Vesicle assays

Halevy R, Rozek A, Kolusheva S, Hancock RE, Jelinek R. Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay. Peptides 2003 24 1753-1761. 

An alternative method to overcome this issue is to perform uptake studies by using membrane vesicles prepared from cDNA-expressing and control cells [5]. Vesicle preparation and vesicle assays are, however, labor intensive. BCRP, MRPs, and BSEP inside-out vesicles prepared from insect cells by using baculovirus system are currently available from BD Biosciences, Solvo, and GenoMembrane. 

Lipoproteins have also been used for studying protein-stimulated phospholipid transfer between lipoproteins (Ihm et al., 1982), and between lipoproteins and vesicles (Damen et al., 1981, 1982). Care must be exercised when high density lipoprotein is used as a substrate in an exchange reaction with vesicles, since vesicles may be disrupted and net transfer of lipid results (Damen et al., 1981). A high density lipoprotein-vesicle assay with appropriate precautions has been employed by Damen et al. (1981). 

A complex relationship exists between the initial rate of lipid transfer and the concentration of acceptor and donor particles (Van den Besselaar et al., 1975 Wirtz et al., 1979). An example of this is illustrated in Figure 1. At a given concentration of acceptor, a low concentration of donor results in a low rate of lipid transfer. When the concentration of donor is increased, so is the transfer rate until a maximum in the transfer rate is reached. A further increase in the donor concentration results in a decline in the transfer rate. In a typical assay, acceptor lipid is present in excess of donor lipid. For example, about four times as much exchangeable acceptor as donor lipid is used in the small unilamellar vesicle-multilamellar vesicle assay (Crain and Zilversmit, 1980b). This minimizes the back-transfer of labeled lipid from the acceptor to the donor particle during the exchange reactions while still maintaining rapid rates of lipid transfer. 

Alkamides also displayed marked inhibitory activity in vitro in the 5-lipoxygenase (porcine leukocytes) and cyclooxygenase (microsomes from ram seminal vesicles) assays. Dodeca-2 ,4 ,8Z,10 /Z-tetraenoic acid isobutylamides inhibited cyclooxygenase-1 at a concentration of 50 p g/ml by 54.7% and 5-lipoxygenase at a concentration of 50 liM by 62.2% [56]. 

The vesicle assay previously used ves a good survey of transporter activity under a fixed set of conditions. However it is not suited to exploration of transport activity as a function of the sign and magmtude of the transmembrane potential. Bilayer conductance, or single channel recording techniques are required (21). In this techmque, a lipid bilayer is formed across a small hole in a Teflon barrier, by direct application of the lipid in decane to the hole. Under favorable conditions, Ae lipid tl s to a bilayer membrane which electrically isolates the two halves of the cell. T icdly KCl is used as an electrol, and electrical contact is made via Ag/AgQ wires directly inserted into the solutions. A high impedance operational amplifier circuit (bUayer clamp) can then be used to apply a fixed transmembrane potential and to monitor the current which flows as a function of time. The two sides of the membrane are independently accessible, so different sequences of transporter addition, control of pH, and other variables are in principle j ssible. 

Rodent 5-7-day Hershberger assay change in weight of prostate and seminal vesicles in castrated rats. 

The application of modified electrodes for the assay of antibodies in senun preparations using redox indicators encapsuled into antigene marked liposomes attached to an electrode surface was suggested First model studies towards this goal make use of ferricyanide ions entrapped in synthetic vesicles. 

Subsequently, proteolytic fragments of the rabbit renal 25-kDa amiloride-binding protein were micro-sequenced and found to have high sequence homology with rat and human NAD(P)H quinone oxidoreductase. Indeed, enzymatic assays revealed that renal brush border membrane vesicles contain significant NADPH quinone oxidoreductase activity. Presumably NAD(P)H quinone oxidoreductase coincidentally binds amiloride analogs with the same rank order as the Na /H exchanger [39]. 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

Nichols, J. W. and Pagano, R. E. (1983). Resonance energy-transfer assay of protein-mediated lipid transfer between vesicles. J. Biol. Chem. 258, 5368-5371. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

Membranes and models membrane organization (e.g. membrane domains, lipid distribution, peptide association, lipid order in vesicles, membrane fusion assays, etc.)... 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

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