Structural features of absorptive cell and microvillus membrane preparations from rat small intestine

GASTROENTEROLOGY 1986;91:1401-14

Structural Features of Absorptive Cell and Microvillus Membrane Preparations From Rat Small Intestine DAVID J. BJORKMAN, CAROL H. ALLAN, SUSAN J. HAGEN, and JERRY S. TRIER Departments of Medicine, Harvard Digestive Disease

Brigham and Women’s Hospital Center, Boston. Massachusetts

Absorptive cells of the small intestine are highly polarized cells with distinct microvillus membrane [MVM) and basolateral plasma membrane domains. We compared membrane structure in the following preparations of rat small intestine commonly used for in vitro study ofMVMfunction: epithelial sheets, isolated epithelial cells, and four different MVM vesicle preparations, using electron microscopy of thin sections and freeze fracture replicas. We also quantitated mean vesicle diameter of the four MVM preparations by quasielastic light scattering and determined their actin content. EpitheJiaJ sheets maintained their plasma membrane polarity as judged by intramembrane particle (IMP) distribution for at least 30 min after isolation. In contrast, the plasma membrane of isolated cells showed redistribution of IMPS, indicating considerable loss of polarity in the few minutes required for cell recovery. The P-face 1MPs in MVM prepared by Ca + + precipitation were randomly distributed but became aggregated after exposure to potassium thiocyanate, which removed approximately 50% of core actin. The P-face JMPs in Mg+ + precipitated MVM were aggregated whether or not core actin was depleted Received December 26, 1985. Accepted May 13, 1986. Address requests for reprints to: Jerry S. Trier, M.D., Gastroenterology Division, Brigham and Women’s Hospital. 75 Francis Street, Boston, Massachusetts 02115. This work was supported by research grants AM36835 and AM34854 from the National Institutes of Health. Dr. Bjorkman was supported in part by National Research Service Institutional Training Program AM07533 and National Research Service Award AM07349. The authors thank Dr. Joan E. Staggers and Dr. Martin C. Carey for their help in performing studies using quasielastic light scattering. This work was presented in part at the Annual Meeting of the American Gastroenterological Association, New York, May 1985 (Gastroenterology 1985;88:1328). 0 1986 by the American Gastroenterological Association 0016/5085/86/$3.50

and Harvard

Medical

School:

and the

with potassium thiocyanate. The shape and size of MVM vesicJes difered considerably with different preparative techniques. The extremely rapid loss of plasma membrane polarity of isolated intestinal epithelial cells and the striking structural heterogeneity of MVM vesicles prepared by commonly used techniques should be considered in the interpretation of functional studies with these preparations. Absorptive cells of the small intestine are highly polarized epithelial cells with two structurally and functionally distinct plasma membrane domains, the apical or microvillus membrane (MVM) and basolatera1 membrane (BLM) (1). A number of cell and membrane preparations have been used to study functional and compositional aspects of intestinal epithelial plasma membranes including isolated epithelial sheets (z), isolated epithelial cells (3-6), and isolated MVM vesicles (7-10). Microvillus membrane vesicles have proven particularly useful for studying the composition of the apical plasma membrane of absorptive cells and its (11). Over the years a interaction with nutrients variety of techniques has been employed to prepare isolated MVM vesicles, including cell disruption followed by density gradient centrifugation (7,11), free flow electrophoresis (12)) immunoabsorbent chromatography (13), and precipitation of contaminating cell membranes other than MVM with the divalent cations Caf + (8,9) or Mg+ + (10). The Caf + and Mg+ + “precipitation” methods are now most widely used, although recently it has been shown that subsequent treatment of isolated MVM with potassium thiocyanate (KSCN) appears to solubilize Abbreviations used in this paper: BLM, basolateral membrane; IMP, intramembrane particle; KSCN, potassium thiocyanate; MVM, microvillus membrane; QLS, quasielastic light scattering; SDS, sodium dodecyl sulfate.

