J. Membrane Biol. 21,375-395 (1975) 9 by Springer-Verlag New York Inc. 1975 Sugar Transport by Renal Plasma Membrane Vesicles Characterization of the Systems in the Brush-Border Microvilli and Basal-Lateral Plasma Membranes R. Kinne, H. Murer, E. Kinne-Saffran, M. Thees, and G. Sachs Max-Planck-Institute for Biophysics, Frankfurt, Germany and Laboratory of Membrane Biology, University of Alabama, Birmingham, Alabama 35294 Received 3 December 1974; revised 17 January 1975 Summary. Uptake studies of D- and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brushborder vesicles D-glucose but not L-glucose transport was Na+-dependent," was inhibited by phlorizin, and showed a transitory vesicle/medium ratio > 1, in the presence of an initial Na + gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorizin as compared to the brush-border fraction. The asymmetry of an epithelial tissue, where the luminal and basal surfaces of the component cells possess different transport properties, seems to be the mechanism whereby vectorial net transfer of a substance is achieved. In renal proximal tubule, the relative contribution of the brushborder membrane and the basal-lateral membrane to transepithelial transport processes is difficult to assess in the intact organ. More recently, progress has been made by simultaneous microperfusion of both sides of the tubule in the intact kidney in vivo (Fr6mter, Muller & Knauf, 1969), perfusion of the isolated tubule in vitro (Tune & Burg, 1971), or experiments on the specialized cyst structure of the flounder tubule (Kleinzeller & McAvoy, 1973). Simplification of kidney system became feasible with development of methods capable of separating brush-border microvilli and basal-lateral membranes (Heidrich, Kinne, Kinne-Saffran & Hannig, 1972). Studies of Address correspondence to: Dr. G. Sachs, Laboratory of Membrane Biology, University of Alabama, Birmingham, Alabama 35294. 25~ 376 R. Kinne, H. Muter, E. Kinne-Saffran, M. Thees, and G. Sachs transport in subcellular vesicles derived from bacterial membranes (Kaback, 1972), sarcoplasmic reticulum (Hasselbach, Fiehn, Makinose & Migala, 1969), neural vesicles (Hazelbauer & Changeux, 1974), red cell ghosts (Benes, Kolinsk~i & Kotyk, 1972), adipocytes (Illiano & Cuatrecasas, 1971), ascites cells (Colombini&Johnstone, 1974), intestinal brush borders (Hopfer, Nelson, Perotto & Isselbacher, 1973), and unfractionated renal membranes (Busse, Elsas &Rosenberg, 1972), for example, suggested further investigation of the transport properties of the separated fractions. Accordingly, the transport characteristics of isolated brush-border microvilli and basal-lateral membranes for D- and L-glucose have been determined. The data indicate that the isolated membrane fragments differ not only as shown recently in their enzyme content, but also in their transport properties. Some of these data have been presented in a preliminary form (Kinne, Kinne-Saffran & Murer, 1973). Materials and Methods Membrane Purification Partially purified renal membranes were obtained by differential centrifugation from rat kidney following homogenization in isotonic sucrose as previously described (Pockrandt-Hemstedt, Schmitz, Kinne-Saffran & Kinne, 1970). These membranes were then separated into brush-border and basal-lateral membrane fractions using the Desaga FF4 free-flow electrophoresis machine as detailed before (Heidrich et al., 1972). Criteria of Purity The fractious were routinely assayed as previously described for enzymes shown to be characteristic of brush-border microvilli, basal-lateral membranes, mitochondria and endoplasmic reticulum, namely alkaline phosphatase, (Na+K+)-ATPase, succinic dehydrogenase and glucose-6-phosphatase (Heidrich et aL, 1972). Protein was determined after precipitation of the membranes with 5 % ice-cold TCA by the Lowry procedure, with bovine serum albumin as a standard (Lowry, Rosebrough, Farr & Randall, 1951). Transport Studies The membrane fractions obtained after electrophoretic separation were suspended by homogenization with a teflon glass homogenizer (10 strokes, 1,200rpm) in 15 ml of a solution containing 100 mM mannitol, 1 mM Tris-HEPES1 buffer, pH 7.4, and centrifuged for 20 min at 30,000 • g at 4 ~ The pellets were resuspended in 1 ml of the same buffer, using a syringe fitted with a fine needle, centrifuged once more, resuspended in the same solution and diluted to a protein concentration of about 10 mg/ml. The incubation medium contained, unless otherwise stated, 100mM mannitol, 100raM NaC1, 1mM Tris-HEPES buffer, pH 7.4, 1 mM D-glucose containing 5 ~CiH3-D-glucose, 1 mM L-glucose containg 2 vtCi C14-L-glucose. The stop solution contained 150mM 1 Tris-N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonicacid.