Membranes isolation

As yet, models for fluid membranes have mostly been used to investigate the conformations and shapes of single, isolated membranes, or vesicles [237,239-244], In vesicles, a pressure increment p between the vesicle s interior and exterior is often introduced as an additional relevant variable. An impressive variety of different shapes has been found, including branched polymer-like conformations, inflated vesicles, dumbbell-shaped vesicles, and even stomatocytes. Fig. 15 shows some typical configuration snapshots, and Fig. 16 the phase diagram for vesicles of size N = 247, as calculated by Gompper and Kroll [243]. 

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

Phloretin is the aglycon of phlorizin and inhibits the facilitated diffusion of glucose catalyzed by GLUT1 or GLUT4. It has been used to terminate the uptake of glucose in timed assays with isolated membranes or reconstituted transporters. 

The patch-clamp technique is based on the formation of a high resistance seal (109-10lon) between the tip of a glass micropipette and the cell membrane it touches (gigaohm-seal). This technique allows recordings of ionic currents through single ion channels in the intact cell membrane and in isolated membrane patches at a... 

Further progress in understanding membrane instability and nonlocality requires development of microscopic theory and modeling. Analysis of membrane thickness fluctuations derived from molecular dynamics simulations can serve such a purpose. A possible difficulty with such analysis must be mentioned. In a natural environment isolated membranes assume a stressless state. However, MD modeling requires imposition of special boundary conditions corresponding to a stressed state of the membrane (see Refs. 84,87,112). This stress can interfere with the fluctuations of membrane shape and thickness, an effect that must be accounted for in analyzing data extracted from computer experiments. 

Li, J., Kelly, J.F., Chernushevich, I., Harrison, D.J., and Thibault P., Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectro-spray mass spectrometry, Anal. Chem. 72, 599, 2000. 

Aoki, J., Suzuki, H., Sugiyama, Y., Quantitative prediction of in vivo biliary excretion clearance across the bile canalicular membrane from in vitro transport studies with isolated membrane vesicles. Abstract of Millennial World Congress of pharmaceutical Sciences, San Francisco, April 16-20, 2000, p. 92. 

Dantzler, W.H., Brokl, O.H. and Wright, S.H. (1989). Brush-border TEA transport in intact proximal tubules and isolated membrane vesicles. Am. J. Physiol. 256 F290-F297. 

To build up a stable cell model according to this concept would mean to isolate membrane proteins and lipids and try to put them together as mother nature does. This idea to use membrane proteins for membrane stabilization does not yet seem to be realizable and therefore simpler possiblities for constructing stable membrane and cell models are desirable. 

Although this correlation uses rather crude data it gives a clear indication that the actual antagonist/receptor binding is the same at both the cardiac and bronchial sites and indicates that cardioselectivity is a function of distribution to the micro-environment of the receptor site. Some experimental evidence supporting this conclusion is now available from biochemical studies on isolated membrane fragments derived from heart and lung. ... 

C. Dilcso, E. Vazsonyi, M. Adam, I. Szabo, I. Bdrsony, J.G.E. Gardeniers, and A. van den Berg. Porous silicon bulk micromachining for thermally isolated membrane formation . Sensors and Actuators A60 (1997), 235-239. 

LIVER Use of isolated perfused liver in studies of biological transport processes, 192, 485 measurement of unidirectional calcium ion fluxes in liver, 192, 495 preparation and specific applications of isolated hepatocyte couplets, 192, 501 characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles, 192, 517 preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes, 192, 534. 

The answer is E. Anesthetics are highly lipid-soluble and experiments with isolated membranes indicate that these molecules can dissolve in the hydrophobic center of the membrane bilayer. This causes a measurable increase in the membrane fluidity by disrupting the packed structure of phospholipids tails. This is considered to be the main, direct mechanism by which this class of drugs inhibits neurotransmission (pain sensations) in neurons. Hallucinogens and opiates may also affect membrane fluidity, but their effects occur by indirect mechanisms, resulting from changes in the protein or lipid composition of the membranes. 

CYPlAl has also been expressed in yeast (Eugster et al., 1990 Sengstag et al., 1994) and EROD activities of 223 pmol/(mg min) have been reported. The turnover numbers for EROD in yeast-expressed CYPlAl (1.4/min) were substantially lower than observed for human lymphoblast-expressed CYPlAl (7.6/min). Apparent KmS were quite similar (92 nM and 87 nM in yeast and human lymphoblasts, respectively). Modified CYPlAl has also been expressed in E. coli (Guo et al., 1994). The expression level of CYPlAl in E. coli (per mg membrane protein) was comparable to that obtained with human lymphoblasts (about 30 pmol/mg in both systems). The turnover number for EROD for E. co//-expressed CYPlAl was quite low in isolated membranes, but could be increased to 8/min if the protein was purified and reconstituted. The apparent for E. coli-exptessed CYPlAl EROD activity, 580 nM, was about 6-fold higher than that for the yeast- or human lymphoblast-expressed enzymes. It is not clear whether this difference is due to the base substitutions necessary to obtain expression in E. coli, or some other cause. 

Along a more biological approach, D. Chapman 116) has described the biosynthetic incorporation of diacetylene acids into the biomembranes of Acholeplasma laidlawii A, an unsaturated fatty acid auxotroph bacterium. As much as 90% of the membrane lipid acyl chains were found to consist of C20-diynoic acid. Upon irradiation with UV-light, the cells and isolated membranes become coloured, due to the crosslinking of lipids by photopolymerization. 

To study transport in the absence of complicating metabolic processes, it often is advantageous to work with isolated membrane vesicles rather than with whole cells. Cytoplasmic membrane vesicles can be obtained from either eukaryotic or bacterial cells after homogenization or osmotic lysis. Transport proteins that have been solubilized with detergents also can be reincorporated into synthetic phospholipid vesicles (fig. 17.27). 

Murer, H. Kinne, R. (1980). The use of isolated membrane vesicles to study epithelial transport processes. J. Memb. Biol. 55, 81-95. 

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