GASTROENTEROLOGYVol. 91.No. 6

1402 BJORKMAN ET AL.

residual cytoskeletal core material and results in substantial further enrichment of MVM markers such as sucrase, alkaline phosphatase, and the Na + , D-glucose cotransporter (14,15). Thin sections of MVM preparations have often been examined by transmission electron microscopy to assess purity, and selected MVM preparations have been examined with negative staining (16) and freeze fracture (17,18) techniques. However, detailed comparisons of the structure of MVM prepared by the most widely used isolation methods are not available. Similarly, rigorous studies of the structure of the plasma membrane in isolated epithelial sheet and cell preparations are very limited (19). There is evidence that the chemical composition of MVMs prepared from intestine by the Mg+ + and Ca++ precipitation methods differ (10,ll). Moreover, differences in ion permeability have been noted in MVMs prepared from rabbit intestine (20) and rat kidney cortex (21) by the Mg+ + and Ca+ + precipitation methods. Since alterations in membrane structure, which might occur during the isolation of epithelial sheets and cells and MVM vesicles, may reflect alterations in membrane chemical composition and influence the results of functional studies that use such preparations, definition of their structural features is important. In this study we provide an analysis of the structure of specific plasma membrane domains in isolated epithelial sheets and cells from the small intestine and compare the structural features of several of the more commonly used absorptive cell MVM preparations. For this we used conventional transmission electron microscopy of thin sections to assess overall structure, and freeze fracture replicas to examine intramembrane particle (IMP) distribution in the plasma membrane. Additionally, for MVMs, we used quasielastic light scattering (QLS) to assess vesicle size and biochemical methods to estimate actin content as an index of contamination of membranes with residual cytoskeletal components of the microvillus core.

Materials and Methods Animals For all experiments, male Sprague-Dawley rats weighing 250-300 g (Charles River Breeding Laboratories, North Wilmington, Mass.) were fasted overnight and anesthetized with ether.

Isolated

Epithelial

solution at pH 7.4 and 37°C. The inferior vena cava was cut and the rat was perfused via the left ventricle with oxygenated calcium-magnesium-free Hank’s solution containing 30 mM EDTA, pH 7.4, at 37°C at a flow rate of 55-60 ml/min for 2.5-3 min. The proximal jejunum was everted on a 4 mm diameter glass rod and vibrated with a Buchler stirrer with several 0.5 s bursts at high amplitude into oxygenated calcium-magnesium-free Hank’s solution at 4°C until epithelial sheets sloughed off. The sheets were rinsed with oxygenated Hank’s solution (1.3 mM CaC12, 0.4 mM MgC12, and 0.4 mM MgS04), pH 7.4, at 4’C, and aliquots were then processed as outlined below for thin section and freeze fracture electron microscopic studies. Additional aliquots were suspended in oxygenated Fluosol (FC-43, Alpha Therapeutics, Los Angeles, Calif.) with 2 mM adenosine diphosphate (Sigma Chemical Co., St. Louis, MO.), pH 7.4, capped, and processed for electron microscopy after 30 min of incubation at 37°C.

Isolated

Epithelial

Cells

Isolated epithelial cells were prepared by minor modifications of the method of Weiser (4). All solutions were warmed to 37°C and oxygenated with 100% OZ. The small bowel was severed 70 cm distal to the pylorus and flushed with 50 ml of rinse solution (0.154M NaCl, 1 mM

dithiothreitol, pH 7.3), quickly excised, filled with solution A (1.5 mM KCl, 96 mM NaCl, 27 mM Na-citrate, 8 mM KH2P04, pH 7.3), and incubated for 15 min at 37°C in a bath containing continuously oxygenated solution A. Solution A was then drained, the bowel was filled with solution B (1.5 mM EDTA, 0.5 mM dithiothreitol in phosphate buffered saline, pH 7.3), and incubated in a bath of continuously oxygenated solution B at 3 7°C for 4 min. The intraluminal solution B was discarded, and the bowel was again filled with solution B and incubated for an additional 6 min. The cell suspension in solution B was collected in a syringe, passed through 20 pm nitex mesh (Tetko, Inc., Elmsford, N.Y.), centrifuged at 900 g for 3 min, washed with phosphate buffered saline, and centrifuged again. The pellet was resuspended in phosphate buffered saline, and passed through a 21 gauge needle. As with epithelial sheets, some aliquots were fixed for electron microscopy; others were incubated in oxygenated Fluosol/2 mM adenosine diphosphate, pH 7.4, or oxygenated Krebs-Ringer phosphate buffer12 mM adenosine diphosphate, pH 7.4, at 37°C for 30 min before fixation. Just before fixation one drop of cell suspension from each aliquot was mixed with one drop of 0.5% trypan blue in phosphate buffered saline on a glass slide and examined at a magnification of 450x with a light microscope. At least 100 cells were counted to determine the percentage of unstained cells. Representative samples from the remaining bowel were excised after each solution B exposure, fixed, and processed for light microscopy to determine the region of the villus devoid of epithelial cells.

