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[26] I s o l a t i o n o f L u m e n a l a n d C o n t r a l u m e n a l Membrane Vesicles from Kidney

Plasma

By EVAMARIAKtNN~-SAFFRANand ROLF K. H. KaNN~ Introduction One of the most striking morphological and functional features of epithelial cells is their polarity. This polarity extends from a specific arrangement of intracellular organelles and cytoskeletal elements to a specific organization and lipid and protein composition of the two areas of the plasma membranes 1-~ covering the opposing faces of epithelial cells. The elucidation of this polarity has for the last two decades fascinated biochemists and physiologists; their studies have yielded a more or less complete picture of the status quo of the polarity. This picture was crucial for the description of transcellular transport as a series of consecutive steps of transport reactions whose characteristics achieve the vectorial movement of solutes not only across the plasma membranes but also across cells in epithelial cell layers. More recently polarity of epithelial cells has attracted the interest of cell biologists who are studying in the most general sense differentiation of cells, and, in particular, the establishment of cell membrane polarity by sorting of specific membrane components to one cell side or the other. Epithelial cells have thus become a very useful model for studies on the basic mechanism of cell membrane targeting and the control thereof.4. 5 Isolation of membranes derived from a defined pole of the epithelial cell therefore often constitutes an essential step in the investigation of membrane components, membrane properties, and their regulation. This chapter is intended to provide some guidelines to isolate lumenal and contralumenal membrane fractions from renal epithelia. Experience has shown that the isolation of lumenal membranes is usually easier to achieve, probably because this cell membrane represents that cell pole which has undergone the highest degree of differentiation. Thus a separation of this membrane from the contralumenal membrane and from intracellular membranes can in most instances be accomplished based on the relatively simple principle of "differential precipitation. ''6 The isolation of contralu1 p. R. Dragstein, R. Blumenthal, and J. S. Handler, Nature (London) 292, 718 (1981). 2 j. S. R o d m a n , L. Seidman, and M. G. Farquhar, J. CellBiol. 102, 77 (1986). 3 K. Simons and S. D. Fuller, Annu. Rev. CellBiol. 1, 243 (1986). 4 K. Simons and G. van Meet, Biochemistry 27, 6197 (1986). 5 K. Simons, Kidneylnt. 23, $201 (1987). A. G. Booth and A. J. Kenny, Biochem. J. 142, 575 (1974).

METHODS IN ENZYMOLOGY, VOL. 191

Copyright© 1990by AcademicPress,Inc. All r/ghtsof reproductionin any formre.fred.

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menal membranes is still difficult, requires a higher degree of sophistication, and is therefore prone to higher van'ability. The scope of this chapter is limited to technical aspects of vesicle isolation; functional, biochemical, and physiological aspects of the approaches used to study epithelial transport employing vesicles can be found in some recent reviews.7-~3 Purification of Lumenal M e m b r a n e s from Proximal Tubule Lumenal membranes are usually membranes which contain a high amount of glycoproteins or glyeofipids; carbohydrate chains contain sialic acid residues as their terminal component and are, therefore, negatively charged at neutral pH. This high surface charge density forms the basis for the purification of these membranes by differential precipitation employing divalent cations such as magnesium or calcium in a low ionic strength medium. In the following, flow diagrams will be presented which describe two procedures that have been applied successfully to isolate brush borders from rat kidney cortex and hog kidney cortex. Some modifications are necessary for other species, such as higher divalent cation concentrations for nonmammalian kidneys ~4or a third precipitation step? 5

Method 1: Purification of Brush Border Membranes from Rat Kidney Cortex (Isolation by Differential Precipitation)~6 Rat kidney cortex slices (outer layer of cortex) One-half millimeter thick, 3 g from 10 rats Homogenize as a 10% suspension (w/w) for 2 rain at 4* in a buffer containing 10 m M mannitol, 2 m M Tris-HC1, pH 7.1, with a blender (Waxing, ESGE, Polytron) Filter through cheesecloth. Under mixing, add CaC12 (prepared in distilled water) to a final concentration of 10 raM. Keep on ice. After 15 rain dilute 1 : 1 with mannitol buffer (4*) containing 10 mMCaCI2 7 E. Kinne-Saffran and R. Kinne, Ann. N.Y. Acad. Sci. 341, 48 (1980). s j. E. Lever, CRC Crit. Rev. Biochem. 7, 1987 (1980). 9 H. Murer and R. Kinne, J. Membr. Biol. 55, 81 (1980). t0 H. Muter, J. Biber, P. Gmaj, and B. Stieger, Mol. Physiol. 6, 55 (1984). m~R. K. H. Kinne, Comp. Biochem. Physiol. 90A, 721 (1988). J2 H. Murer and P. Gmaj, Kidneylnt. 30, 171 (1986). m3R. D. Mamelok, D. F. Groth, and S. B. Prusiner, Biochemistry 19, 2367 (1980). m4j. Eveloff, M. Field, R. Kinne, and H. Murer, J. Comp. Physiol. 135, 175 (1980). ~5N. A. Wolffand R. Kinne, J. Membr. Biol. 102, 131 (1988). ~6C. Evers, W. Haase, H. Murer, and R. Kinne, Membr. Biochem. 1, 203 (1978).

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Centrifuge at 500 g for 12 min Remove supernatant as completely as possible Discard pellet Centrifuge supernatant at 15,000 g for 12 rain Crude brush border membrane fraction Resuspend pellet in 15 ml mannitol buffer with a Potter-Elvehjem homogenizer (10 strokes at 1000 rpm), clearance 0.1 ram. Add CaCI: to a final concentration of 10 mM. Keep on ice. After 15 min dilute 1 : 1 with mannitol buffer containing 10 mM CaC12 Centrifuge at 750 g for 12 min Discard pellet Centrifuge supernatant at 15,000 g for 12 rain Purified brush border membranes Resuspend in 60 ml of buffer as required for further experiments (for transport studies use 100 mM mannitol, 20 mM Tris-HEPES, pH 7.4) with a glass-Teflon homogenizer (10 strokes at 1000 rpm). Centrifuge at 48,000 g for 20 min Homogenize pellet in 0.6 ml buffer (sterilized by filtration through a 0.2-gin membrane filter) as required for further experiments by sucking the suspension 10 times through a steel needle (26 gauge) into a I ml plastic syringe Brush border membrane vesicles All centrifugation and homogenization steps should be performed at 4 °. There are several aspects which have to be considered at the various steps. These are reviewed below. Step 1: Tissue Selection and Pretreatment. If the starting material is not homogeneous, it is important to enrich the cell type of interest. In the kidney, dissection of the various kidney regions is necessary. Thin cortical slices ( - 3 - 5 mm thick) prepared from the outer layer contain mainly $2 segments (or pars convoluta) of the superficial proximal tubule. The outer medulla contains $3 segments (or pars recta) of the proximal tubule. Since it has been shown that these two segments differ in their transport properties ~7-26 careful separation is necessary. In species such as the ~7R. Kinne, in "Renal Biochemistry: Cells, Membranes, Molecules" (R. K. H. Kinne, ed.), p. 99. Elsevier, Amsterdam, 1985. 18R. J. Turner and A. Moran, Am. J. Physiol. 242, F406 (1982). t9 R. J. Turner and A. Moran, J. Membr. Biol. 70, 37 (1982). 20 K. E. Jergensen and M. I, Sheikh, Biochern. J. 223, 803 (1984). 2~ K. E. Jergensen and M. I. Sheikh, Biochem. Biophys. Acta 814, 23 (1985). 22 K. E. Jergensen and M. I. Sheikh, Biochim. Biophys. Acta 860, 632 (1986).

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flounder, in which the kidney contains hematopoietic tissue, tubular cells and hematopoietic cells have to be separated. In general, the tissues used in this preparation procedure should be devoid of red blood cells, since erythrocyte ghosts tend to copurify with lumenal membranes.6 This can be achieved, for example, by blotting the tissue slices briefly on dry filter paper. In most instances it has turned out to be advantageous to freeze the tissue at - 70" for at least 24 hr before initiating the experiment. Purity and yield of the membranes increase markedly if frozen instead of freshly obtained tissue is used. The homogenization step can be carded out in almost any blender of the appropriate volume. Small membrane vesicles can also be obtained by nitrogen cavitation.27 As long as the ratio between tissue wet weight and homogenization buffer is kept at 1/10 the initial amount of tissue can be varied from 0.3 to 200 g. Addition of Divalent Cations. The concentration of divalent cations and the kind of cation must be chosen for the studies to be done after the separation. Some tissues, especially from lower vertebrates, require up to 30 m M calcium to obtain tight membrane vesicleS. 2a'29 Membrane-bound phospholipases may be activated by the addition of calcium; this activation leads to the formation of lysophospholipids, which increase membrane permeability for small cations such as protons. 3° Furthermore, divalent cations are trapped inside the vesicles and tightly bound to the former cytoplasmic face of the membrane. About 50 nmol Ca2+/mg protein has been found in hog kidney brush border membranes isolated using l0 m M calcium for the differential precipitation, a~ Thus for studies on the calcium dependence of membrane processes the use of magnesium and EGTA is recommended.32 It also should be noted that the most reproducible manner for calcium addition is to add a defined volume of the tissue homogenate to a beaker which already contains the appropriate amount of 1 M calcium chloride

23 U. Kragh-Hansen, H. Reigaard-Petersen, C. Jacobsen, and M. I. Sheikh, Biochem. J. 220, 15 (1984). 24 n . R~igaard-Petersen and M. I. Sheikh, Biochem. £ 220, 25 (1984). 2s H. l~igaard-Petersen, C. Jacobsen, and M. I. Sheikh, Am £ Physiol. 253, FI5 (1987). 26 H. R~,aard-Petersen, C. Jacobsen, and M. I. Sheikh, Am. £ Physiol. 254, F628 (1988). 27 j. E. Lever, CRC Crit. Rev. Biochem. 7, 187 (1980). 2s j. Eveloff, R. Kinne, E. Kinne-Saffran, H. Muter, P. Silva, F. H. Epstein, J. Stoff, and W. B. Kinter, Pfluegers Arch. 378, 87 (1978). 29 N. A. Wolff, R. Kinne, B. Elger, and L. Goldstein, J. Comp. Physiol. B. 157, 573 (1987). ao I. Saboli~ and G. Burckhardt, Biochim. Biophys. Acta 772, 140 (1984). 31 S. M. Grassl, E. Heinz, and R. Kinne, Biochim. Biophys. Acta 736, 178 (1983). 32J.-T. Lin, Z.-J. Xu, C. Lovelace, E. E. Windhager, and E. Heinz, Am. J. Physiol. 257, F126 (1989).

