534
GASTROINTESTINAL SYSTEM
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[34] P r e p a r a t i o n o f B a s o l a t e r a l ( S i n u s o i d a l ) a n d Canalicular Plasma Membrane Vesicles for the Study of Hepatic Transport Processes By P~TER J. MEIER and JAMES L. BOYER Hepatocytes represent highly polarized secretory cells that exhibit eificient transport of a wide variety of endogenous and exogenous compounds from blood into bile. In order to maintain these vectorial transport processes hepatocytes localize distinct membrane transport systems on their various surface domains. While sinusoidal and lateral surfaces (i.e., the "basolateral" pole of hepatocytes) provide for etficient exchange of various ions, organic solutes, and proteins with blood plasma, the bile canalicular or apical pole of the cell is separated from the plasma space by tight junctions and is highly specialized for the primary secretion of bile. In order to be able to separately study basolateral and canalicular membrane transport processes without interference of intracellular metabolic events, it is necessary to selectively isolate basolateral (blLPM) and/or canalicular (cLPM) liver plasma membrane vesicles. Since the original description of isolation of plasma membranes from rat liver by Neville,mnumerous major technical modifications have been introduced in order to improve yield and purification of membranes as well as reproducibility of the procedure.2-4 Most of these techniques involve mild homogenization of the liver to prevent bile canaliculi from fragmentation followed by isolation of a "mixed" plasma membrane fraction from an initial low-speed (i.e., "crude nuclear") pellet. This fraction is enriched in intact bile canaliculi and lateral membrane sheets with some attached sinusoidal membrane fragments. 5-s Extensive studies by Evans and co-workers have shown that up to 30 rag "bile canalicular enriched" plasma membrane protein can be isolated from a total of 100-120 g liver D. M. Neville, Jr., J. Biophys. Biochem. Cytol. 8, 413 (1960). 2 p. Emmelot, C. J. Bos, R. P. van Hoeven, and W. J. van Blitterswijk, this series, Vol. 31, p.76. a N. N. Aronson, Jr., and O. Touster, this series, Vol. 31, p. 90. 4 W. H. Evans, in "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work, eds.), Vol. 7, Part I. Elsevier/North-Holland Biomedical, Amsterdam, 1978. 5 W. H. Evans, FEBSLett. 3, 237 (1969). 6 M. H. Wisher and W. H. Evans, Biochem. J. 146, 375 (1975). 7 C. S. Song, W. Rubin, A. B. Rifldnd, and A. Kappas, J. CellBiol. 41, 124 (1969). 8 A. L. Hubbard, D. A. Wall, and A. Ma, J. Cell Biol. 96, 217 (1983).
METHODS 1N ENZYMOLOGY, VOL. 192
Copyright © 1990 by A ~ c Press, Inc. All rights of reproduction in any form reserved.
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PREPARATIONOF blLPM AND cLPM
535
by a rate zonal centrifugation technique.4-6 Furthermore, subsequent tight homogenization followed by density-gradient centrifugation in sucrose resulted in "light" and "heavy" plasma membrane subfractions of preferential canalicular and basolateral origin, respectively. 6,9 However, these studies required specialized zonal rotors and lengthy ultracentrifugation steps (up to 16 hr). In addition, modest enrichments of characteristic marker enzyme activities (i.e., 5'-nuclcotidase, leudnaminopeptidas¢) indicate that the presumptive canalicular membrane subfraction, as isolated in these studies, was still contaminated with basolateral membranes to a large extent and/or the respective enzyme activities were partially inactivated during the prolonged centrifugation steps. Although recent alternative isolation methods have been proposed, these procedures are associated either with insufficient purification,3,1°-16 low yields, 12'14'15'17'18 o r they require antibodies against specific canalicular membrane components, s,~s Hence, for in vitro transport studies isolation procedures are still required that result in both high yields and high purification of basolateral and canalicular membrane subfractions within reasonable working hours. The procedure described here represents an extensive modification of the Evan's procedure using a combination of rate zonal flotation and highspeed discontinuous sucrose gradient centrifugation techniques./9 The method permits the simultaneous isolation of bILPM and cLPM vesicles from the same homogenate in a yield sufficient for a large number of individual transport studies to be carried out in both plasma membrane subfractions. Isolation Procedure The method is essentially based on two sequential centrifugation steps (Fig. 1). First, rate zonal flotation is used to prepare a "mixed" plasma membrane fraction that exhibits the same composition and morphology as the bile canalicular enriched fraction described by others. 5-8 Second, after 9 W. H. Evans, Biochim. Biophys. Acta 604, 27 (1980). 1oG. Toda, H. Oka, T. Oda, and Y. Ikeda, Biochim. Biophys. Acta 413, 52 (1975). i1 M. M. Fisher, D. L. Bloxam, M. Oda, M. J. Phillips, and I. M. Yousef, Pro¢. Soc. Exp. Biol. Med. 150, 177 (1975). 12B. F. Scharschmidt and E. B. Kccffe, Biochim. Biophys. Acta 646, 369 (1981). 13E. G. Loten and J. C. Redshaw-Loten, Anal. Biochem. 154, 183 (1986). ,4 p. Gierow, M. Sommarin, Ch. L. Larsson, and B. Jergil, Biochem. J. 235, 685 (1986). 15R. E. Poupon and W. H. Evans, FEBSLett. 38, 134 (1979). 16R. J. Epping and F. L. Bygrave, Biochem. J. 223, 733 (1984). 17j. L. Boyer, R. M. Allen, and Oi Cheng Ng, Hepatology (Baltimore) 3, 18 (1983). is L. M. Roman and A. L. Hubbard, J. CellBiol. 98, 1497 (1984). 19p. j. Meier, E. S. Sztul, A. Reiben, and J. L. Boyer, J. CellBiol. 98, 991 (1984).
536
GASTROINTESTINAL SYSTEM
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"~ IO0 (:J liver 1:8 HOMOGENATE (traM NoHC03-,pHT.5) J 1500gxl5min ,[crude nuclear pellet 5 voI. 56%sucrose (w/w)
ii~
20,O00rpmx2hrs
' ~""1 = l"I mixed
.....
f
TZ-28 zonal rotor (Sorvall R)
(44/36.5%interface)
tight homogenization
1 =ram - [ ~ 31%I 40,O00rpm x 3 hrs = I SW 41 rotor I
/
FIG. 1. Flow diagram for preparation of basolateral (blLPM) and canalicular (cLPM) plasma membrane vesicles from rat liver.