Sheets

Epithelial sheets were prepared by ethylenediaminetetraacetic acid (EDTA) perfusion as described by Bjerknes and Cheng (2). The jejunum was flushed with 20-50 ml of oxygenated calcium-magnesium-free Hank’s

Microvillus

Membrane

Vesicles

Microvillus membrane vesicles were prepared at 4°C by four different methods. For the CaC12 precipitation technique we made minor modifications of the method

December 1986

ABSORPTIVE CELL AND MVM ISOLATES

described by Schmitz el al. (8) as modified by Kessler et al. (9). The small bowel was flushed with 50 ml of cold normal saline and rapidly removed. After additional rinsing and blotting with gauze to remove mucus and debris, the intestinal mucosa from 4 rats was removed by gentle scraping with a glass slide, collected in 120 ml of buffer 1 (50 mM mannitol, 2 mM Tris/HCl, pH 7.1) on ice, and homogenized with 10 strokes of a motorized glass/Teflon (Potter-Elvehjem) homogenizer at high speed and then in a Waring blender (Waring Products Div., Dynamics Corp. of America, New Hartford, Conn.) at maximum speed for 4 min. After an aliquot of this crude homogenate was obtained for protein and enzyme analyses, CaCl, was added to a final concentration of 10 mM and the preparation was homogenized for an additional 2 min in the blender. The mixture was placed on ice for 15 min and then spun at 12,000 g for 5 min. The supernatant was spun at 48,000 g for 20 min. The resultant pellet was suspended in 10 ml of buffer 2 (50 mM mannitol, 10 mM HEPESiTris, pH 7.5) and homogenized 10 strokes with the Potter-Elvehjem homogenizer. Aliquots of this homogenate were obtained for protein, enzyme, vesicle size, and actin analyses. The remainder was spun at 48,000 g for 20 min and the pellet was fixed for thin section or freeze fracture electron microscopic studies as outlined below. For the MgClz precipitation method of Hauser et al. (lo), the identical procedure was followed except that 5 mM ethyleneglycol-bis(P-aminoethylether)-N,N’-tetraacetic acid was added to buffers 1 and 2 and that MgClz instead of CaCl, was added to a final concentration of 10 mM. Aliquots of vesicles obtained by CaCl, or MgClz precipitation for analysis were then treated with KSCN as described by Hopfer et al. (14) and modified by Peerce and Wright (15). For this, the vesicle pellet was resuspended in 10 ml of buffer 3 (10 mM HEPES/Tris, pH 7.5) and homogenized 10 strokes with a Potter-Elvehjem homogenizer. KSCN was then added to a concentration of 0.6 M, and the mixture was homogenized an additional 10 strokes, placed on ice for 20 min, diluted 1: 10 with buffer 3, and placed on ice for an additional 20 min. After centrifugation at 6000 g for 10 min, the supernatant was spun at 38,000 g for 30 min. The resultant pellet was rehomogenized in 10 ml of buffer 2, aliquots were obtained for protein, enzyme, vesicle size, and actin analyses, and the remainder was spun at 48,000 g for 20 min and fixed for thin section or freeze fracture electron microscopic study. Light and

Electron

Microscopy

Epithelial sheets, isolated cells, and vesicle pellets were fixed in 2% glutaraldehyde in 0.1 M cacodylate, pH 7.4, for 2 h, embedded in 5% agar, and trimmed into 2 mm X 3 mm x 4 mm blocks. Agar blocks for thin sectioning were postfixed with 1% 0~0, for 1 h, stained en bloc for 20 min in 2% uranyl acetate, dehydrated with a graded series of alcohol solutions, and embedded in epoxy resin. One micrometer sections for light microscopy were cut with glass knives and stained with toluidine blue. Thin sections were cut with diamond knives and examined and photographed in a Philips 300 electron microscope.

1403

For freeze fracture electron microscopy, sections of agar blocks were cut 150 pm thick on a vibratome (Lancer, St. Louis, MO.). These sections were rinsed overnight in 0.1 M cacodylate buffer, pH 7.4, cryoprotected with 20% glycerol, frozen between gold discs in semisolid Freon 22, and stored in liquid nitrogen. Discs were fractured at -110°C and rapidly replicated with platinum and carbon without etching using a Balzers 300 apparatus (Balzers, Hudson, N.H.). Replicas were rinsed in absolute methanol overnight, cleaned in commercial bleach, and mounted on formvar coated grids for transmission electron microscopy. Quantitation

of Intramembrane

Particles

Intramembrane particle area1 density (IMPs/~m2 of membrane area) and distribution were evaluated on convex membrane faces of vesicles, that is the putative protoplasmic leaflet (P-face) of the plasma membrane, but not on concave fracture faces (the putative external or E-face membrane leaflets] with a computerized image analyzer (Videoplan, Carl Zeiss, Oberkochen, West Germany) using micrographs magnified 70,000 times. Only isolated sheets and cells in which basolateral and microvillus membrane fracture faces from the same cell could be analyzed were used. At least 12 cells from four preparations of epithelial sheets and isolated cells were studied for each time group. Vesicles from at least three preparations made by each method were analyzed. The IMP area1 density for vesicles prepared by each method was determined by quantitating IMPs//Lm’ from a minimum of 230 convex vesicle fracture faces. Other