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solution. Rapid swirling distributes the calcium evenly in the suspension. Pipetting a concentrated calcium solution On top of the (often foaming) homogenate is not as reproducible; addition of solid calcium salts leads to high local concentrations which affect the selectivity of precipitation. Preparation of the Crude Brush Border Membrane Fraction. In some tissue homogenates the lumenal membrane vesicles are so small that a large fraction of them is lost in the supernatant at this centrifugation step. The speed of centrifugation and the centrifugation time must be adjusted accordingly.29 The recovery of smaller fragments is also the aim of the additional centrifugation step described by Vannler et al. (second method, see below), a3 However, since in the latter procedure mitochondrial and lumenal membranes eosediment, the subsequent alkaline treatment is required. 34 This treatment introduces further cations into the vesicles which might interfere with subsequent investigations. Purified Brush Border Membranes. In some tissues the purity of the brush border membrane fraction obtained after two steps of calcium precipitation is not satisfactory with regard to the contamination with lysosomes and basolateral plasma membranes. In this instance a third precipitation step can be added or other means of separation, such as density gradient centrifugation,35,36must be employed. Final Steps of Suspension. The aims of the final steps are the suspension of the membrane vesicles in the desired buffer and the obtainment of a homogeneous suspension of vesicles. Because for transport experiments highly concentrated vesicle suspensions are used, careful homogenization using a fine needle and a syringe is important for good reproducibility. The inclusion of air into the syringe during the homogenization should be avoided since it leads to loss of membrane material and potentially to disruption of membrane vesicles.

Method 2: Purification of Brush Border Membranes on a Preparative Scale by Differential Precipitation and Alkaline Treatment (Modified after Vannier et al?3 and Meldolesi et al?4) Slices I Homogenize hog kidney cortex slices (3-5 mm thick, 200 g) at 4 ° in 1200 ml buffer (10 mM mannitol, 2 mM Tris, pH 7.1, adjusted with 33C. Vannier, D. Louvard, S. Maroux, and P. Desnuelle, Biochim. Biophys. Acta 455, 185 (1976). J. Meldolesi, J. D. Jamieson, and G. E. Palade, J. Cell Biol. 49, 109 (1971). 35L. P. Karniski and P. S. Aronson, Am. J. Physiol. 253, F515 (1987). R. Kinne and G. Sachs, in "Physiology of Membrane Disorders" (T. E. Andreoli, J. F. Hoffman, D. D. Fanestil, and S. G. Schultz, eds.), p. 83. Plenum, New York, 1986.

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HC1) in a Waring blender (full speed) twice, 1 min each time, with a 2-min interval Filter through cheesecloth Add CaC12 (final concentration, l0 mM) Keep on ice for 15 min with occasional stirring Centrifuge at 1500 g for 12 min Discard pellet, centrifuge supernatant at 30,000 g for 3 hr Crude brush border membrane fraction Suspend pellet in 150 ml salt solution (170 mM NaC1, 2 mM Tris, pH 7.1, adjusted with HC1) by homogenizing in a Waring blender (full speed for 20 sec). Add 350 ml 200 mM Tris/HC1, pH 7.8, and mix Centrifuge at 10,000 g for l0 min Discard pellet Centrifuge supernatant at 105,000 g for 1 hr, discard supernatant Purified brush border membranes Continue as described in Method 1 to obtain vesicles The yield of membranes is about 10% if the first method is employed and about 27-30% if the second is used. The purity of the two final membrane fractions is comparable. The brush border membrane vesicles are almost exclusively oriented right side out37; their intravesicular space as determined by o-glucose uptake is about 2-4/zl/mg protein. Typical transport properties have been reviewed recently.7,~°-~2 Isolation of Contralumenal (Basolateral) Membrane Vesicles from Rat Kidney Cortex Two methods have been successfully applied to enrich basolateral plasma membrane with acceptably low contamination by lumenal membranes. In both methods a crude plasma membrane fraction is first prepared in which basolateral membranes are enriched compared to brush border membranes and intracellular organelles. Then, either the small difference in apparent density or electrophoretic mobility is employed for the final purification. Small differences in density are effective in shallow regions of PercoU gradients~3,3s,39; the different electrophoretic mobilities become evident during free-flow dectrophoresis.4°-42 37W. Haase, A. Schifer, H. Muter, and R. Kinne, Biochem. J. 172, 57 (1978). 3s V. Scalera, Y.-H. Huang, B. Hildman, and H. Murer, Membr. Biochem. 4, 49 (|981). 39 K. Inui, T. Okano, M. Takano, S. Kitazawa, and R. Hofi, Biochim. Biophys. Acta 647, 150 (1981).

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Method 3: Purification of Basolateral Plasma Membranes from Rat Kidney Cortex Using Percoll Density Gradient Centrifugation3s Slices One-half millimeter thick, 1.5 g from five rats Homogenize at 4* in 35 ml sucrose buffer (250 m M sucrose, 10 m M triethanolamine/HC1, pH 7.6, and 0.1 m M phenylmethylsulfonylfluoride) with 20 strokes in a Potter-Elvehjem homogenizer at 1200 rpm (clearance = 0.1 cm) Dilute 1 : 2 with sucrose buffer Centrifuge at 2500 g for 15 min Discard pellet Centrifuge at 20,500 g for 20 min Remove supernatant carefully, add 5 ml of sucrose buffer on top of the pellet, suspend fluffy layer of pellet by swirling and scraping Discard supernatant and residual pellet Fluffy layer of second pellet Add 20 ml sucrose buffer Homogenize in #ass-Teflon homogenizer with 20 strokes at 1200 rpm Crude plasma membranes Add Percoll [8% (v/volume of membrane suspension)] Centrifuge at 48,000 g for 30 min, switch off automatic brake, recover fractions between 13 and 17 ml of the gradient from the top Dilute 1 : 10 with sucrose-free buffer (10 m M triethanolamine/HC1, pH 7.4, 0.1 m M phenylmethylsulfonylfluoride) Centrifuge at 48,000 g for 30 min Purified basolateral membranes Continue as described in Method 1 to obtain vesicles In basolateral membrane fractions obtained after Percoll density gradient centrifugation, Na+,K+-ATPase, a marker enzyme for basolateral membranes, is usually enriched 20-fold and brush border membranes are enriched 2- to 3-fold. 38 The yield is about 5% of the enzyme activity. The following points deserve further description. The homogenization step is 40H. G. Heidrich,R. Kinne, E. Kinne-Saffran,and K. Hannig,J. CellBiol. 54, 232 (1972). 4~R. A. Reynolds,H. Wald, P. D. MeNamara,and S. Segal,Biochim. Biophys.Acta 61}1,92 (1980). 42M. S. Medow,K. S. Roth, K. Ginkinger,and S. Segal,Biochem. J. 214, 209 (1983).

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critical with regard to purity and yield of the final membrane fraction. The aim of the homogenization is to obtain relatively large sheets of basolateral plasma membranes, which can be enriched in the crude membrane fraction and then separated on the PercoU gradient. Clearance of the pestle, speed of rotation, as well as forces applied during the vertical movement of the homogenization tube have to be relatively gentle and should be standardized as much as possible. Different batches of Percoll differ slightly in the density profile of their gradients established during the centrifugation. Colored density beads should, therefore, be used to determine whether the gradient can effectively separate blue and orange marker beads. For optimal membrane separation the blue markers (d --- 1.038) are found between 19 and 25 ml and the orange marker (d = 1.043) at 31-32 ml. Removal of Percoll from the fractions requires additional centrifugation. For the upper 18 ml of the gradient a 1 : 10 dilution in sucrose-free buffer followed by centrifugation at 48,000 g for 30 min yields tightly packed pellets of Percoll on top of which the membranes can be retrieved as a fluffy layer. In the following a combination of differential centrifugation and freeflow electrophoresis is depicted. This method is a modification of the electrophoretic separation described below4° in that a plasma membrane fraction is used which is already highly enriched in basolateral membranes compared to brush border membranes.

Method 4: Purification of Basolateral Plasma Membranes from Rat Kidney Cortex Using Free-Flow Electrophoresis39 Ra kidney cortex slices One-half millimeter thick, - 6 g wet from 20 rats Homogenize in 30 ml sucrose buffer (250 m M sucrose, 10 m M triethanolamine/HC1 adjusted to pH 7.4 with NaOH) with eight strokes in a Potter-Elvehjem homogenizer at 300 rpm (clearance = 0.025 cm) Centrifuge at 1475 g for l0 min Discard supernatant Resuspend pellet in 2 M sucrose (l ml/g starting tissue) Homogenize with three strokes in a Potter-Elvehjem homogenizer at 1000 rpm Centrifuge at 13,300 g for l0 min Discard pellet Dilute supernatant to isotonicity by the addition of 7 vol of distilled water

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Homogenize with three strokes in a Potter-Elvehjem homogenizer Centrifuge at 33,000 g for 15 min Upper fluffy layer of pellet Resuspend in half of the supernatant Homogenize with three strokes in a Potter-Elvehjem homogenizer Centrifuge at 33,000 g for 15 min Resuspend upper fluffy layer in sucrose buffer (1 ml/g starting tissue) Homogenize with three strokes in a Potter-Elvehjem homogenizer Centrifuge at 33,000 g for 15 min Suspend upper fluffy layer of pellet in electrophoresis buffer (250 m M sucrose, 8.5 m M triethanolamine titrated with acetic acid to pH 7.4) Crude basolateral membrane fraction Adjust protein concentration to 3.5-5.5 mg protein/ml Homogenize with five strokes in tight-fitting Potter-Elvehjem homogenizer at 1000 rpm Inject into free-flow electrophoresis chamber Free-flow electrophoresis Adjust chamber buffer flow to 2 ml/hr/fraction, 875 V, chamber temperature 4.5 ° Determine distribution of protein and marker enzymes Combine fractions with highest Na+,K+-ATPase activity, centrifuge at 33,000 g for 20 min Resuspend pellet in vesicle buffer (see Method 1) Basolateral plasma membranes This method has a yield of 25%, the final membrane fraction exhibits a 16-fold enrichment of Na+,K+-ATPase, and the specific activity of marker enzymes for brush border enzymes is lower than in the starting material? ~ The aforementioned method can also be used to isolate brush border membranes and basolateral membranes simultaneously in high purity from rat renal cortex?° For this purpose a slightly different procedure is used to prepare the crude plasma membrane fraction that is to be subjected to free-flow electrophoresis. This crude plasma membrane fraction contains both lumenal and contralumenal membranes but has low mitochondrial, lysosomal, .and endoplasmic contamination. This method was the first to be described for the preparation of highly purified brush border membranes and basolateral membranes.4° It played a pivotal role in the discovery of a variety of transport systems and enzymes in renal membranes and the establishment of the polarity of the proximal tubule cell with regard to transport systems and enzymes. ~7