tight homogenization of mixed LPM, basolateral and canalicular vesicles are separated by high-speed centrifugation through discontinuous sucrose gradients. Animals. Male Sprague-Dawley rats (200-250 g) are housed in a constant temperature-humidity environment with alternating 12-hr fight and dark cycles for at least 4 days prior to use. During this time all animals are allowed access to food and water ad libitum. On the day of the experiment, the rats are killed by decapitation between 7:30 and 8:30 AM. Solutions. NaHCO3 (1 mM), pH 7.4; 0.25 M sucrose (8.1%, w/w); 31% sucrose (w/w; d = 1.1318); 34% sucrose (w/w; d = 1.1463); 36.5% sucrose (w/w; d = 1.1587); 38% sucrose (w/w; d = 1.1663); 44% sucrose (w/w; d = 1.1972); 56% sucrose (w/w; d = 1.2623). All solutions are prepared 24 to 48 hr before use and stored at 4 °. Densities of the sucrose solutions are adjusted at room temperature by refractometry (Abbe-3L refractometer;, Bausch and Lomb, Rochester, NY). Step 1: Isolation of Mixed LPM. Routinely, 10-12 fed animals are killed and their fives (110-115 g) rapidly removed and chilled on ice. Ten-gram portions of fiver are cut into small pieces, washed three times in 80 ml cold 1 m M NaHCO3 with a loose (type A) Dounce homogenizer (seven up-and-down strokes). The homogenate is further diluted to 1800-
[34]
PREPARATIONOF blLPM AND cLPM
537
2000 ml with cold NaHCO3 and filtered twice through two layers of 60-grade cheesecloth. Centrifugation at 1500 g for 15 min (GSA rotor, E. I. Du Pont de Nemours and Co., Inc., Sorvall Instruments Division, Newton, CT) gives a "crude nuclear pellet" that is resuspended in 5.5 vol of 56% sucrose and stirred for 15 min to disrupt membrane aggregates (Fig. 1). The sample is then loaded onto a 100-ml cushion of 56% sucrose with a variable speed pump into the zonal rotor TZ-28 (Sorvall Instruments) and overlayed with 400 ml of 44% sucrose and 200 ml of 36.5% sucrose, respectively. Finally, the rotor is filled to its total volume capacity (1350 ml) with 0.25 M (8.1%) sucrose. During the entire loading procedure the rotor is run at 3000 rpm. The completed discontinuous sucrose gradient system is centrifuged at 20,000 rpm for 120 min (Fig. 1). After slow deceleration to a complete stop, 70 15-ml fractions are collected from the bottom of the rotor and routinely analyzed for turbidity (absorbance at 700 nm). The bulk of plasma membrane fragments is normally contained in fractions of the third turbidity peak (44/36.5% sucrose interface). 19 These fractions are combined and diluted with NaHCO 3 to 1000 ml. The suspension is then centrifuged at 7500 g for 15 min. The resulting pellet is gently resuspended (by vortex mixing) in 250 ml NaHCO 3, and the material representing the mixed LPM resedimented at 2700 g for 15 min.
Step 2: Separation of Basolateral (blLPM) and Canalicular (cLPM) Subfractions. Mixed LPM are diluted with 0.25 M sucrose to a total volume of 25 ml and homogenized with a tight type B glass-glass Dounce homogenizer by 50 strokes (1 stroke equals up and down). Mixed LPM (3.5 ml) are layered on top of a three-step sucrose gradient consisting of 4 ml 38%, 2.5 ml 34%, and 2.5 ml 31% sucrose (Fig. 1). The tubes are centrifuged at 40,000 rpm (195, 700 g) for 3 hr in a Beckman SW41 rotor Beckman Instruments, Inc., Palo Alto, CA). This results in three distinct bands and a pellet (Fig. 1). The material on top of the 31% sucrose layer represents cLPM whereas the membranes at the 34/38% interface correspond to blLPM (Fig. 1). These two LPM subfractions are carefully collected with a plastic Pasteur pipet, diluted in 0.25 M sucrose, and sedimented at 105,000 g for 60 min. The pellets are resuspended in the appropriate buffer medium by repeated (20 times in and out) suctioning through a 25-gauge needle. Alternatively, vesiculation of the membrane fragments can be performed by 30 strokes in a tight-fitting glass-glass or Teflon-glass homogenizer. The vesiculated membranes are then stored in liquid nitrogen for up to 4 weeks without loss of transport functions.
Properties of Isolated blLPM and cLPM Subfractions Morphology. Transmission electron microscopy revealed that both LPM subfractions are composed of membrane vesicles, although blLPM
538
GASTROINTESTINAL SYSTEM
[34]
still contained some unbroken lateral membrane sheets (Fig. 2a). In addition, mitochondrial membrane fragments were occasionally found in the blLPM subfraction, a finding consistent with the slight enrichment over homogenate of succinate cytochrome c reductase activity (Table I). Correspondingly, intravesicular volumes are approximately twofold lower in blLPM ( - 1 / d mg-l protein) than in cLPM (~2/~1 mg--' protein) vesicles as estimated from equilibrium uptakes o f ['4C]glucose. 2° Freeze-fracture analysis indicated that 72% of blLPM 2' and 77% of cLPM vesicles2° are oriented fight side out, i.e., the outer vesicle surface corresponds to the extracellular membrane area in vivo. However, since the basolateral subfraction is regularly contaminated with canalicular vesicles to approximately 10%'9 the true fight-side-out orientation of basolateral vesicles might range between 60 and 65%. Recoveries. Following the above-outlined procedure it is possible to simultaneously isolate approximately 0.19 mg blLPM and 0.10 nag cLPM protein/g liver from the same homogenate (Table I). Since the capacity of the method extends to a total of 115 g liver (wet wt), the maximum yields of blLPM and cLPM protein range around 22 and 12 rag, respectively. These are the highest recoveries of simultaneously isolated basolateral and canalicular LPM reported so far. Degree of Purification. Table I summarizes the protein recovery and enzymatic characteristics of the isolated LPM subfractions. Compared to the homogenate blLPM are slightly enriched with mitochondria, but threeto fivefold deenriched with respect to microsomes, lysosomes and Golgi complex. In contrast, cLPM are virtually free of mitochondrial fragments, but contain slightly more microsomal, lysosomal, and Golgi membranes than the blLPM subfraction. Since endoplasmic reticulum represents 24%, Golgi complex 1%, lysosomes 2%, and mitochondria 16% of total homogehate protein,22 it can be calculated that the total contamination of blLPM and cLPM with these intracellular organelles accounts for approximately 38% ( 2 4 X 0 . 3 + l × 0 . 2 + 2 X 0 . 3 + 1 6 X 1 . 9 ) and 23% ( 2 4 X 0 . 7 + 1 X 0.4 + 2 X 1.1 + 16 X 0.2) of total protein, respectively. In regard to domain-specific plasma membrane markers the blLPM subfraction is 24- to 45-fold enriched over homogenate in Na+,K+-ATPase activity, contains this enzyme's a-subunit, 23 and localizes glucagon-stimu2o p. j. Mier, A. St. Meier-Abt, C. Barrett, and J. L. Boyer, J. Biol. Chem. 259, 10614 (1984). 21 R. H. Moseley, P. J. Meier, P. S. Aronson, and J. L. Boyer, Am. J. Physiol. 250 G35 (1986). 22 D. M. Neville, in "Biochemical Analyis of Membranes" (A. H. Maddy, ed.), p. 27. Chapman & Hall, London, 1976. 23 E. S. Sztul, D. Biemesderfer M. J. Caplan, M. Kashgadan, and J. L. Boyer, J. Cell Biol. 104, 1239 (1987).
[34]
PREPARATIONOF blLPM AND cLPM
539
FIo. 2. Electron micrographs of(A) isolated basolateral (blLPM) and canaficular (cLPM) rat liver plasma membrane subfractions. (Bars, 1/~m; X 14,000)
latable adenylate cyclase activity (Table I). In contrast, these marker enzymes could not be detected in highly purified cLPM that are additionally characterized by 50- to 90-fold enrichments of classical canalicular marker enzyme activities such as alkaline phosphatase, aminopeptidase (leucylnaphthylamidase), 7-glutamyltranspeptidase, and 5'-nucleotidase (Table I)fl 9 These results indicate that the isolated cLPM are virtually devoid of basolateral (sinusoidal) contaminants. In contrast, based on the fourfold enrichment of the canalicular specific marker enzyme aminopeptidase (leucylnaphthylamidase), the blLPM subfraction is contaminated with canalicular membranes to approximately 10%. Comments
Suitability of Marker Enzyme Activities for Assessment of the Separation between blLPM and cLMP. In order to correctly delineate the functional polarity of the hepatocellular surface domains with respect to ion transport processes, it is necessary to routinely test the purification of isolated blLPM and cLPM vesicles. For this purpose marker enzymes must be used that have unequivocally been demonstrated to be selectively localized at either the canalicular or the basolateral pole of hepatocytes. Aminopeptidase (leucylnaphthylamidase) has been shown to be specific for the canalicular membrane by both enzyme activity measurements as well as
540
GASTROINTESTINAL SYSTEM
[34]
TABLE I PROTEIN RECOVERY A N D ENZYMATIC CHARACTERIZATION OF BASOLATERAL (blLPM) A N D CANALICULAR (cLPM) SUBFRACTIONSa
blLPM Protein recovery (mg/g fiver) (193) Marker enzymes for intracellular organellesb Succinate cytochrome c reductase (mitoehondria) (55) NADPH cytochrome c reductase (microsomes) (32) Acid phospfiatase (lysosomes) (8) UDPgalactosyltransferase ((3o1#) (8) Plasma membrane enzyme markers~ Na+,K+-ATPase activity (53) Alkaline phosphatase (12) Leucylnaphthylamidase (33) 7-Glutamyltranspeptidase(7) 5'-Nucleotidase (5) Glucagon-stimulatable adenylate cyclase (3)* a Subunit of Na+,K+-ATPasd
cLPM
0.19±0.06 0.10±0.03 Relative specific activities" 1.9±0.9 0.2±0.2 0.3±0.2 0.2±0.3
0.2±0.4 0.7±0.4 1.1±0.3 0.4±0.4
34 ± 11 12 ± 4 4_ 3 15 ± 5 11 ± 5 Present Present
Not detectabled 71 + 21 42+ 9 6 0 + 12 64+ 5 Not detectable Not detectable
"Results are given as the means + SD with the number of experiments in parentheses.
b Marker enzyme activities were determined according to the following procedures: Succinate and NADPH eytoehrome c reductuses [G. L. Sottocasa, B. Kuylenstiema, L. Ernster, and A. Bergstrand, J. Cell Biol. 32, 415 (1967)], acid phosphatase [T. L. Rothstein and J. J. Blum, J. Cell Biol. 57, 630 (1973)], UDPgalactosylWansferase [B. Fleischer and M. Smigel, J. Biol. Chem. 253, 1632 (1978)], Na+,K+-ATPase [B. F. Scharschmidt, E. B. Keeffe, N. M. Blankenship, and R. IC Oeimer, J. Lab. Clin. Med. 93, 790 (1979)], alkaline phosphatase [E. B. Keeffe, B. F. Seharsehmidt, N. M. Blankenship, and R. K. Oekner, J. Clin. Invest. 64, 1590 (1979)], leueylnaphthylamidase [J. A. Goldbarg and A. M. Rutenburg, Cancer (Philadelphia) 2, 283 (1958)], p-glutamyltranspeptidase [M. Orlowski and A. Meister, Biochim. Biophys. Acta 73, 676 (1963)], 5'-nucleotidase [J. Avruch and D. F. H. Wallaeh, Biochim. Biophys. Acta 233, 334 (1971)]. c Relative specific activity -- specific activity ratios, LPM subfraetions/homogenate. a Absence of any detectable Na+,K+-ATPase activity in cLPM has been achieved in 2 out of 3 in a total of over 500 membrane preparations. e A. F. Stewart, K. L. Insogna, D. Goltzman, and A. E. Broadus, Proc. Natl. Acad. Sci. U.S.A. 80, 1454 (1983). f Determined by immunoblotting using monoclonal antibodies [E. Sztul, M. Caplan, D. Biemesdeffer, L. Barrett, M. Kashgafian, and J. L. Boyer, £ Cell Biol. 104, 1239 (1987).
immunolocalization studies, u Therefore, this enzyme is well suited for estimating the contamination of blLPM with cLPM. In contrast, according to histochemical and various subeellular fractionation studies, the Na+,K+-ATPase is selectively localized at the basolateral mem24 L. M. Roman and A. L. Hubbard, J. CellBiol. 98, 1488 (1984).
[34]
PREPARATIONOF blLPM AND cLPM
541
brane? 5,17,~9,25-27 In addition, recent monoclonal antibody studies have also found the ot subunit of Na+,K+-ATPase to be exclusively localized at the basolateral membrane domain. 23Although two other immunolocalization studies found the t~ subunit at the eanalicular membrane as well,2s,29 further purification of the antibody preparation resulted in a complete loss of canalicular immunoreactivity in one study.2sa9- Since in isolated plasma membrane subfractions enzyme activity as well as the c~ subunit of Na+,K+,ATPase are associated with blLPM, but not with cLPM,23 the absence of any detectable Na+,K+-ATPase activity is an adequate criterion to exclude significant basolateral contamination of c L P M . 29b Thus, measurements of aminopeptidase and Na+,K+-ATPase activities are useful tests for routine screening of the degree of cross-contamination of liver plasma membrane subfractions. Reproducibility of the Isolation Method. Based on the determination of leucine aminopeptidase and Na+,K+-ATPase activities, blLPM with approximately 10% canalicular contamination and cLPM devoid of measurable Na+,K+-ATPase activity can be prepared in two out of three experiments. Successful separation appears to most critically depend on (1) the use of fed animals, (2) rat weights of 200 g, and (3) tight homogenization of the mixed LPM. The latter step is especially important since insufficient dissociation of canalicular membranes from lateral membrane sheets considerably decreases the final yield of cLPM. Although we recommend 50 strokes (I stroke equals up and down) with a tight type B glass-glass Dounce homogenizer, alternative methods (e.g., sonication, motor-driven Teflon-glass homogenizer) can also be used. It should be realized, however, that too-harsh homogenization conditions can damage the membrane integrity which can best be controlled by determining the recovery of marker enzyme activities in the isolated LPM subfractions. Practicability of the Procedure. Although the described procedure involves zonal centrifugation, the handling of the TZ-28 reorienting density gradient zonal rotor (Sorvall) is considerably easier than the use of classical high-speed zonal rotors. For example, the TZ-28 rotor does not require an 2s B. L. Blitzer and J. L. Boyer, J. Clin. Invest. 62, 1101 (1978). 26 p. S. Latham and M. Kashgarian, Gastroenterology 76, 988 (1979). 27M. Inoue, R. Kinne, T. Tran. L. Biempica, and I. M. Arias, J. Biol. Chem. 258, 5183 (1983). 28 S. Takemura, IC Omori, K. Tanaka, IC Omori, S. Matsuura, and Y. Taslfiro, J. Ceil Biol. 99, 1502 (1984). 29D. B. Schenk and H. L Leffert, Proc. Natl. Acad. Sci. U.S.A. 80, 5281 (1983). 29,y. Fashiro, K. Omori, A. Yamamoto, J. Histochem. Cytochem. 36, 221 (1988). 29bM. Sellinger, C. Barrett, Ph. Malle, E. R. Gordon, and J. L. Boyer, Hepatology (Baltimore) 11, 223 (1990).
542
GASTROINTESTINAL SYSTEM
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ultracentrifuge and can be unloaded statically (nonturning position). The total centrifugation time of the procedure is 6½ hr permitting high-yield isolation of both LPM subfractions in 1 day (total preparation time - 11 hr). Furthermore, the procedure can easily be scaled down to one rat liver (-10 g net wt). For this purpose the initial crude nuclear pellet is resuspended in 2.2 vol of 70% (w/w) sucrose. After stirring for 15 min, 12 ml of the suspension is filled into SW 28 rotor (Beckman Instruments, Inc., Palo Alto, CA) tubes and ovedayered with 10 ml 44% and 10 ml 36.5% (w/w) sucrose. The tubes are filled to the top with 0.25 M sucrose and the gradient system centrifuged at 26,000 rpm for 90 min. Mixed LPM are recovered from the 44/36.5% sucrose interface and subjected to the same washing, tight homogenization, and high-density sucrose centrifugation steps are described above. By this means approximately 1 mg cLPM and 3 mg blLPM protein can be isolated from one liver with virtually the same degree of purification as demonstrated for the high-capacity zonal centfifugation method (Table I).