Assay

Procedures

Vesicle aliquots obtained as outlined above were stored at -15°C. Protein was determined by the Bio-Rad protein assay method (Bio-Rad, Richmond, Calif.) because the trace amounts of KSCN remaining in rinsed vesicle preparations interfered with the method described by Lowry et al. (22). Sucrase activity was measured by the method of Messer and Dahlqvist (23) as modified by Grand et al. (24), and alkaline phosphatase activity was determined by the method of Bessey et al. (25). To estimate actin content, solubilized samples of MVM containing 25 /Ig of protein were electrophoresed in concert with actin standards (rabbit muscle actin, Sigma Chemical Co.) using 10% sodium dodecyl sulfate (SDS)polyacrylamide gel by the method of Laemmli (26) and stained with Coomassie Blue. Bands migrating with the actin standards were cut out and extracted with 25% pyridine, and their protein content was estimated according to the method of Fenner et al. (27). We expressed the estimated actin content of KSCN-treated vesicles as the percentage of the estimated actin content of CaC12 or MgCl, precipitated vesicles not treated with KSCN from the same animals. Immunoblotting was performed using the technique of Towbin et al. (28). Proteins were transferred to nitrocellulose paper (HAWP 304 FO, Millipore, Bedford, Mass.). After a brief incubation in STTAB (150 mM NaCl, 10 mM Tris-HCl pH 7.7, 3 mM NaN3, 0.1% Triton X-100, 0.1%

1404 BJORKMAN ET AL.

GASTROENTEROLOGY

Vol. 91, No. 6

bovine albumin) buffer (29), the nitrocellulose paper was treated with rabbit antiactin antibody [kindly provided by Dr. Keigi Fujiwara) at a concentration of 1 pg/ml in STTAB buffer for 2 h followed by “‘I-protein A (Amersham, Arlington Heights, Ill.) for 1 h. Autoradiograms were prepared using Kodak X-OMAT film (Eastman Kodak Company, Rochester, N.Y.). Microvillus membrane vesicle size was determined by QLS using a Spectra Physics ionized argon laser (model 164, Spectra-Physics, Mountain View, Calif.) at 514.5 nm and a Langley-Ford autocorrelator (model 1096, Langley Ford Instruments, Amherst, Mass.). Microvillus membrane samples were equilibrated at 37°C and a sample time of 1 X 10e5 s was used.

Statistical

Analysis

Statistical significance of all data was determined using a two-tailed Student’s t-test for independent samples. Data are expressed as mean 2 1 SD.

Results Isolated

Epithelial

Sheets

Using the method outlined we were able to isolate epithelial sheets up to 8 mm X 8 mm in size.

Figure 2. Representative freeze fracture replica of an isolated epithelial sheet. Fracture faces of microvillus membrane (short arrow] and lateral membrane (long arrow] are shown. The arrow in the lower right-hand corner of this and subsequent replicas indicates the direction of shadowing. Magnification x 5200.

Figure

1. Electron micrograph of a representative isolated epithelial sheet. Cell junctions are intact and cytoplasmic organelles are well preserved. Vacuoles are prominent in the basal cytoplasm and there is some expansion of the intercellular space (arrows). Magnification X 5600.

Light microscopy revealed that these sheets included both villus and crypt epithelium as noted previously (2). Electron microscopy (Figure 1) showed excellent morphologic preservation of individual epithelial cells, suggesting that they remained viable to the time of fixation. Cell junctions remained intact but the intercellular space was focally expanded. There was some cytoplasmic vacuolization, which was greatest in the basal cytoplasm (Figure 1). This has been noted before in isolated epithelial cell preparations and has been postulated to represent pinocytosis of medium (19). Incubation for 30 min in Fluosol provided sheets that were morphologically similar except for some increase in the cytoplasmic vacuolization and further expansion of the intercellular space. Freeze fracture replicas of isolated epithelial sheets (Figure 2) confirmed the presence of intact tight junctions and provided wide expanses of plasma membrane for study. As in intact small

