J. MembraneBiol. 55, 81-95 (1980) The Journal af Membrane Biology Topical Review The Use of Isolated Membrane Vesicles to Study Epithelial Transport Processes Heini Murer and Rolf Kinne Institut de Chimiephysiologique, UniversitY,CH-1700Fribourg, Switzerland, Max Planck-Institutf/Jr Biophysik,D-6 Frankfurt/M,WestGermany,and Albert EinsteinCollegeof Medicine, Bronx, NewYork 10461 Summary. Epithelia are multicompartment and multi- component systems performing transcellular and paracellular transport in a very complex manner. One way to get a deeper understanding of the function of such a complex system is to dissect it into the single components and then, after having defined the components under well-controlled conditions, to try to describe the behavior of the whole system on the basis of the properties of the single components. This article deals with the analysis of isolated plasma membranes derived from the luminal and con- traluminal face of epithelial cells, predominantly renal proximal tubular and small intestinal cells. It is aimed to give an overview of methods used to isolate and separate plasma membranes, to study their transport properties as membrane vesicles, and also to address the question of how information gained with the iso- lated membranes corresponds to observations made in the intact cell using other, notably electrophys- iologieal, measurements. The review also critically evaluates the limitations of the approach and thereby tries to set the work on isolated membranes in the proper perspective within the field of transport phys- iology. Luminal and Contraluminal Membranes are Separable Entities The rationale in attempts to isolate the luminal and the contraluminal areas of the cellular envelope of epithelial cells is the assumption of a different func- tion of the two membranes, which is reflected in a slightly, but appreciably, different composition of the membranes. The different membrane composition may be reflected in various biochemical parameters: different buoyant density - determined by the lipid-to- protein ratio - and different surface properties such as surface receptors, surface charge density, and sur- face hydrophobicity - a property which is related to the different degree of glycosylation of membrane proteins and membrane lipids. The luminal and con- traluminal membranes may also differ in their resis- tance to osmotic shock or shearing forces. Thereby fragments of different sizes may be formed from the two membranes during the homogenization necessary to break up the cells. This technically induced differ- ence can form the basis for separation; it is unique for plasma-membranes. Membranes, in contrast to mitochondria, lysosomes, and nuclei, do not reside inside the cell as organelles with a predetermined size and shape; their size and shape are determined during the preparation of the so-called cell homogenate. This homogenization is aimed to free all cellular elements from their neighboring components for future separa- tion. Historically, the first methods described for the isolation of brush border fragments were based mainly on the relative rigidity of the brush border during cell homogenization as contrasted to the labil- ity of the basal-lateral plasma membranes. Thus large brush border membrane fragments were obtained which differed sufficiently enough from other or- ganelles in their sedimentation rate to make their sep- aration by density gradients possible [6, 26, 46, 62, 100]. Nowadays the method used most frequently is based on the different reactivity of the luminal mem- brane and the other cellular organelles with magne- sium or calcium. It can be called differential precipita- tion in analogy to differential centrifugation. The brush border membranes, and probably other luminal membranes of epithelial cells, have the property to compensate within their membrane surface the two charges of the divalent cations. On other membranes the surface density of such charge-compensating groups is smaller; therefore, the divalent cations cross- link the intracellular organelles and basal-lateral plasma membranes and a coprecipitate is formed [8, 20, 41, 84]. 0022-2631/80/0055-0081/$03.00 9 1980 Springer-Verlag New York Inc.