Suitability of Isolated blLPM and cLPM Vesicles for in Vitro Transport Studies. Table II summarizes several receptors and transport systems identified and partially characterized in the two isolated LPM subfractions. The findings demonstrate that both vesicle preparations are well suited for investigating hepatocellular membrane transport functions. Furthermore, the results illustrate the polar nature of hepatocyte plasma membrane transport processes. This supports the concept that the mechanisms and driving forces involved in overall transport of solutes from blood into bile are similar to vectorial transport mechanisms in other epithelial cells. Comparison with Other Isolation Procedures In addition to the described technique, other methods have recently been developed for isolation of highly purified basolateral and canalicular LPM vesicles. Although we have no direct experience with these alternative procedures, a short description of the various principles and techniques involved might help in choosing the most adequate method for a given purpose. Procedures of Inoue and co-workers27,3°describe the isolation of sinusoidal or canalicular membrane vesicles. Sinusoidal vesicles are prepared from a postnuclear supernatant by sucrose-Ficoll density gradient centrifugation. Seventy percent of the vesicles are oriented right side out and 3oM. Inoue, R. Kinne, T. Tran, and I. M. Arias, Hepatology (Baltimore) 2, 572 (1982).
[34]
PREPARATIONOF b l L P M A N D c L P M TABLE II RECEPTORS AND TRANSPORT SYSTEMS IDENTIFIED IN
BASOLATERAL(blLPM) AND CANALICULAR (cLPM) VESICLESa
Intact IgA2 receptor b ("secretory component") Bile acid-binding polypeptides~ ATP-dependent Ca 2+ uptake d Na +/alanine cotransport 2° Na+/taurocholate cotransport 2o Na+,K+.dependent glutamate uptake ~ Na+/H + antiport 21 CI-/HCO3- exchangef OH-/SO4 2- exchanges HCO3-/SO4 2- exchangeh
blLPM
cLPM
+
--
M r 54,000
M r 100,000
Mr 48,000 +
--
+ +
+ -
-
+
+ +
+ +
-
a +, Present; - , not detectable. b Determined by immunoblotting using a monospecilic antibody.~9 c Identified by photoaifinity labeling of blLPM and cLPM with the sodium salt of the photolabile derivative (7,7-azo-3,12-dihydroxy-5-[3H]cholan-24oyl)-2-aminoethanesulfonic acid [W. Kramer, U. Bickel, H. P. Buscher, W. Gerok, and G. Kurz, Eur. J. Biochem. 129, 13 (1982); St. Ruelz, G. Fricker, G. Hugentobler, K. Winterhalter, G. Kurz, and P. J. Meier, J. Biol. Chem. 262, 11324 (1987)]. d C. Evers, G. Hugentobler, R. Lester, P. Gmaj, P. J. Meier, and H. Murer, Biochim. Biophys. Acta 939, 542 (1988). e N. Ballatori, R. H. Moseley, and J. L. Boyer, J. Biol. Chem. 261, 6216 (1986). f P. J. Meier, R. Knickelbein, R. H. Moseley, J. W. Dobbins, and J. L. Boyer, J. Clin. Invest. 75, 1256 (1985). s G. Hugentobler and P. J. Meier, Am. J. Physiol. 251, 6656 (1986). h p. j. Meier, J. Valantinas, G. Hugentobler, and I. Rahm, Am. J. Physiol. 253, 6461 (1987).
543
544
GASTROINTESTINAL SYSTEM
[34]
exhibit an internal volume of 1 gl mg-~ protein. The recovery is 0.3 mg protein/g liver with a maximum yield of 7.5 mg protein. Na+,K+-ATPase activity is 20-fold enriched over homogenate. Total contamination with intracellular organelles approximates 34%. The preparation time is less than 5 hr. Canalicular vesicles are isolated from the crude nuclear pellet using nitrogen cavitation and Ca2+ precipitation techniques. Virtually 100% of the vesicles are reported'to be oriented right side out (intravesicular volume I gl mg-t protein). Recovery and maximal yields are 0.12 mg/g liver and 3.0 mg, respectively. Canalicular marker enzyme activities are 50to 60-fold enriched over homogenate, whereas the relative specific activity for Na+,K+-ATPase is low (2.5 ×). Contamination with intracellular organelles is around 15%. Isolation time is 4 to 5 hr. Comments. Since sinusoidal vesicles are prepared from a postnuclear superuatant while canalicular vesicles are isolated from the crude nuclear pellet, this method should theoretically also permit isolation of both LPM subfractions from the same homogenate. However, comparative studies from such simultaneously isolated sinusoidal and canalicular vesicles have not been reported so far and, therefore, the practicability and total preparation time required for isolation of both LPM subfractions are not known (rough estimation: 7 to l0 hr). Furthermore, the contamination of the LPM subfractions with Golgi-derived membranes has not been assessed. Nevertheless, these vesicle preparations have been successfully used to assess the polar distribution of a variety of plasma membrane functions, including insulin and asialoglycoprotein receptors (sinusoidal),3~ Na+/H + antiport (sinusoidal),32 and transport of taurocholate3°,33 and glutathione~-37 and the multidrug resistance cation pump (Gp 170; canaficular). 37a The method of Bfitzer and Donovan3s involves Percoll gradient centrifugation and permits the preparation of 0.64 mg/g fiver basolateral vesicle protein within 4 hr. The maximal yield is between 12 and 16 rag. Seventy3~ I. M. Arias, in "Progress in Liver Diseases" (H. Popoer and F. Schaffner, eds.), Vol. 8, p.
145. Grune & Stratton, Orlando, Florida, 1986. 32 I. M. Arias and M. Forgac, £. Biol. Chem. 259, 5406 (1984). 33 M. Inoue, R. Kinne, T. Tran, and I. M. Arias, J. Clin. Invest. 73, 659 (1984). M. Inou¢, R. Kinne, T. Tran, and I. M. Arias, Eur. £. Biochem. 138, 491 (1984). 35 M. Inoue, R. Kinne, T. Tran, and I. M. Arias, Eur. J. Biochem. 134, 467 (1983). 36 T. Akerboom, M. Inoue, H. Sies, R. Kinne, and I. M. Arias, Fur. £. Biochem. 141, 211 (1984). 37 M. Inoue, T. M. Akerboom, H. Sies, R. Kinne, T. Tram, and I. M. Arias, J. Biol. Chem. 259, 4998 (1984). aTay. Kamimoto, Z. Gaitmaitan, J. Hsu, and I. M. Arias, J. Biol. Chem. 264, 11693 (1989). 3s B. L. Blitzer and C. B. Donovan, J. Biol. Chem. 259, 9295 (1984).
[34]
PREPARATIONOF blLPM AND eLPM
545
five percent of the vesicles are oriented right side out. Na+,K+-ATPase activity is 28-fold enriched over homogenate. Total contamination with intracellular organdies approximates 46%. Comments. This method is fast and does not require ultracentrifugation. However, it requires removal of the Percoll before transport studies can be performed and results in an approximately 30% contamination of basolateral vesicles with endoplasmic reticulum. The vesicles have so far mainly been used for characterization of the basolateral bile acid uptake systems3s-4° as well as the ATP-dependent Ca + pumps.4~ The highest purification of canalicular membrane vesicles (153-fold enrichment of aminopeptidase) has been reported by Hubbard et al., 8,~8 whose isolation procedure involves immunoadsorption on anti-aminopeptidase antibody-coated Staphylococcus aureus cells. Although the procedure has not been used on a preparative scale (protein yield --~45/zg), it could theoretically be scaled up 10- to 100-fold provided high enough amounts of antibodies are available. Acknowledgment Theses studies were supported by the Swiss National Science Foundation (Grants 3.983.0.84 and 3.992.0.87) and United States Public Health Service Grants DK-25636 and DK-36854.
39B. L. Blitzer and L. Lyons, Am. J. Physiol. 249 G34 (1985). 4oB. L. Blitzr, Ch. Terzakis, and K. A. Scott, J. Biol. Chem. 261, 12042 (1986). 4] B. L. Bitzer, B. R. Hosteler, and K. A. Scott, J. Clin. Invest. 83, 1319 (1989).