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Method 5: Simultaneous Purification of Lumenal and Contralumenal Membranes by Free-FlowElectrophoresis4° Rat kidney cortex slices One-half millimeter thick, 3 g from 10 rats Homogenize in 10 ml sucrose buffer (250 mM sucrose, 10 mM triethanolamine/HC1, pH 7.6, adjusted with NaOH) in loose-fitting PotterElvehjem homogenizer, 10 strokes by hand and 3 strokes at 300 rpm Centrifuge at 700 g for 10 min Discard pellet Centrifuge supernatant at 700 g for 10 min Discard pellet Centrifuge supernatant at 16,000 g for 20 min Upper fluffy layer of pellet Resuspend in 5 ml of supernatant by swirling and scraping Add residual supernatant Homogenize gently in loose-fitting Potter-Elvehjem homogenizer with three strokes at 800 rpm Centrifuge at 16,000 g for 20 rain Resuspend upper fluffy layer of pellet in 10 ml sucrose buffer, homogenize gently with three strokes, add 10 ml of sucrose buffer Centrifuge at 16,000 g for 20 rain Resuspend upper fluffy layer of pellet in eleetrophoresis buffer (250 mM sucrose, 8.5 mM triethanolamine titrated with acetic acid to pH 7.4) Crude plasma membrane fraction J, D. free-flow electrophoresis as described in Method 4 Brush border membranes Basolateral membranes Free-flow electrophoresis is also sometimes used to further purify basolateral membranes enriched by Percoll gradient centrifugation.43 The degree of vesiculation in isolated basolateral membranes is usually lower than in brush border membrane fractions, resulting in smaller intravesicular spaces (2 #l/mg protein). The orientation is random; some preparations contain a slightly higher amount of inside-out vesicles. Further transport properties have been reviewed recently. 1°,~9

43 B. Hagenbuch and H. Murer, Pfluegers Arch. 4117, 149 (1986).

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Purity of Lumenal and Contralumenal Membrane Preparation Purity of lumenal and contralumenal membrane preparations is usually established by the use of marker enzymes assumed to be localized exclusively in one or the other membrane. Despite the fact that targeting of membrane proteins into the basolateral plasma membrane may also include some incorporation of enzymes destined for the lumenal membrane,4,5 the assumption of an almost exclusive localization of marker enzymes is an appropriate approximation for the purpose of purity determinations under physiological conditions. In pathophysiological situations--such as short ischemia, for example--the distribution of marker enzymes changes due to lateral diffusion of the membrane proteins across the tight junctions. 44 Enrichment of marker enzymes therefore partly loses its value as a criterion of purity. It is noteworthy that differential precipitation of brush border membranes leads also to an enrichment of lysosomes and peroxisomes in the final membrane preparation, 16a fact that has to be considered when studying functions that are exerted both by microvilli and lysosomes. The basolateral membranes isolated by Percoll density gradient centrifugation also show an enrichment of lumenal membranes, as It might be of interest to consider the latter contamination on a quantitative basis. The highest enrichment achieved for basolateral membranes from renal cortex is about 20-fold, indicating that in the starting material about 5% of the protein constitutes basolateral plasma membranes. For brush border membranes an enrichment of about 15-fold is the average, thus about 7% of the cell protein represents brush border membranes. A threefold enrichment of the marker enzyme alkaline phosphatase in a basolateral membrane fraction therefore suggests that 21% of the protein in this fraction is derived from lumenal membranes. Taking into account the twofold higher degree of vesiculation of the brush border membranes, about 42% of the overall intravesicular space could represent intravesicular space surrounded by lumenal membranes. This very high degree of "functional" cross-contamination relative to the minor contamination derived from enzymatic measurements demonstrates that any enrichment of lumenal marker enzymes in contralumenal membrane fractions should be avoided. Otherwise wrong conclusions on the properties and distribution of transport systems can result. An interesting example for such functional cross-contamination are studies on the sodium dependence of phosphate transport in basolateral plasma membranes.43 44B. A. Molitoris,C. A. Hoilien,R. Dalai,D. J. Ahnen,P. D. Wilson,and J, Kim,J. Membr. Biol. 106, 233 (1988).

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46 1

The methods described above can also be used to isolate brush border membranes from S3 segments (or pars recta) of proximal tubules. For this purpose the cortex and outer medulla are carefully dissected and the latter is used as starting material) s-26 Isolation of Plasma M e m b r a n e Vesicles from M e d u l l a r y Thick Ascending Limb The renal outer medulla contains in its inner stripe, among other cells, 45 the thick ascending limb of Henle's loop (TALH). This tubular segment has a very high Na+,K+-ATPase concentration in the contralumenal membrane and its lumenal membrane is rich in Na+-K+-2C1 - cotransporter and K + channel activity.~ The membrane vesicles isolated from this region of the kidney have, therefore, been used so far to study mainly the molecular mechanism of these three transport elements.

Lumenal Plasma Membranes Lumenal plasma membranes derived from TALH cells can be identified by functional criteria, such as Na+-K+-2CI- cotransport activity47 or capacity to bind with high affinity and specificity to inhibitors of this transport system, such as bumetanide. ~ Since only about 20% of the starting material represent TALH cells,45 cell separation 49,5° has to precede some of the isolation methods described below. 5~

Method 6: Enrichment of Lumenal Plasma Membranes from TALH Cells by Differential Centrifugation Isolated TALH cells47,49 Prepare 24 mg protein from two cell preparations using six rabbits; store at - 7 0 * in 4 ml sucrose buffer (250 m M sucrose, 10 m M triethanolamine, pH 7.6, adjusted with HNO3) Homogenize at 4 ° in a total volume of 15 ml sucrose buffer upon thawing with 40 strokes in a tight-fitting Potter-Elvehjem homogenizer at 1000 rpm 45 B. Kaissling and W. Kriz, Adv. Anat. Embryol. Cell Biol. 56, 1 (1979). R. Greger, Physiol. Rev. 65, 760 (1985). 47 j. Eveloffand R. Kinne, £ Membr. Biol. 72, 173 (1983). 4a B. Forbush III and H. C. Palfrey, J. Biol. Chem. 258, 11787 (1983). 49 j. Eveloff, W. Haase, and R. Kinne, J. Cell Biol. 87, 672 (1980). 5o M. E. Chamberlin, A. LeFurgey, and L. J. Mandel, Am. J. PhysioL 247, F955 (1984). sl R. Kinne, this series.

