Vesicle membranes

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. 

Even though dynein, kinesin, and myosin serve similar ATPase-dependent chemomechanical functions and have structural similarities, they do not appear to be related to each other in molecular terms. Their similarity lies in the overall shape of the molecule, which is composed of a pair of globular heads that bind microtubules and a fan-shaped tail piece (not present in myosin) that is suspected to carry the attachment site for membranous vesicles and other cytoplasmic components transported by MT. The cytoplasmic and axonemal dyneins are similar in structure (Hirokawa et al., 1989 Holzbaur and Vallee, 1994). Current studies on mutant phenotypes are likely to lead to a better understanding of the cellular roles of molecular motor proteins and their mechanisms of action (Endow and Titus, 1992). 

Histopathological features are dominated by the large number of centrally-placed muscle nuclei, sometimes affecting more than 90% of muscle fibers. The nuclei form long chains in the middle of the fiber and are surrounded by cytoplasm, which contains mitochondria and membranous vesicles, but no myofibrils. This morphological appearance has prompted comparison with myotubes, and in fact centronuclear myopathies are sometimes referred to as myotubular myopathies. This is a misnomer, however, since although the affected fibers retain some of the structural features of myotubes, and maturational arrest may play a role in their formation, the vast majority of such fibers are fully differentiated histochemically into either type 1 or type 2. 

The mechanisms involved in the establishment of lipid asymmetry are not well understood. The enzymes involved in the synthesis of phospholipids are located on the cytoplasmic side of microsomal membrane vesicles. Translocases (flippases) exist that transfer certain phospholipids (eg, phosphatidylcholine) from the inner to the outer leaflet. Specific proteins that preferentially bind individual phospholipids also appear to be... 

Q Studies using v- and t-SNARE ptoteins reconsti-mted into separate lipid bilayer vesicles have indicated that they form SNAREpins, ie, SNARE complexes that hnk two membranes (vesicles). SNAPs and NSF are required for formation of SNAREpins, but once they have formed they can apparently lead to spontaneous fusion of membranes at physiologic temperamre, suggesting that they are the minimal machinery required for membrane fusion. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Primarily using isolated plasma membrane vesicles as an experimental preparation, the functional properties of Na /H exchangers have been elucidated. The important kinetic properties include (1) stoichiometry (one-for-one) (2) reversibility (3) substrate specificity (monovalent cations Na, H, Li, NH4, but not K, Rb, Cs, choline) (4) modes of operation (Na -for-H, Na -for-Na Li " -for-Na, Na -for-NH4 ) (5) existence of an internal site for allosteric activation by (6) reversible inhibition by amiloride (Af-amidino-5-amino-6-chloropyr-azine carboxamide) and its 5-amino-substituted analogs and (7) competitive nature... 

Biochemical studies of plasma membrane Na /H exchangers have been directed at two major goals (1) identification of amino acids that are involved in the transport mechanism and (2) identification and characterization of the transport pro-tein(s). To date, most studies have been performed on the amiloride-resistant form of Na /H" exchanger that is present in apical or brush border membrane vesicles from mammalian kidney, probably because of the relative abundance of transport activity in this starting material. However, some studies have also been performed on the amiloride-sensitive isoform present in non-epithelial cells. 

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]. 

Other blockers of epithelial Cl -channels are of the aryl-amino-benzoate type or phenoxy-acetic-acid type [70]. Very few systematic surveys comparing different classes of blockers in one type of Cl -channel are available at this stage. One such study has been performed in membrane vesicles from kidney cortex [80]. In this study IAA-94 and NPPB (cf. Fig. 2) turned out to be the most potent blocker of conductive Cl -flux. In another systematic survey the Cl -conductance of the sweat duct was examined, and it was found that dichloro-DPC (Fig. 2) was the most potent inhibitor of the transepithelial Cl -conductance [90]. 

Kaczorowski G, L Shaw, R Laura, C Walsh (1975) Active transport in Escherichia coli B membrane vesicles. Differential inactivating effects from the enzymatic oxidation of P-chloro-L-alanine and P-chloro-D-alanine. J Biol Chem 250 8921-8930. 

Garcia Ruiz, C., Fernandez Checa, J. and Kaplowitz, N. (1992). Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J. Biol. Chem. 267, 2256-2264. 

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). 

Z. Varanini, R. Pinton, M. G. De Biasi, S. Astolfi, and A. Maggioni, Low molecular weight humic substances stimulate H -ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots. Plant Soil I53 6 (1993). 

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