GASTROINTESTINAL SYSTEM
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[34] P r e p a r a t i o n o f B a s o l a t e r a l ( S i n u s o i d a l ) a n d Canalicular Plasma Membrane Vesicles for the Study of Hepatic Transport Processes By P~TER J. MEIER and JAMES L. BOYER Hepatocytes represent highly polarized secretory cells that exhibit eificient transport of a wide variety of endogenous and exogenous compounds from blood into bile. In order to maintain these vectorial transport processes hepatocytes localize distinct membrane transport systems on their various surface domains. While sinusoidal and lateral surfaces (i.e., the "basolateral" pole of hepatocytes) provide for etficient exchange of various ions, organic solutes, and proteins with blood plasma, the bile canalicular or apical pole of the cell is separated from the plasma space by tight junctions and is highly specialized for the primary secretion of bile. In order to be able to separately study basolateral and canalicular membrane transport processes without interference of intracellular metabolic events, it is necessary to selectively isolate basolateral (blLPM) and/or canalicular (cLPM) liver plasma membrane vesicles. Since the original description of isolation of plasma membranes from rat liver by Neville,mnumerous major technical modifications have been introduced in order to improve yield and purification of membranes as well as reproducibility of the procedure.2-4 Most of these techniques involve mild homogenization of the liver to prevent bile canaliculi from fragmentation followed by isolation of a "mixed" plasma membrane fraction from an initial low-speed (i.e., "crude nuclear") pellet. This fraction is enriched in intact bile canaliculi and lateral membrane sheets with some attached sinusoidal membrane fragments. 5-s Extensive studies by Evans and co-workers have shown that up to 30 rag "bile canalicular enriched" plasma membrane protein can be isolated from a total of 100-120 g liver D. M. Neville, Jr., J. Biophys. Biochem. Cytol. 8, 413 (1960). 2 p. Emmelot, C. J. Bos, R. P. van Hoeven, and W. J. van Blitterswijk, this series, Vol. 31, p.76. a N. N. Aronson, Jr., and O. Touster, this series, Vol. 31, p. 90. 4 W. H. Evans, in "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work, eds.), Vol. 7, Part I. Elsevier/North-Holland Biomedical, Amsterdam, 1978. 5 W. H. Evans, FEBSLett. 3, 237 (1969). 6 M. H. Wisher and W. H. Evans, Biochem. J. 146, 375 (1975). 7 C. S. Song, W. Rubin, A. B. Rifldnd, and A. Kappas, J. CellBiol. 41, 124 (1969). 8 A. L. Hubbard, D. A. Wall, and A. Ma, J. Cell Biol. 96, 217 (1983).
METHODS 1N ENZYMOLOGY, VOL. 192
Copyright © 1990 by A ~ c Press, Inc. All rights of reproduction in any form reserved.
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PREPARATIONOF blLPM AND cLPM
535
by a rate zonal centrifugation technique.4-6 Furthermore, subsequent tight homogenization followed by density-gradient centrifugation in sucrose resulted in "light" and "heavy" plasma membrane subfractions of preferential canalicular and basolateral origin, respectively. 6,9 However, these studies required specialized zonal rotors and lengthy ultracentrifugation steps (up to 16 hr). In addition, modest enrichments of characteristic marker enzyme activities (i.e., 5'-nuclcotidase, leudnaminopeptidas¢) indicate that the presumptive canalicular membrane subfraction, as isolated in these studies, was still contaminated with basolateral membranes to a large extent and/or the respective enzyme activities were partially inactivated during the prolonged centrifugation steps. Although recent alternative isolation methods have been proposed, these procedures are associated either with insufficient purification,3,1°-16 low yields, 12'14'15'17'18 o r they require antibodies against specific canalicular membrane components, s,~s Hence, for in vitro transport studies isolation procedures are still required that result in both high yields and high purification of basolateral and canalicular membrane subfractions within reasonable working hours. The procedure described here represents an extensive modification of the Evan's procedure using a combination of rate zonal flotation and highspeed discontinuous sucrose gradient centrifugation techniques./9 The method permits the simultaneous isolation of bILPM and cLPM vesicles from the same homogenate in a yield sufficient for a large number of individual transport studies to be carried out in both plasma membrane subfractions. Isolation Procedure The method is essentially based on two sequential centrifugation steps (Fig. 1). First, rate zonal flotation is used to prepare a "mixed" plasma membrane fraction that exhibits the same composition and morphology as the bile canalicular enriched fraction described by others. 5-8 Second, after 9 W. H. Evans, Biochim. Biophys. Acta 604, 27 (1980). 1oG. Toda, H. Oka, T. Oda, and Y. Ikeda, Biochim. Biophys. Acta 413, 52 (1975). i1 M. M. Fisher, D. L. Bloxam, M. Oda, M. J. Phillips, and I. M. Yousef, Pro¢. Soc. Exp. Biol. Med. 150, 177 (1975). 12B. F. Scharschmidt and E. B. Kccffe, Biochim. Biophys. Acta 646, 369 (1981). 13E. G. Loten and J. C. Redshaw-Loten, Anal. Biochem. 154, 183 (1986). ,4 p. Gierow, M. Sommarin, Ch. L. Larsson, and B. Jergil, Biochem. J. 235, 685 (1986). 15R. E. Poupon and W. H. Evans, FEBSLett. 38, 134 (1979). 16R. J. Epping and F. L. Bygrave, Biochem. J. 223, 733 (1984). 17j. L. Boyer, R. M. Allen, and Oi Cheng Ng, Hepatology (Baltimore) 3, 18 (1983). is L. M. Roman and A. L. Hubbard, J. CellBiol. 98, 1497 (1984). 19p. j. Meier, E. S. Sztul, A. Reiben, and J. L. Boyer, J. CellBiol. 98, 991 (1984).
536
GASTROINTESTINAL SYSTEM
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"~ IO0 (:J liver 1:8 HOMOGENATE (traM NoHC03-,pHT.5) J 1500gxl5min ,[crude nuclear pellet 5 voI. 56%sucrose (w/w)
ii~
20,O00rpmx2hrs
' ~""1 = l"I mixed
.....
f
TZ-28 zonal rotor (Sorvall R)
(44/36.5%interface)
tight homogenization
1 =ram - [ ~ 31%I 40,O00rpm x 3 hrs = I SW 41 rotor I
/
FIG. 1. Flow diagram for preparation of basolateral (blLPM) and canalicular (cLPM) plasma membrane vesicles from rat liver.
