal substrate for routine measurements of transfer activity because some transfer proteins do not readily accelerate the transfer of phospholipids from these membranes. Van Meer et al. (1980) found that a very high concentration of the phosphatidylcholine-specific transfer protein was necessary to exchange the phosphatidylcholine of intact red blood cells. Erythrocyte ghosts are a more active substrate for this protein (Bloj and Zilversmit, 1976). However, the nonspecific transfer protein from bovine liver accelerates the exchange of phospholipid between intact erythrocytes and phosphatidylcholine vesicles (Crain and Zilversmit, 1980c). 

Roseman and Thompson (1980) used a higher concentration of pyrene-labeled phospholipids (3.8 mol%) in phosphatidylcholine vesicles to measure the rate of spontaneous transfer of these fluorescent phospholipids. An equation was derived that relates the sum of the monomer and eximer intensities in the donor and acceptor vesicles to the extent of lipid transfer. This assay requires a calibration procedure that relates the change in the concentration of the fluorescent derivative in the bilayer to the change in the monomer and eximer fluorescent intensities. Oxygen... 

The universal character of calorimetric measurements also pays in the elucidation of the reversible transformations undergone by bilayer-forming phospholipids. The transitions of phosphatidylcholine and similar congeners between their vesicular and micellar states depending on their concentrations, the presence of simple detergents, and the temperature are quite sharp and accompanied by sensible heat effects that allow for their thermodynamic characterization. In a particularly illustrative example, the dissolution and reconstitution of lipid vesicles from Escherichia coli native polar lipid fraction by octyl-jS-o-glucoside as analyzed by ITC was reported (Figure 15). ... 

The capacity of release of PL molecules from sonicated vesicles was measured by means of the translocation rate of PL molecules across a dialysis membrane. DPPC vesicles, about 2 ymol Pi, were added to the upper cell of the dialysis apparatus. The amount of PL translocated was measured by determining the inorganic phosphate content in the lower cell and it was found to increase linearly with the time. Fig. 1 shows the PL translocation rate from vesicles with different chain length when variable amounts of myristic acid were added to the upper cell of the dialysis apparatus. Temperature was 50°C. The translocation rate abruptly increased at a "critical" myristic acid concentration, dependent on the phospholipid chain length. Similar results were obtained when myristic acid was cosonicated with PL molecules to form mixed vesicles. The abrupt increase occurred with vesicles either in the solid or in the liquid--crystalline state. However the "critical" value of myristic acid... 

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