Inside-out vesicles

Fig. 9. Transfer of mannitol bound to inside-out vesicles to the cytoplasmic volume. (A) Unphos-phorylated ll ". (B) Phosphorylated 11 . It is assumed that the phosphoryl group transfer from the enzyme to the sugar can only take place when the sugar is bound the cytoplasmic-facing binding site, E-Pcyt Mtl (see also Fig. 6).

Figure 8-14 SDS-polyacrylamide gel electrophoresis of human erythrocyte ghosts. (A) From untreated cells. (B) From cells digested externally with chymotrypsin. (C) Inside-out vesicles prepared from cells pretreated with chymotrypsin. (D) The same inside-out vesicles after further treatment with chymotrypsin. (E) Polypeptides released hy the chymotryptic treatment of the inside-out vesides. The peptides are numbered according to the system of Steck232 Hb, hemoglobin. From Luna et al233...

Most results on calcium transport have been obtained using cytoplasmic membrane vesicles, which may be prepared in inside-out or right-side-out configurations. Inside-out vesicles may be obtained by the disruption of E. coli cells in a French press. These then accumulate Ca2+ in an energy-dependent fashion, provided ATP or an oxidizable substrate is available. Addition of phosphate enhances the uptake of calcium as calcium phosphate is precipitated inside the tell, thus accounting for the lack of exchangeability of the calcium.186... 

Inside-out vesicles can be perfectly used to study single efflux processes, because preloading of cells is not necessary and because of well-defined study conditions with transporter directly facing compound concentration in the incubation medium in contrast to double-transfected cell lines (3.1.2.4.L). Therefore, these studies allow establishing structure activity relationships (SAR) for one efflux transporter only. 

The preparation of plasma membrane vesicles from liver canalicular membrane is highly enriched with the canalicular (apical) isoform MRP2 (Buchler et al. 1996). Methods for the isolation of hepatocyte canalicular membranes from liver tissue have been described in detail (Bohme et al. 1994 and Boyer and Meier et al. 1990). The percentage of inside-out-oriented vesicles in these preparations amounts to 32%. Alternatively, transfected I ILK and MDCK cells are often used to study ATP-dependent transport into inside-out vesicles (Cui et al. 1999 Leier et al. 2000). 

Several polypeptide components of PS II and OEC have been isolated from thy-lakoids and PS II preparations capable of O2 evolution, after the initial isolation by Kuwabara and Murata [31] of a 33-34 kDa polypeptide (see, for a review. Ref. 10). On the basis of several criteria, such as the extraction by different reagents and the accessibility to antibodies in thylakoids or in inside-out vesicles prepared from thylakoids, a tentative and certainly incomplete picture has been proposed... 

The evidence that water is split on the inner side of thylakoids is convincing early experiments by Fowler and Kok [33] and more recent ones [6] have shown that the protons generated by water splitting are detected inside the thylakoid lumen. Furthermore, it has been shown that the 24 and 18 kDa polypeptides are accessible to antibodies only in so-called inside-out preparations these polypeptides can be extracted in salt solutions from the inside-out vesicles, and subsequently rebound to them [34,35]. 

Rs. rubrum, with its single antenna complex, is an ideal object to probe the membrane-surface exposed regions of the antenna polypeptides. Chromatophores (inside-out vesicles, the cytoplasmic side of the membrane outside) have been chemically modified with the hydrophilic marker diazobenzenesulfonate [24], In addition, protease treatment of chromatophores [25-27] and of spheroplasts (periplasm outside) was carried out [27]. The surface location of the B880 complex was further investigated with antibodies raised to either the intact complex or to the individual polypeptides [27]. In addition, the secondary structures of the polypep-... 