tight homogenization of mixed LPM, basolateral and canalicular vesicles are separated by high-speed centrifugation through discontinuous sucrose gradients. Animals. Male Sprague-Dawley rats (200-250 g) are housed in a constant temperature-humidity environment with alternating 12-hr fight and dark cycles for at least 4 days prior to use. During this time all animals are allowed access to food and water ad libitum. On the day of the experiment, the rats are killed by decapitation between 7:30 and 8:30 AM. Solutions. NaHCO3 (1 mM), pH 7.4; 0.25 M sucrose (8.1%, w/w); 31% sucrose (w/w; d = 1.1318); 34% sucrose (w/w; d = 1.1463); 36.5% sucrose (w/w; d = 1.1587); 38% sucrose (w/w; d = 1.1663); 44% sucrose (w/w; d = 1.1972); 56% sucrose (w/w; d = 1.2623). All solutions are prepared 24 to 48 hr before use and stored at 4 °. Densities of the sucrose solutions are adjusted at room temperature by refractometry (Abbe-3L refractometer;, Bausch and Lomb, Rochester, NY). Step 1: Isolation of Mixed LPM. Routinely, 10-12 fed animals are killed and their fives (110-115 g) rapidly removed and chilled on ice. Ten-gram portions of fiver are cut into small pieces, washed three times in 80 ml cold 1 m M NaHCO3 with a loose (type A) Dounce homogenizer (seven up-and-down strokes). The homogenate is further diluted to 1800-
[34]
PREPARATIONOF blLPM AND cLPM
537
2000 ml with cold NaHCO3 and filtered twice through two layers of 60-grade cheesecloth. Centrifugation at 1500 g for 15 min (GSA rotor, E. I. Du Pont de Nemours and Co., Inc., Sorvall Instruments Division, Newton, CT) gives a "crude nuclear pellet" that is resuspended in 5.5 vol of 56% sucrose and stirred for 15 min to disrupt membrane aggregates (Fig. 1). The sample is then loaded onto a 100-ml cushion of 56% sucrose with a variable speed pump into the zonal rotor TZ-28 (Sorvall Instruments) and overlayed with 400 ml of 44% sucrose and 200 ml of 36.5% sucrose, respectively. Finally, the rotor is filled to its total volume capacity (1350 ml) with 0.25 M (8.1%) sucrose. During the entire loading procedure the rotor is run at 3000 rpm. The completed discontinuous sucrose gradient system is centrifuged at 20,000 rpm for 120 min (Fig. 1). After slow deceleration to a complete stop, 70 15-ml fractions are collected from the bottom of the rotor and routinely analyzed for turbidity (absorbance at 700 nm). The bulk of plasma membrane fragments is normally contained in fractions of the third turbidity peak (44/36.5% sucrose interface). 19 These fractions are combined and diluted with NaHCO 3 to 1000 ml. The suspension is then centrifuged at 7500 g for 15 min. The resulting pellet is gently resuspended (by vortex mixing) in 250 ml NaHCO 3, and the material representing the mixed LPM resedimented at 2700 g for 15 min.
Step 2: Separation of Basolateral (blLPM) and Canalicular (cLPM) Subfractions. Mixed LPM are diluted with 0.25 M sucrose to a total volume of 25 ml and homogenized with a tight type B glass-glass Dounce homogenizer by 50 strokes (1 stroke equals up and down). Mixed LPM (3.5 ml) are layered on top of a three-step sucrose gradient consisting of 4 ml 38%, 2.5 ml 34%, and 2.5 ml 31% sucrose (Fig. 1). The tubes are centrifuged at 40,000 rpm (195, 700 g) for 3 hr in a Beckman SW41 rotor Beckman Instruments, Inc., Palo Alto, CA). This results in three distinct bands and a pellet (Fig. 1). The material on top of the 31% sucrose layer represents cLPM whereas the membranes at the 34/38% interface correspond to blLPM (Fig. 1). These two LPM subfractions are carefully collected with a plastic Pasteur pipet, diluted in 0.25 M sucrose, and sedimented at 105,000 g for 60 min. The pellets are resuspended in the appropriate buffer medium by repeated (20 times in and out) suctioning through a 25-gauge needle. Alternatively, vesiculation of the membrane fragments can be performed by 30 strokes in a tight-fitting glass-glass or Teflon-glass homogenizer. The vesiculated membranes are then stored in liquid nitrogen for up to 4 weeks without loss of transport functions.
Properties of Isolated blLPM and cLPM Subfractions Morphology. Transmission electron microscopy revealed that both LPM subfractions are composed of membrane vesicles, although blLPM
538
GASTROINTESTINAL SYSTEM
[34]
still contained some unbroken lateral membrane sheets (Fig. 2a). In addition, mitochondrial membrane fragments were occasionally found in the blLPM subfraction, a finding consistent with the slight enrichment over homogenate of succinate cytochrome c reductase activity (Table I). Correspondingly, intravesicular volumes are approximately twofold lower in blLPM ( - 1 / d mg-l protein) than in cLPM (~2/~1 mg--' protein) vesicles as estimated from equilibrium uptakes o f ['4C]glucose. 2° Freeze-fracture analysis indicated that 72% of blLPM 2' and 77% of cLPM vesicles2° are oriented fight side out, i.e., the outer vesicle surface corresponds to the extracellular membrane area in vivo. However, since the basolateral subfraction is regularly contaminated with canalicular vesicles to approximately 10%'9 the true fight-side-out orientation of basolateral vesicles might range between 60 and 65%. Recoveries. Following the above-outlined procedure it is possible to simultaneously isolate approximately 0.19 mg blLPM and 0.10 nag cLPM protein/g liver from the same homogenate (Table I). Since the capacity of the method extends to a total of 115 g liver (wet wt), the maximum yields of blLPM and cLPM protein range around 22 and 12 rag, respectively. These are the highest recoveries of simultaneously isolated basolateral and canalicular LPM reported so far. Degree of Purification. Table I summarizes the protein recovery and enzymatic characteristics of the isolated LPM subfractions. Compared to the homogenate blLPM are slightly enriched with mitochondria, but threeto fivefold deenriched with respect to microsomes, lysosomes and Golgi complex. In contrast, cLPM are virtually free of mitochondrial fragments, but contain slightly more microsomal, lysosomal, and Golgi membranes than the blLPM subfraction. Since endoplasmic reticulum represents 24%, Golgi complex 1%, lysosomes 2%, and mitochondria 16% of total homogehate protein,22 it can be calculated that the total contamination of blLPM and cLPM with these intracellular organelles accounts for approximately 38% ( 2 4 X 0 . 3 + l × 0 . 2 + 2 X 0 . 3 + 1 6 X 1 . 9 ) and 23% ( 2 4 X 0 . 7 + 1 X 0.4 + 2 X 1.1 + 16 X 0.2) of total protein, respectively. In regard to domain-specific plasma membrane markers the blLPM subfraction is 24- to 45-fold enriched over homogenate in Na+,K+-ATPase activity, contains this enzyme's a-subunit, 23 and localizes glucagon-stimu2o p. j. Mier, A. St. Meier-Abt, C. Barrett, and J. L. Boyer, J. Biol. Chem. 259, 10614 (1984). 21 R. H. Moseley, P. J. Meier, P. S. Aronson, and J. L. Boyer, Am. J. Physiol. 250 G35 (1986). 22 D. M. Neville, in "Biochemical Analyis of Membranes" (A. H. Maddy, ed.), p. 27. Chapman & Hall, London, 1976. 23 E. S. Sztul, D. Biemesderfer M. J. Caplan, M. Kashgadan, and J. L. Boyer, J. Cell Biol. 104, 1239 (1987).
[34]
PREPARATIONOF blLPM AND cLPM
539
FIo. 2. Electron micrographs of(A) isolated basolateral (blLPM) and canaficular (cLPM) rat liver plasma membrane subfractions. (Bars, 1/~m; X 14,000)
latable adenylate cyclase activity (Table I). In contrast, these marker enzymes could not be detected in highly purified cLPM that are additionally characterized by 50- to 90-fold enrichments of classical canalicular marker enzyme activities such as alkaline phosphatase, aminopeptidase (leucylnaphthylamidase), 7-glutamyltranspeptidase, and 5'-nucleotidase (Table I)fl 9 These results indicate that the isolated cLPM are virtually devoid of basolateral (sinusoidal) contaminants. In contrast, based on the fourfold enrichment of the canalicular specific marker enzyme aminopeptidase (leucylnaphthylamidase), the blLPM subfraction is contaminated with canalicular membranes to approximately 10%. Comments
Suitability of Marker Enzyme Activities for Assessment of the Separation between blLPM and cLMP. In order to correctly delineate the functional polarity of the hepatocellular surface domains with respect to ion transport processes, it is necessary to routinely test the purification of isolated blLPM and cLPM vesicles. For this purpose marker enzymes must be used that have unequivocally been demonstrated to be selectively localized at either the canalicular or the basolateral pole of hepatocytes. Aminopeptidase (leucylnaphthylamidase) has been shown to be specific for the canalicular membrane by both enzyme activity measurements as well as
540
GASTROINTESTINAL SYSTEM
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TABLE I PROTEIN RECOVERY A N D ENZYMATIC CHARACTERIZATION OF BASOLATERAL (blLPM) A N D CANALICULAR (cLPM) SUBFRACTIONSa
blLPM Protein recovery (mg/g fiver) (193) Marker enzymes for intracellular organellesb Succinate cytochrome c reductase (mitoehondria) (55) NADPH cytochrome c reductase (microsomes) (32) Acid phospfiatase (lysosomes) (8) UDPgalactosyltransferase ((3o1#) (8) Plasma membrane enzyme markers~ Na+,K+-ATPase activity (53) Alkaline phosphatase (12) Leucylnaphthylamidase (33) 7-Glutamyltranspeptidase(7) 5'-Nucleotidase (5) Glucagon-stimulatable adenylate cyclase (3)* a Subunit of Na+,K+-ATPasd
cLPM
0.19±0.06 0.10±0.03 Relative specific activities" 1.9±0.9 0.2±0.2 0.3±0.2 0.2±0.3
0.2±0.4 0.7±0.4 1.1±0.3 0.4±0.4
34 ± 11 12 ± 4 4_ 3 15 ± 5 11 ± 5 Present Present
Not detectabled 71 + 21 42+ 9 6 0 + 12 64+ 5 Not detectable Not detectable
"Results are given as the means + SD with the number of experiments in parentheses.
