Membrane vesicle preparation

Martin, R.J., Kusel, J.R. and Pennington, A.J. (1990) Surface-properties of membrane-vesicles prepared from muscle-cells of Ascaris suum. Journal of Parasitology 76, 340-348. 

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]. 

Fujii R, Mutoh M, Sumizawa T, Chen Z, Yoshimura A, Akiyama S. Adenosine triphosphate-depen-dent transport of leukotriene C4 by membrane vesicles prepared from cisplatin-resistant human epidermoid carcinoma tumor cells. J Natl Cancer Inst 1994 86 1781-1784. 

Membrane vesicles, prepared from CHO cells, have been used to determine the kinetic parameters associated with P-gp efflux (97,98). Factors such as the ATP hydrolysis rate associated with transport of various substrates have been studied along with the Michaelis-Menten parameters of efflux for various substrates. 

Fig. 5.—Stimulation of UDP-gIucose glucan Synthetase Activities by Conditions that Lead to Induction of a Transmembrane, Electrical Potential.169 The experiment was performed by using membrane vesicles prepared from developing cotton-fibers incorporation of radioactivity from UDP-D-[,4C]glucose into total /3-D-glucans was measured. Anion and cation concentrations were 50 mM valinomycin (VAL) was present at 5 u.M and UDP-glucose at 0.1 mM 1 /xCi per irmol. ...

After preincubation of the brush border membrane vesicle preparation for 2 h, [2 14 C]urate uptake is initiated by adding 200 pi of incubation medium to 20 pi of the membrane suspension. The incubation medium has the following composition (mmol/1) 150 mannitol, 2 MgS04, 50 potassium phosphate buffer, pH 6.0 or 7.5, 0.02 [2-14 C]urate, and various concentrations of the inhibitor. At 10 s after the addition of the incubation medium, 200 pi portions of the suspension are pipetted onto the center of prewetted cellulose acetate filters kept under suction. The vesicles retaining on the filter are washed immediately with 5 ml of an ice-cold solution containing 150 mmol/1 mannitol and 50 mmol/1 potassium phosphate buffer, pH 6.0 or 7.5, which is used at the same pH as the incubation medium. Preincubations and incubations are performed at 23 1 °C. Each experiment is performed in triplicate. Corrections are made for the radioactivity bound to the filters in the absence of membrane vesicles. The term of the OH gradient-dependent urate uptake is defined as the difference between the uptakes in the incubation medium at pH 6.0 and that at pH 7.5. The OII gradient-dependent urate uptake at 10 s is assumed to present an initial velocity. 

Selection of the cell line or tissue type influences not only the technique for membrane vesicle preparation but also the resulting percentage of inside-out-oriented plasma membrane vesicles. A sufficient amount of inside-out-oriented vesicles is essential, since only this fraction, with the ATP-binding domains oriented to the outer surface, mediates ATP-dependent transport of a labeled substrate into the vesicle. 

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. 

Gastric membrane vesicle preparations enriched with H /K -ATPase have also been used to examine PPIs. The inhibition of hydrogen ion transport by PPIs is measured by use of the initial rate of acridine orange quenching as an index of acidification. However, steady-state acidification, as measured by aminopy-rine accumulation, is inhibited with greater potency and this is consistent with the accumulation of PPIs in the intravesicular acidic space (38). 

Daniell LC (1992) Determination of the intravesicular ionized sodium concentration in a cell-free brain membrane vesicle preparation using the fluorescent indicator, SBFI. Anal Biochem 202 239-244... 

Non-bilayer-forming lipids are also required for protein translocation across the membrane of E. coli. The only non-bilayer-forming lipid in E. coli mutants lacking PE is CL. Protein translocation into inverted membrane vesicles prepared from PE-lacking cells (now enriched in CL) is reduced with divalent cation-depletion but can be enhanced by inclusion of Mg or Ca [ 1 ]. Protein translocation in the absence of divalent cations was restored by incorporation of non-bilayer PE (18 1 acyl chains) but not by bilayer-prone PE (14 0 acyl chains). These results indicate that lipids with a tendency to form non-bilayer stmctures provide a necessary environment for translocation of proteins across the membrane. 

Flavopiridol was also shown to increase the activity of ATPase in plasma membrane vesicles prepared from a cell line GLC4/ADR which over-expresses multidrug resistance protein (MRP) [26]. The production and activity of MRP is dependent on ATP and so the increase in ATPase activity, stimulated by flavopiridol, reduces the influence of MRP and made the cells more susceptible to cytotoxic agents. 

A frequently used model for studying the role of ions in the 5-HT transport process employs plasma membrane vesicles prepared from porcine blood platelets [12-17]. 

A similar mechanism of binding and cotranslocation of 5-HT", Na and Cl was demonstrated by using plasma membrane vesicles prepared from a synaptosomal fraction of mouse cerebral cortex [18,19]. The stoichiometry of transport in this case however was 5-HT Na Cr=l 2 l. 

The vesicles have now been further characterized. Light saturation curves showed that the PS II is in the form PS Ila. Two-dimensional gel electrophoresis of the LHC-II polypeptides demonstrated that the ratio 27/25 kDa for BS is lower than for previous PS II membrane vesicle preparations, indicating a large peripheral subpopulation of LHC-II. 

Glavinas H, Mehn D, Jani M, Oosterhuis B, Heredi-Szabo K, Krajcsi P (2008) Utilization of membrane vesicle preparations to study drug-ABC transporter interactions. Expert Opin Drug Metab Toxicol 4 721-732. 

Membrane vesicles prepared from Thiobacillus ferrooxidans contained three enzymes of the iron oxidation system. Delipidation of these vesicles by aqueous acetone decreased the enzymatic activities. They were restored by incubation of the lipid-depleted vesicles with a dispersion of ornithine-containing lipid together with coenzyme Qg. A possible role of the ornithine-containing lipid in the iron oxidation system has been suggested (25). 

A/B phosphorylation by membrane vesicle preparations from maize chloropiasts is affected by the osmolarity of the medium (Fig, 6) just as had been reported for spinach preparations. Also, removal of CFi by NaCl-EDTA abolishes the A/B phosphorylating capacity but has little or no effect on osmotic responsiveness of maize thylakoid preparations (Fig. 6). Osmotic responsiveness is necessary, but not sufficient, for A/B phosphorylation. The other component of the system, as we have divided it, is CFi. 

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