The aqueous polymer two-phase partition technique pioneered by Albertsson et al. [11] not only provides a method to separate right-side-out from inside-out vesicles (Section 2.2), but also allows the partial separation of appressed and non-appressed membrane fractions. The inside-out vesicles which partition to the lower phase were depleted in PS II activity [59]. Significantly, they were derived from the appressed membranes of the grana stacks as judged by electron microscopy [60] and their mode of formation [61], Futhermore, analysis of the Chl-protein content revealed a substantial depletion of PS I complex, and an enrichment of PS II complex and LHC II in the appressed membrane fraction [62]. In 1980, An-dersson and Anderson postulated that PS II and PS I are mainly laterally segregated, with PS I excluded from the appressed grana partitions, where most PS II-LHC II complexes are located [62,63] (Fig. 5). 

As adenylate cyclase is probably oriented within the membrane with receptors exposed on the outside and catalytic components on the inside, the formation of vesicles on homogenisation result in the expression of artifactual properties. Thus inside-out vesicles reduce or prevent hormone interaction and right-side-out vesicles effectively reduce total enzyme activity if the vesicles were impervious to the exogenous substrate. 

These ion movements are shown schematically in Figure 11. It should be noted that the orientation of the protonmotive force is reversed from that in vivo. The proton-pumping Mg -ATPase will be described in a later section. This calcium transport system is not inhibited significantly by ruthenium red, the classical inhibitor for calcium uptake in mitochondria. However, uptake of Ca by these inside-out vesicles of E. coli is inhibited dramatically by a dimeric, mixed-valence complex of Ru" ", [(NH3)3RuCl3Ru(NH3)3]. The mode of action remains to be established. 

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. 

Figure 18.4 Binding from water to the transporter (1) is divided into two steps membrane partitioning (2) and transporter binding (3). These processes are fast and can be considered (to a first approximation) as equilibrium processes in inside-out vesicles as well as in cells under steady-state conditions in a Cytosensor this is indicated by the double arrows. The free energy of binding of the drug from water to the transporter (AGj jj) was...

A more direct confirmation of the inside-out character of the B3 vesicles was provided by freeze-fracture electron microscopy. Representative replicas ofthe T2 and B3 vesicles are shown in Fig. 19 (D). As expected for the B3 inside-out vesicles, large distinct particles appear in the EF faces next to the intra-thylakoid space [Fig. 19 (D), left], while closely packed small particles appear in the PF faces next to the stroma [Fig. 19 (D), right]. A large number of vesicular faces were examined and the results revealed that on average the B3 fraction contained 74% inside-out and 26% rightside-out vesicles while the T2 fraction contained 89% rightside-out and only 11% inside-out vesicles. 

Freeze-fracture electron microscopy of thylakoid membranes has clearly revealed an asymmetric lateral distribution of the various photosynthetic complexes in the granal and stromal membranes, i.e., the distribution of the protein complexes in the membrane is nonrandom. This lateral asymmetry was further substantiated by the results of electron microscopy of the inside-out vesicles discussed in Section Vll. These findings by electron microscopy are summarized by the model shown in Fig. 21 (A). It is a transverse cross section of the thylakoids shown earlier in Fig. 13 (D) and (D ), with the various photosynthetic protein complexes appropriately placed in the granal and stromal regions. 

B Andersson and JM Anderson (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593 427-440 P-" Albertsson (1985) Partition of Cell Particles and Macromolecules (3rd edition) John Wiley H-E "kerlund, B Andersson and P-" Albertsson (1976) Isolation of photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition. Biochim Biophys Acta 449 525-535 P Grber, A Zickler and H-E "kerlund (1978) Electric evidence for the isolation of inside-out vesicles from spinach chloroplasts. FEES Lett 96 233-237... 

Fig. 3. Oxygen-evolution activity as a function of Cl- concentration in the PS-II inside-out vesicles without (o) and with ( ) the 23-kOa poiypeptide present. Figure source Andersson, Critchiey, Ryrie, Jansson, Larson and Anderson (1984) Modification of the chloride requirement for photosynthetic oxygen evolution. FEBS Left 168 115.

Kondo T,Dale GL, Beutler E (1981) Studies on glutathione transport utilizing inside-out vesicles prepared from human erythrocytes. Biochim Biophys Acta 645 132 Srivastava SK, Beutler E (1969) The transport of oxidized glutathione from human erythrocytes. J Biol Chem 244 9... 