b Marker enzyme activities were determined according to the following procedures: Succinate and NADPH eytoehrome c reductuses [G. L. Sottocasa, B. Kuylenstiema, L. Ernster, and A. Bergstrand, J. Cell Biol. 32, 415 (1967)], acid phosphatase [T. L. Rothstein and J. J. Blum, J. Cell Biol. 57, 630 (1973)], UDPgalactosylWansferase [B. Fleischer and M. Smigel, J. Biol. Chem. 253, 1632 (1978)], Na+,K+-ATPase [B. F. Scharschmidt, E. B. Keeffe, N. M. Blankenship, and R. IC Oeimer, J. Lab. Clin. Med. 93, 790 (1979)], alkaline phosphatase [E. B. Keeffe, B. F. Seharsehmidt, N. M. Blankenship, and R. K. Oekner, J. Clin. Invest. 64, 1590 (1979)], leueylnaphthylamidase [J. A. Goldbarg and A. M. Rutenburg, Cancer (Philadelphia) 2, 283 (1958)], p-glutamyltranspeptidase [M. Orlowski and A. Meister, Biochim. Biophys. Acta 73, 676 (1963)], 5'-nucleotidase [J. Avruch and D. F. H. Wallaeh, Biochim. Biophys. Acta 233, 334 (1971)]. c Relative specific activity -- specific activity ratios, LPM subfraetions/homogenate. a Absence of any detectable Na+,K+-ATPase activity in cLPM has been achieved in 2 out of 3 in a total of over 500 membrane preparations. e A. F. Stewart, K. L. Insogna, D. Goltzman, and A. E. Broadus, Proc. Natl. Acad. Sci. U.S.A. 80, 1454 (1983). f Determined by immunoblotting using monoclonal antibodies [E. Sztul, M. Caplan, D. Biemesdeffer, L. Barrett, M. Kashgafian, and J. L. Boyer, £ Cell Biol. 104, 1239 (1987).
immunolocalization studies, u Therefore, this enzyme is well suited for estimating the contamination of blLPM with cLPM. In contrast, according to histochemical and various subeellular fractionation studies, the Na+,K+-ATPase is selectively localized at the basolateral mem24 L. M. Roman and A. L. Hubbard, J. CellBiol. 98, 1488 (1984).
[34]
PREPARATIONOF blLPM AND cLPM
541
brane? 5,17,~9,25-27 In addition, recent monoclonal antibody studies have also found the ot subunit of Na+,K+-ATPase to be exclusively localized at the basolateral membrane domain. 23Although two other immunolocalization studies found the t~ subunit at the eanalicular membrane as well,2s,29 further purification of the antibody preparation resulted in a complete loss of canalicular immunoreactivity in one study.2sa9- Since in isolated plasma membrane subfractions enzyme activity as well as the c~ subunit of Na+,K+,ATPase are associated with blLPM, but not with cLPM,23 the absence of any detectable Na+,K+-ATPase activity is an adequate criterion to exclude significant basolateral contamination of c L P M . 29b Thus, measurements of aminopeptidase and Na+,K+-ATPase activities are useful tests for routine screening of the degree of cross-contamination of liver plasma membrane subfractions. Reproducibility of the Isolation Method. Based on the determination of leucine aminopeptidase and Na+,K+-ATPase activities, blLPM with approximately 10% canalicular contamination and cLPM devoid of measurable Na+,K+-ATPase activity can be prepared in two out of three experiments. Successful separation appears to most critically depend on (1) the use of fed animals, (2) rat weights of 200 g, and (3) tight homogenization of the mixed LPM. The latter step is especially important since insufficient dissociation of canalicular membranes from lateral membrane sheets considerably decreases the final yield of cLPM. Although we recommend 50 strokes (I stroke equals up and down) with a tight type B glass-glass Dounce homogenizer, alternative methods (e.g., sonication, motor-driven Teflon-glass homogenizer) can also be used. It should be realized, however, that too-harsh homogenization conditions can damage the membrane integrity which can best be controlled by determining the recovery of marker enzyme activities in the isolated LPM subfractions. Practicability of the Procedure. Although the described procedure involves zonal centrifugation, the handling of the TZ-28 reorienting density gradient zonal rotor (Sorvall) is considerably easier than the use of classical high-speed zonal rotors. For example, the TZ-28 rotor does not require an 2s B. L. Blitzer and J. L. Boyer, J. Clin. Invest. 62, 1101 (1978). 26 p. S. Latham and M. Kashgarian, Gastroenterology 76, 988 (1979). 27M. Inoue, R. Kinne, T. Tran. L. Biempica, and I. M. Arias, J. Biol. Chem. 258, 5183 (1983). 28 S. Takemura, IC Omori, K. Tanaka, IC Omori, S. Matsuura, and Y. Taslfiro, J. Ceil Biol. 99, 1502 (1984). 29D. B. Schenk and H. L Leffert, Proc. Natl. Acad. Sci. U.S.A. 80, 5281 (1983). 29,y. Fashiro, K. Omori, A. Yamamoto, J. Histochem. Cytochem. 36, 221 (1988). 29bM. Sellinger, C. Barrett, Ph. Malle, E. R. Gordon, and J. L. Boyer, Hepatology (Baltimore) 11, 223 (1990).
542
GASTROINTESTINAL SYSTEM
[34]
ultracentrifuge and can be unloaded statically (nonturning position). The total centrifugation time of the procedure is 6½ hr permitting high-yield isolation of both LPM subfractions in 1 day (total preparation time - 11 hr). Furthermore, the procedure can easily be scaled down to one rat liver (-10 g net wt). For this purpose the initial crude nuclear pellet is resuspended in 2.2 vol of 70% (w/w) sucrose. After stirring for 15 min, 12 ml of the suspension is filled into SW 28 rotor (Beckman Instruments, Inc., Palo Alto, CA) tubes and ovedayered with 10 ml 44% and 10 ml 36.5% (w/w) sucrose. The tubes are filled to the top with 0.25 M sucrose and the gradient system centrifuged at 26,000 rpm for 90 min. Mixed LPM are recovered from the 44/36.5% sucrose interface and subjected to the same washing, tight homogenization, and high-density sucrose centrifugation steps are described above. By this means approximately 1 mg cLPM and 3 mg blLPM protein can be isolated from one liver with virtually the same degree of purification as demonstrated for the high-capacity zonal centfifugation method (Table I).
