Membrane vesicle systems, transporter

In eukaryotes there is also evidence that Met(O) is actively transported. It has been reported that Met(O) is transported into purified rabbit intestinal and renal brush border membrane vesicles by a Met-dependent mechanism and accumulates inside the vesicles against a concentration gradient102. In both types of vesicles the rate of transport is increased with increasing concentrations of Na+ in the incubation medium. The effect of the Na+ is to increase the affinity of Met(O) for the carrier. Similar to that found in the bacterial system, the presence of Met and other amino acids in the incubation medium decreased the transport of Met(O). These results suggest that Met(O) is not transported by a unique carrier. 

Heijn, M., Oude Elferink, R. and Jansen, P. (1992). ATP-dependent multispecific organic anion transport system in rat erythrocyte membrane vesicles. Am. J. Physiol. 262, 104-110. 

P. Askerlund and C. Larsson, Transmembrane electron transport in plasma membrane vesicles loaded with an NADH-generating system or ascorbate. Plant Phy-.i-iol. 96 1178 (1991). 

Although several allelochemicals (primarily phenolic acids and flavonoids) have been shown to inhibit mineral absorption, only the phenolic acids have been studied at the physiological and biochemical levels to attempt to determine if mineral transport across cellular membranes can be affected directly rather than indirectly. Similar and even more definitive experiments need to be conducted with other allelochemicals that are suspected of inhibiting mineral absorption. Membrane vesicles isolated from plant cells are now being used to elucidate the mechanism of mineral transport across the plasma membrane and tonoplast (67, 68). Such vesicle systems actively transport mineral ions and thus can serve as simplified systems to directly test the ability of allelochemicals to inhibit mineral absorption by plant cells. 

Mucosal brush border membrane vesicles and basolateral membrane vesicles can be isolated to study solute uptake across specific enterocyte boundaries. These more isolated vesicle systems allow for investigation of solute transport across a particular membrane barrier and permit separation of membrane trans-... 

Figure 11.1 Schematic representation of iron uptake mechanisms, (a) The transferrin-mediated pathway in animals involves receptor-mediated endocytosis of diferric transferrin (Tf), release of iron at the lower pH of the endocytic vesicle and recycling of apoTf. (b) The mechanism in H. influenzae involves extraction of iron from Tf at outer membrane receptors and transport to the inner membrane permease system by a periplasmic ferric binding protein (Fbp). From Baker, 1997. Reproduced by permission of Nature Publishing Group.

It is important to establish an in vitro system which will allow in vivo transport across the bile canalicular membrane to be predicted quantitatively. By comparing the transport activity between in vivo and in vitro situations in isolated bile canalicular membrane vesicles, it has been shown that there is a significant correlation for nine types of substrates [90]. Here, in vivo transport activity was defined as the biliary excretion rate, divided by the unbound hepatic concentration at steady-state, whereas in vitro transport activity was defined as the initial velocity for the transport into the isolated bile canalicular membrane vesicles divided by the medium concentration [90]. Collectively, it is possible to predict in vivo canalicular transport from in vitro experiments with the isolated bile canalicular membrane vesicles. 

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]. 

M. Takano, K. Inui, T. Okano, H. Satio, and R. Hori. Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles. Biochim Biophys Acta 773 113-124 (1984). 

Figure 1.6 Vesicular transport of proteins within the cell. Vesicles from the endoplasmic reticulum [A] carry protein to the Golgi complex, they are repackaged in the Golgi from which they leave to form primary lysosomes [B] or fuse with the plasma membrane this is to add proteins or to be secreted from the cell [C]. In the Golgi, new vesicles are formed to transport the proteins to the plasma membrane (e.g. transport proteins or proteins for export) or the lysosomes. This system transports, safely, dangerous hydrolytic enzyme to the lysosomes and it also protects membrane proteins, or proteins for export, from degradation in the cytosol.