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Cell Homogenate

(see Tissue homogenate below) Outer strip of outer medulla52 Obtain 10 g wet wt from four or five rabbits Mince in 5 ml sucrose buffer (see above) Homogenize at 4 ° in a total of 60 ml buffer with 15 strokes in a loose-fitting Dounce (#ass-glass) homogenizer. Filter homogenate through two layers of cheesecloth Tissue Homogenate Centrifuge at 700 g for 10 min, discard pellet Centrifuge supernatant at 16,000 g for 20 min Save supernatant Resuspend upper white fluffy layer of pellet in 12-15 ml sucrose buffer by homogenizing with 10 strokes in a loose-fitting PotterElvehjem homogenizer Centrifuge at 16,000 g for 20 min Repeat twice Centrifuge combined four supernatants at 100,000 g for 60 min Resuspend pellet in small volume of vesicle buffer [100 m M sucrose, 1 mmol Mg(NOa)2, 20 m M triethanolamine adjusted to pH 7.4 with H2SO4] by repeated passage through a 26-gauge needle Crude lumenal membrane fraction The yield of membrane protein is about 1.5 mg if isolated TALH cells are used and 25 mg if renal tissue is employed. The transport properties of the two membrane fractions are similar; bumetanide (10-4 M) inhibits chloride-dependent sodium uptake by about 50%.47.52 No enrichment of Na+,K÷-ATPase is observed, indicating that the membrane fraction is not contaminated with basolateral membranes. As expected from the anatomical arrangement, membranes derived directly from renal outer medulla contain appreciable amounts of brush border membranes. This cross-contamination is avoided when isolated TALH cells are used. The intravesicular volume of the membrane preparations is about 2 al/mg membrane protein. Further transport properties of these vesicles are described in more detail in some recent publications.47,52-54 52R. Kinne, E. Kinne-Saffran, B. Sch01ermann, and H. Schfitz, Pfluegers Arch. 407, S168 (1986). 53B. KSnig, S. Ricapito, and R. Kinne, PfluegersArch. 399, 173 (1983). 54R. Kinne, E. Kinne-Saffmn, H. Sehiitz, and B. SehOlermann, £ Membr. Biol. 94, 279 (1986).

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Another method to enrich lumenal plasma membranes from TALH cells, one employing density gradient centrifugation in addition to differential centrifugation, is compiled below.

Method 7: Preparation of Lumenal Membranes from Red Outer Medulla Using Metrizamide Density Gradient Centrifugation52-~ Red Outer Medulla Prepare 0.6 g/rabbit kidney Homogenize at 4 ° in 10 ml sucrose buffer (250 m M sucrose, 50 m M KC1, 2 m M MgC12, 1 m M EGTA, 10 m M 4-morpholinopropanesulfonic acid (MOPS), pH 7.2, adjusted with triethanolamine) in a tightfitting Potter-Elvehjem homogenizer with five strokes at 1000 rpm Centrifuge at 6000 g for 15 rain Save supernatant Resuspend pellet in original volume of sucrose buffer Rehomogenize with five strokes in Potter-Elvehjem homogenizer Centrifuge at 6000 g for 15 rain Discard pellet Combine supernatants Centrifuge at 48,000 g for 30 rain Resuspend pellet in the same buffer as above except for 25 m M imidazole/acetic acid instead of MOPS buffer Crude microsomes Adjust protein concentration to 3 - 4 mg/ml Layer 1 ml of crude microsomes on continuous metrizamide gradient [5 - 15% (w/v), on top of a 1-ml cushion of 30% metrizamide] Centrifuge for 16 hr at 106,000 g Collect the fraction between 3 and 5 ml Dilute in 25 m M imidazole acetate, 1 m M EDTA (pH 7.4) Centrifuge at 140,000 g for 90 min Resuspend in a small volume of aforementioned buffer by repeatedly drawing and expelling the suspension with a micropipet Crude lumenal membrane fraction In the metrizamide gradient lumenal and contralumenal membranes are 55 D. A. Klaerke, S. J. D. Karlish, and P. L. Jergensen, J. Membr. Biol. 95, 105 (1987). 56 C. Burnham, S. J. D. Karlish, and P. L. Jergensen, Biochim. Biophys. Acta 821, 461 (1985).

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only partially separated. The "lumenal" membrane fraction shows Na+-K+-2C1- cotransporter and K + channel activity, these activities have been reconstituted, and the regulation of the K + channel has been studied in some detail. 55,56 Furosemide inhibition of barium-insensitive S6Rb uptake is about 30%. Basolateral membranes of the thick ascending limb cells are characterized by a very high Na+,K+-ATPase activity.57,5sThis marker enzyme has been successfully employed in isolating membrane fractions highly enriched in this enzyme and thus predominantly derived from these cells.