Suitability of Isolated blLPM and cLPM Vesicles for in Vitro Transport Studies. Table II summarizes several receptors and transport systems identified and partially characterized in the two isolated LPM subfractions. The findings demonstrate that both vesicle preparations are well suited for investigating hepatocellular membrane transport functions. Furthermore, the results illustrate the polar nature of hepatocyte plasma membrane transport processes. This supports the concept that the mechanisms and driving forces involved in overall transport of solutes from blood into bile are similar to vectorial transport mechanisms in other epithelial cells. Comparison with Other Isolation Procedures In addition to the described technique, other methods have recently been developed for isolation of highly purified basolateral and canalicular LPM vesicles. Although we have no direct experience with these alternative procedures, a short description of the various principles and techniques involved might help in choosing the most adequate method for a given purpose. Procedures of Inoue and co-workers27,3°describe the isolation of sinusoidal or canalicular membrane vesicles. Sinusoidal vesicles are prepared from a postnuclear supernatant by sucrose-Ficoll density gradient centrifugation. Seventy percent of the vesicles are oriented right side out and 3oM. Inoue, R. Kinne, T. Tran, and I. M. Arias, Hepatology (Baltimore) 2, 572 (1982).
[34]
PREPARATIONOF b l L P M A N D c L P M TABLE II RECEPTORS AND TRANSPORT SYSTEMS IDENTIFIED IN
BASOLATERAL(blLPM) AND CANALICULAR (cLPM) VESICLESa
Intact IgA2 receptor b ("secretory component") Bile acid-binding polypeptides~ ATP-dependent Ca 2+ uptake d Na +/alanine cotransport 2° Na+/taurocholate cotransport 2o Na+,K+.dependent glutamate uptake ~ Na+/H + antiport 21 CI-/HCO3- exchangef OH-/SO4 2- exchanges HCO3-/SO4 2- exchangeh
blLPM
cLPM
+
--
M r 54,000
M r 100,000
Mr 48,000 +
--
+ +
+ -
-
+
+ +
+ +
-
a +, Present; - , not detectable. b Determined by immunoblotting using a monospecilic antibody.~9 c Identified by photoaifinity labeling of blLPM and cLPM with the sodium salt of the photolabile derivative (7,7-azo-3,12-dihydroxy-5-[3H]cholan-24oyl)-2-aminoethanesulfonic acid [W. Kramer, U. Bickel, H. P. Buscher, W. Gerok, and G. Kurz, Eur. J. Biochem. 129, 13 (1982); St. Ruelz, G. Fricker, G. Hugentobler, K. Winterhalter, G. Kurz, and P. J. Meier, J. Biol. Chem. 262, 11324 (1987)]. d C. Evers, G. Hugentobler, R. Lester, P. Gmaj, P. J. Meier, and H. Murer, Biochim. Biophys. Acta 939, 542 (1988). e N. Ballatori, R. H. Moseley, and J. L. Boyer, J. Biol. Chem. 261, 6216 (1986). f P. J. Meier, R. Knickelbein, R. H. Moseley, J. W. Dobbins, and J. L. Boyer, J. Clin. Invest. 75, 1256 (1985). s G. Hugentobler and P. J. Meier, Am. J. Physiol. 251, 6656 (1986). h p. j. Meier, J. Valantinas, G. Hugentobler, and I. Rahm, Am. J. Physiol. 253, 6461 (1987).
543
544
GASTROINTESTINAL SYSTEM
[34]
exhibit an internal volume of 1 gl mg-~ protein. The recovery is 0.3 mg protein/g liver with a maximum yield of 7.5 mg protein. Na+,K+-ATPase activity is 20-fold enriched over homogenate. Total contamination with intracellular organelles approximates 34%. The preparation time is less than 5 hr. Canalicular vesicles are isolated from the crude nuclear pellet using nitrogen cavitation and Ca2+ precipitation techniques. Virtually 100% of the vesicles are reported'to be oriented right side out (intravesicular volume I gl mg-t protein). Recovery and maximal yields are 0.12 mg/g liver and 3.0 mg, respectively. Canalicular marker enzyme activities are 50to 60-fold enriched over homogenate, whereas the relative specific activity for Na+,K+-ATPase is low (2.5 ×). Contamination with intracellular organelles is around 15%. Isolation time is 4 to 5 hr. Comments. Since sinusoidal vesicles are prepared from a postnuclear superuatant while canalicular vesicles are isolated from the crude nuclear pellet, this method should theoretically also permit isolation of both LPM subfractions from the same homogenate. However, comparative studies from such simultaneously isolated sinusoidal and canalicular vesicles have not been reported so far and, therefore, the practicability and total preparation time required for isolation of both LPM subfractions are not known (rough estimation: 7 to l0 hr). Furthermore, the contamination of the LPM subfractions with Golgi-derived membranes has not been assessed. Nevertheless, these vesicle preparations have been successfully used to assess the polar distribution of a variety of plasma membrane functions, including insulin and asialoglycoprotein receptors (sinusoidal),3~ Na+/H + antiport (sinusoidal),32 and transport of taurocholate3°,33 and glutathione~-37 and the multidrug resistance cation pump (Gp 170; canaficular). 37a The method of Bfitzer and Donovan3s involves Percoll gradient centrifugation and permits the preparation of 0.64 mg/g fiver basolateral vesicle protein within 4 hr. The maximal yield is between 12 and 16 rag. Seventy3~ I. M. Arias, in "Progress in Liver Diseases" (H. Popoer and F. Schaffner, eds.), Vol. 8, p.
145. Grune & Stratton, Orlando, Florida, 1986. 32 I. M. Arias and M. Forgac, £. Biol. Chem. 259, 5406 (1984). 33 M. Inoue, R. Kinne, T. Tran, and I. M. Arias, J. Clin. Invest. 73, 659 (1984). M. Inou¢, R. Kinne, T. Tran, and I. M. Arias, Eur. £. Biochem. 138, 491 (1984). 35 M. Inoue, R. Kinne, T. Tran, and I. M. Arias, Eur. J. Biochem. 134, 467 (1983). 36 T. Akerboom, M. Inoue, H. Sies, R. Kinne, and I. M. Arias, Fur. £. Biochem. 141, 211 (1984). 37 M. Inoue, T. M. Akerboom, H. Sies, R. Kinne, T. Tram, and I. M. Arias, J. Biol. Chem. 259, 4998 (1984). aTay. Kamimoto, Z. Gaitmaitan, J. Hsu, and I. M. Arias, J. Biol. Chem. 264, 11693 (1989). 3s B. L. Blitzer and C. B. Donovan, J. Biol. Chem. 259, 9295 (1984).
[34]
PREPARATIONOF blLPM AND eLPM
545
five percent of the vesicles are oriented right side out. Na+,K+-ATPase activity is 28-fold enriched over homogenate. Total contamination with intracellular organdies approximates 46%. Comments. This method is fast and does not require ultracentrifugation. However, it requires removal of the Percoll before transport studies can be performed and results in an approximately 30% contamination of basolateral vesicles with endoplasmic reticulum. The vesicles have so far mainly been used for characterization of the basolateral bile acid uptake systems3s-4° as well as the ATP-dependent Ca + pumps.4~ The highest purification of canalicular membrane vesicles (153-fold enrichment of aminopeptidase) has been reported by Hubbard et al., 8,~8 whose isolation procedure involves immunoadsorption on anti-aminopeptidase antibody-coated Staphylococcus aureus cells. Although the procedure has not been used on a preparative scale (protein yield --~45/zg), it could theoretically be scaled up 10- to 100-fold provided high enough amounts of antibodies are available. Acknowledgment Theses studies were supported by the Swiss National Science Foundation (Grants 3.983.0.84 and 3.992.0.87) and United States Public Health Service Grants DK-25636 and DK-36854.
39B. L. Blitzer and L. Lyons, Am. J. Physiol. 249 G34 (1985). 4oB. L. Blitzr, Ch. Terzakis, and K. A. Scott, J. Biol. Chem. 261, 12042 (1986). 4] B. L. Bitzer, B. R. Hosteler, and K. A. Scott, J. Clin. Invest. 83, 1319 (1989).