INTESTINE Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium-sensitive dye techniques, 192, 309 isolation of intestinal epithelial cells and evaluation of transport functions, 192, 324 isolation of enterocyte membranes, 192, 341 established intestinal cell lines as model systems for electrolyte transport studies, 192, 354 sodium chloride transport pathways in intestinal membrane vesicles, 192, 389 advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems, 192, 409 isolation and reconstitution of the sodium-de-pendent glucose transporter, 192, 438 calcium transport by intestinal epithelial cell basolateral membrane, 192, 448 electrical measurements in large intestine (including cecum, colon, rectum), 192, 459... 

In such vesicle systems, the electrons are transported through the membrane. Electron carriers such as quinones or alloxazines in the vesicle wall enhance remarkably the rate of photoinduced charge separation. The vesicle system shown in Fig. 6 contains the surfactant Zn-porphyrine complex (ZnC12TPyP) in the wall 23). 

Liver Uptake Blood Parenchymal cells Isolated, cultured cryopreserved hepatocytes, sinusoidal membrane vesicles, transporter expressions system... 

Excretion Parenchymal cells Bile Canalicular membrane vesicles, transporter expression system... 

Small intestine Uptake Digestive tract Epithelial cells Everted sac, Ussing-chamber experiments using intestinal epithelium, brush border membrane vesicles, Caco-2 cells monolayer, transporter expression system... 

A. In Vitro Transport Systems Using Tissues, Cells, and Membrane Vesicles... 

Murer H, Gmaj P, Steiger B, et al. Transport studies with renal proximal tubular and small intestinal brush border and basolateral membrane vesicles vesicle heterogeneity, coexistence of transport system. Methods Enzymol 1989 172 346-364. 

From the earliest proposal for Na+-coupled transport, it was considered likely that K+ participated in the overall reaction (Crane, 1965). With the use of membrane vesicles where the internal and external K+ levels could be manipulated and replaced by other ions (Colombini and Johnstone, 1974 Murer and Hopfer, 1974 Sigrist-Nelson et al., 1975 Hopfer, 1978), it became clear that the apparent K+ requirement in intact cell systems was likely indirect and due to its requirement by the Na+/K+ ATPase to maintain the electrochemical potential difference for Na+. 

Many ambiguities in the study of Na+ (or H+) coupled solute transport have been clarified by the introduction of isolated membrane vesicles to study transport. The inherent property of membranes to seal up and hence form a closed system for measuring translocation has made these preparations popular with investigators. A large variety of cell types and even greater number of solutes have been used in these systems. 

The first reported instances using isolated plasma membrane vesicles to study Na+-coupled transport were derived from brush borders of the small intestine (Murer and Hopfer, 1974 Sigrist-Nelson et al., 1975) and Ehrlich cells (Colombini and Johnstone, 1974). In rapid succession a number of other systems were established to study translocation of many solutes in many animal cell systems (Schuld-iner and Kaback, 1975 Lever, 1977 Hammerman and Sacktor, 1978 Wright et al., 1983 Saieret al., 1988 see also Table 1). 

Even if membranous vesicles were commonplace on the early Earth and had sufficient permeability to permit nutrient transport to occur, these structures would be virtually impermeable to larger polymeric molecules that were necessarily incorporated into molecular systems on the pathway to cellular life. The encapsulation of macromolecules in lipid vesicles has been demonstrated by hydration-dehydration cycles that simulate an evaporating lagoon [53] or by freeze-thaw cycles [54]. Molecules as large as DNA can be captured by such processes. For instance, when a dispersion of DNA and fatty acid vesicles is dried, the vesicles fuse to form a multilamellar sandwich structure with... 

This, however, introduces a number of complications, and for information on the molecular mechanics of transport it would be desirable to work with a simpler system, such as membrane vesicles, and such experiments are in progress (84). Many unresolved questions in siderophore transport await resolution. 

The urate-anion exchanger system in brush border membrane vesicle, which mediates hydroxyl ion gradient-dependent urate uptake, is the most likely route for the mediation of urate transport in the first step of urate reabsorption in the proximal tubules. Luminal drugs which inhibit urate reabsorption are inhibiting the transport of urate by blocking the urate/anion exchanger. 

Itoh T, Tanno M, Li YH, Yamada H (1998) Transport of phenethicillin into rat intestinal brush border membrane vesicles role of the monocarboxylic acid transporter system. Int I Pharm 172 102-112... 

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