Method 8: Purification of Inside-Out Oriented Basolateral Plasma Membrane Vesiclesfrom Outer Medulla Using Hypaque Gradient Centrifugation 59 Outer medulla Dissect tissue from transverse slices of pig kidney using scissors Add 5 ml sucrose/histidine buffer (250 mM sucrose, 30 mM histidine, pH 7.2) per 1 g tissue Homogenize with five strokes in a very loose-fitting Potter-Elvehjem homogenizer (clearance = 0.05 cm) at 1000 rpm Filter through cheesecloth Homogenize in a Potter-Elvehjem homogenizer (0.01-cm clearance) with 10 strokes at 1000 rpm Centrifuge at 7500 g for 15 min Discard pellet Centrifuge supernatant for 30 rain at 48,000 g Resuspend light upper portion of the pellet in sucrose/histidine buffer Crude microsomes Layer 1 ml of membrane suspended in sucrose/histidine buffer (20 mg protein/ml) on 20 ml continuous Hypaque gradient. Hypaque gradients are prepared from 8% (w/v) Hypaque (prepared by dilution of 60% Hypaque in sucrose/histidine buffer) and 20% Hypaque (w/v) (prepared in 25 mM imidazole) [alternatively a step gradient (16%/20%/23%; 10/10/3 ml) can be used] Centrifuge for 2 hr at 102,000 g 5Tj. L. F. Shaver and C. Stirling, J. Cell Biol. 278, (1978). 5s U. Schmidt and U. C. Dubaeh, PfluegersArch. 306, 219 (1969). s9 K. N. Dzhandzhugazyan and P. L. Jgrgensen, Biochim. Biophys. Acta 817, 165 (1985).

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Collect fractions between 13 and 17 ml from the top or membranes at 0/16% interface Dilute 1 : 10 with sucrose/histidine buffer Centrifuge for 1 hr at 100,000 g Resuspend pellet in desired volume of buffer Right-side-out basolateral plasma membrane vesicles This method yields membranes with a specific activity of Na+,K÷-ATPase of about 10 #mol of P-fl(mg protein, min). The membranes are all oriented right side out since almost all of the Na+,K+-ATPase is cryptic and can be determined only after opening the membrane vesicles with detergents. Similar preparations can be obtained using linear metrizamide gradients. 59 Such vesicles have been employed so far mainly in studies on the mechanism of action of Na+,K+-ATPase; other transport properties of these vesicles are only poorly characterized. Isolation of Plasma M e m b r a n e Vesicles from the Inner Medulla (Renal Papilla) The renal inner medulla, similar to the outer medulla, contains a variety of different tubular segments such as the thin descending limb of Henle's loop, the papillary collecting ducts, a substantial number of capillaries, as well as a significant number of interstitial cells. Plasma membranes derived from this region of the kidney can, therefore, be expected to show a large degree of heterogeneity unless either defined cell populations6° or a sophisticated sequence of several separation procedures is employed. In addition, there are only a few enzymatic markers which identify specific membranes derived from a defined cell type. The cell that has attracted most interest hitherto is the collecting duct, on the one hand because it is the site of action of antidiuretic hormone, on the other hand because of its capability to strongly acidify the urine. In the following sections separation procedures are described which yield purified basolateral plasma membranes of the collecting duct, identified by their vasopressin-sensitive adenylate cyclase activity, and plasma membrane fractions, which probably are derived from the lumenal surface or reserve vesicles thereof (as judged by their proton pump and cAMP-dependent protein kinase activity). The final separation in both procedures occurs via free-flow electrophoresis; the second procedure includes a density gradient which enriches lumenal or contralumenal membranes before the final purification step. 6o j. B. Stokes, C. Grupp, and R. K. H. Kinne, Am. J. Physiol. 253, F251 (1987).

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Method 9: Simultaneous Purification of Lumenal and Contralumenal Membranes from Bovine Papilla by Free-Flow Electrophoresis 6~

White papilla from bovine kidney Mince 6 g wet wt (from two kidneys) in a small volume of ST buffer (250 m M sucrose, 10 m M triethanolamine/HC1, pH 7.6) Dilute to 30 ml with ST buffer Homogenize 10-ml aliquots in a Dounce (glass-glass) homogenizer (five strokes by hand with a loose-fitting pestle) Combine homogenates and filter through two layers of cheesecloth Homogenize filtrate in a Dounce glass-glass homogenizer (15 strokes by hand with a tight-fitting pestle) Centrifuge for 10 min at 700 g Discard pellet Centrifuge supernatant for 10 rain at 10,000 g Save supernatant Rehomogenize pellet (I 5 strokes by hand with a tight-fitting pestle) in 5 ml ST buffer; add 10 ml ST buffer Centrifuge for 10 min at 10,000 g Discard pellet Combine supernatants Centrifuge for 1 hr at 100,000 g Crude plasma membrane fraction Suspend pellet in 4 ml electrophoresis buffer (280 m M sucrose, 8.5 m M acetic acid, 8.5 m M triethanolamine, pH 7.4, adjusted with 2 M NaOH) Homogenize by 10 strokes in a tight-fitting homogenizer Dilute with 10 ml electrophoresis buffer Centrifuge for 10 min at 3000 g Save supernatant Repeat twice Subject supernatant to free-flow electrophoresis Free-flow electrophoresis I Arrange electrophoresis conditions as in Method 4 Combine fractions according to specific activity of Ca2+-ATPase or 6t I. L. Schwartz,L. J. Shlatz,E. Kinne-Saffran,and R. Kinne,Proc.Natl.Acad. Sci. U.S.A. 71, 2595 (1974).