Kondo T, Murao M, Taniguchi N (1982) Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes. Eur J Biochem 125 551 Lu SC, Kuhlenkamp J, Ge JL, Sun WM, Kaplowitz N (1994) Specificity and directionality of thiol effects on sinusoidal glutathione transport in rat liver. Mol Pharmacol 46 578-85... 

Ultrastructural observations suggested that during invasion Plasmodium rhoptries extrude copious membranous material (see ref. 6 and references therein), which may provide a matrix for rhoptry proteins (13). Some rhoptry proteins bind to both erythrocyte inside-out vesicles and to vesicles prepared from phospholipids predominant in the erythrocyte membrane inner leaflet, particularly phosphatidylserine (PS) and phosphatidylinositol (PI) (14,15). One has been shown to intercalate into the erythrocyte membrane (16). These proteins, the RHOP-H complex and SERA, are proposed to integrate differentially into the erythrocyte membrane upon invasion, causing an inward membrane expansion, to produce the parasitophorous vacuole. 

In order to reduce such interferences, successful efforts have been made to isolate the cell membranes, or even their transport-active constituents. One way to achieve this is by preparation of isolated membranes which have a natural tendency to form closed and homogenous vesicles [31,32]. Another approach is by reconstituted systems , i.e. to isolate membrane components involved in specific transport processes, and to incorporate them in artificial lipid membranes, usually liposomes [33,34]. Vesicles have been successfully prepared from various cells and tissues and tested for transport activities. Whenever membranous material has been isolated from other cellular components it tends to form vesicles spontaneously, sometimes with an uniform orientation, right-side-out or inside-out vesicles, respectively. For mixtures of vesicles of the two orientations, methods were developed to separate the two polarities. Furthermore, one can separate vesicles from different cell types or even from different regions of the cell, e.g. brush-border membranes form basal lateral ones [35,36]. 

Investigations on primary active transport are also performed with vesicles. For inside-out vesicles no additional experimental problems arise, since the enzymatic site of the transport system located at the cytoplasmic site of the membrane is freely accessible to the substrate of the exergonic driving reaction from the incubation medium. Right-side-out vesicles, however, have to be prepared in the presence of the driver substrate, e.g. ATP, or a system for its regeneration, e.g. ATP/ADP + phosphoenolpyruvate + pyruvate kinase. 

In Figs. 1 and 2 (A,B), the fluorescence yields, 80 ms after each flash of a series of flashes and the luminescence intensities, 1ms after each flash were measured under the same flash energy. The two types of inside-out vesicles used (BS and 30S) differ in their functional antenna size. The antenna size of the 30S fraction is smaller than the antenna size of the BS fraction which is about the same as that of the inside-out thylakoids from which it originates [12]. In the 30S fraction, less flash energy is given to the photosystem II centers and the fluorescence oscillations in Fig. 2 present the same change observed when the flash energy is decreased [ 6]. The least... 

The shape of the patterns depicted in Fig. 2 strongly depends on the degree of trypsination. After destruction of the oxygen evolution capacity by extensive trypsination each flash induces the same H -release that is assumed to be due to Yj oxidation by as was shown for Tris-washed inside out vesicles (10). ... 

The BS particles were prepared from inside out vesicles from spinage thylakoids that had been broken by a mechanical press treatment. These vesicles were separated further by partition with an aqueous polymer two-phase system (1). The vesicles thus obtained have a diameter of about 0. 2 pm and contain only trace amounts of PS 1 (P700). 

Several studies have suggested a close association between the 33, 23 and 16 kDa extrinsic proteins and the Dl-protein (1). It was therefore of interest to investigate whether the three proteins still were bound to the inner thylakoid surface and associated with PS II after degradation of the Dl-protein. This was done by isolation of inside-out vesicles from control and photoinhibited spinach thylakoids using phase partition technique (4). As revealed by immu-noblotting, inside-out vesicles isolated from photoinhibited thylakoids showed a marked reduction of all three proteins as compared to inside-out vesicles isolated from dark control thylakoids (Fig.l). 

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