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vasopressin-sensitive adenylate cyclase (contralumenal membranes) or nonmitochondrial HCO3--stimulated ATPase (lumenal membranes) Dilute with 0.1 M Tris-HC1 buffer, pH 7.4 Centrifuge for 1 hr at 100,000 g Suspend in desired buffer (see Method 1) Enriched lumenal and contralumenal membranes

Method 1O: Isolation of Lumenal and Contralumenal Membranes from Canine Inner Medulla ~2 Dissected papilla from mongrel dogs Homogenize in ST buffer (250 mM sucrose, 20 mM Tris-HC1, pH 7.4) as a 10% (w/v) suspension by two 2- to 5-see bursts at full speed in a Waring blender and five strokes of a loose-fitting Teflon-glass homogenizer at 2000 rpm Filter homogenate through a double layer of cheesecloth Centrifuge for 10 min at 600 g Discard pellet Centrifuge supernatant for 20 min at 10,000 g Save pellet (Pl0) Crude contralumenal plasma membranes I Centrifuge supernatant for 1 hr at 100,000 g Save pellet (Pl0o) Crude lumenal plasma membranes Further purification of contralumenal membranes Resuspend P~o in 5 ml ST buffer Homogenize by five strokes in a loose-fitting Dounce homogenizer Load on top of a linear sucrose gradient [34-45% (w/w), dissolved in 20 mM Tris-HC1, pH 7.4] Centrifuge for 7.5 hr at 100,000 g Collect fractions between 36 and 41% sucrose Dilute to 8% sucrose with 20 mM Tris-HC1, pH 7.4 Sediment membranes by centrifugation for 1 hr at 100,000 g Save pellet for free-flow electrophoresis as detailed in Method 4 62 R. Iyengar, D. S. Mailman, and G. Sachs, Am. J. Physiol. 234, F247 (1978).

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Further purification of lumenal membranes Resuspend P~ooin 5 ml ST buffer Homogenize by five strokes in a loose-fitting Dounce homogenizer Load on top of an exponential gradient prepared by mixing 7.5% Ficoll in ST buffer and 43% (w/w) sucrose dissolved in 20 m M TrisHCI, pH 7.4 Centrifuge for 45 hr at 100,000 g Collect second protein peak at density 1.15 Dilute fractions to 8% sucrose with 20 m M Tris-HCl buffer, pH 7.4 Sediment membranes by centrifugation for 1 hr at 100,000 g Save pellet for free-flow electrophoresis as detailed in Method 4 Purified lumenal plasma membranes The lumenal membranes obtained by the first method show a 10-fold enrichment for the anion-stimulated ATPase; the method including density gradient centrifugation prior to free-flow electrophoresis yields membranes with a 26-fold enrichment of the anion-stimulated ATPase. Both membrane fractions show considerable intrinsic cAMP-dependent protein kinase activity. The basolateral membrane fractions are enriched 16- to 20-fold in Ca2+-ATPase or vasopressin-sensitive adenylate cyclase, respectively. The lumenal membrane vesicles were found to be osmotically reactive63 and to possess a proton pump which can generate quite high proton gradients across the membrane. ~ Some technical aspects of the isolation procedures presented above require some further comment. Homogenization of papillary tissue is difficult because of its high content in connective tissue. Thorough mincing into small pieces of tissue is therefore essential prior to using the loose-fitting Dounce homogenizer. Filtration through cheesecloth removes connective tissue which would impede the final homogenization in the tight-fitting Dounce homogenizer. During free-flow electrophoresis the membranes show a high tendency to aggregate; occasional suction of the membrane suspension through a 26-gauge needle alleviates this problem. In view of the intense exchange between the lumenal membrane and endosomes in the collecting duct, purified endosomes can also be used to study specific functions of the lumenal membrane. 65 63 R. Kinne and I. L. Schwartz, in "Disturbances in Body Fluid Osmolality" (T. E. Andre,off, J. Grantham, F. C. Rector, eds.), p. 37. Am. Physiol. Soc., Baltimore, 1977. 64 E. Kinne-Saffran and R. Kinne, in "Hydrogen Ion Transport in Epithelia" (J. G. Forte, D. G. Warnock, and F. C. Rector, Jr., ¢ds.), p. 247. Wiley, New York, 1984. 65 A. S. Verkman, W. I. Lencer, D. Brown, and D. A. AusieUo, Nature (London) 333, 268 (1988).

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Concluding Remarks Despite the apparently straightforward methods given above the isolation of lumenal and contralumenal vesicles from defined renal tubular segments can by no means be regarded as routine. There are problems with regard to the purity of the membrane fractions and with regard to their unambiguous assignment to a particular cell type and a particular cell surface. The extent to which these factors may interfere with experiments must be considered in every instance.66 Generally speaking there is no problem obtaining membrane vesicles; the tendency of renal plasma membranes to vesiculate spontaneously is quite strong and transport studies can be performed relatively easily. But here again a cautious approach to techniques, results, and conclusions is advisable) 2 Acknowledgment The authorwishesto thank Mrs. DanielaMagdefesselfor skillfulsecretarialwork. 66 E. Kinne-Saffran and R. Kinne, this series, Vol. 172, p. 3.

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Studies by Optical Methods

By G. SACHS, E. RABON, and S. J. D. KARLISH Introduction The introduction of vesicle technology in the early 1960s by Kaback demonstrated that it was possible to retain transport properties of membranes after homogenization of cells and fractionation. 1 Various types of experimental design, involving the use of ionophores coupled with tracers or radioactive weak bases to measure potential or pH gradients, were applied to bacterial, mitochondrial, or chloroplast vesicles. 2 In the 1970s optical probes gradually replaced radioactive methods in many vesicle and then intact cell applications. This chapter will describe probes of pH and potential frequently utilized for vesicles and cells in the current literature. 3 H. R. Kaback and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 55, 920 (1966). 2 p. Mitchell, Biol. Rev. 41, 455 (1966). 3 B. Chance, M T P l n t . Rev. Sci.: Biochem. 3, 1 (1975).

METHODS IN ENZYMOLOGY, VOL. 191

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Isolation of lumenal and contralumenal plasma membrane vesicles from kidney.

450 KIDNEY [26] [26] I s o l a t i o n o f L u m e n a l a n d C o n t r a l u m e n a l Membrane Vesicles from Kidney Plasma By EVAMARIAKtNN~-SA...
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