ABSORPTIVE CELL AND MVM ISOLATES

December 1986

intestinal epithelium (l), the area1 density of P-face IMPS was much greater in absorptive cell MVM than in BLM (Figures 3 and 4). In sheets fixed immediately after collection, the mean area1 density of IMPS was 2915 + 186 IMPs/pm2 in MVMs and 1533 t 403 IMPS/pm’ in BLMs (p < 0.001). After incubation in Fluosol for 30 min the polarity of IMP distribution in membrane domains was maintained while the mean IMP area1 density increased to 3507 + 207 IMPs/pm’ for MVMs and 2170 +- 232 IMPs/pm’ for BLMs (p < 0.001 for both compared with sheets fixed immediately).

4000 “E i \ 3 & g

I

1 3000

1

% : 6

2000

3 5 F z w

1000

i!

0

1

Epithelial

Cells

Histologic sections of the bowel segment that remained after the first EDTA treatment (from which cells were discarded) revealed that the cells had been removed from the villus tips. Histology after the second EDTA treatment showed that crypt cells remained, indicating that the collected cells were from the sides of the villi. Passage of the cells through Nitex mesh effectively removed sheets and clumps of cells, providing a preparation consisting of isolated cells with only occasional cell couplets. Ninety to ninety-five percent of cells excluded trypan blue immediately after collection. These cells were usually rounded and contained many cytoplasmic vacuoles. However, cytoplasmic organelles such

Figure

3. A. Replica of the P-face of the membrane of’ two microvilli from an isolated epithelial sheet showing the characteristic high IMP density. B. Replica of basolatera1 membrane of an absorptive cell from an isolated epithelial sheet. Although the IMP density of the P-face (P) is greater than that of the E-face (E), it is substantially less than that of the P-face of the microvillus membrane shown in A. Magnification ~111,000.

1 L

MVM

Isolated

1405

BLM

0 TIME SHEETS

MVM

30

BLM

MINUTES

SHEETS

MVM

BLM

0 TIME CELLS

MVM 30

ELM

MINUTES CELLS

Figure 4. The P-face IMP area1 density (mean t SD) in microvillus membranes (MVM) and basolateral membranes (BLM) of absorptive cells of isolated sheets and isolated individual absorptive cells fixed immediately after isolation (0 time] or after incubation for 30 min in oxygenated Fluosol. Intramembrane particle area1 density differed markedly between MVMs and BLMs in isolated sheets (p < O.OOl] but not in isolated cells (p > 0.05) at both time points. Intramembrane particle density increased during Fluosol incubation when compared with immediate fixation in both MVMs and BLMs of both sheets and cells (all p < 0.001).

as mitochondria were well preserved and microvilli remained intact (Figure 5). It should be noted that 7 to a min were required for centrifugation between the actual collection of cells from the intestine and their fixation or assessment by trypan blue exclusion. Further incubation for 30 min in Fluosol increased trypan blue exclusion slightly, to 95%-9a%, whereas incubation in oxygenated Krebs-Ringer phosphate buffer with 2 mM adenosine diphosphate for 30 min resulted in a 15%--30% decrease in trypan blue exclusion. Sections of isolated cells incubated for 30 min in Fluosol demonstrated preservation of intracellular organelles and microvilli, although the extent of cytoplasmic vacuolization was greater than in cells fixed immediately after collection. Freeze fracture replicas of isolated cells revealed that the polarity of the plasma membrane is lost rapidly as judged by IMP distribution. In cells fixed rapidly after isolation, there already was redistribution of P-face IMPS (Figures 4 and 6), resulting in a mean area1 density of 1865 _t 135 IMPS/pm’ in MVM domains and 1838 -+ 375 IMPS/pm2 in the BLM domains (p > 0.05). As in epithelial sheets, incubation in Fluosol for 30 min increased the area1 density of IMPS in both the MVMs and BLMs compared with that of rapidly fixed cells (p < 0.001 from MVMs and BLMs).

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BJORKMAN ET AL.

GASTROENTEROLOGY Vol. 91, No. 6

Figure 5. Electron micrograph of a representative isolated absorptive cell. The cytoplasm is vacuolated (V) but organelles, such as the mitochondria, appear well preserved. N, nucleus. Magnification x 7200.

Microvillus

Membrane

Vesicles

Microvillus membrane vesicles prepared by the four different methods showed differences in size, shape, enzyme enrichment, P-face IMP distribution and area1 density, and actin content. Microvillus membrane vesicles precipitated with CaClz and MgClz had similar enrichment of sucrase and alkaline phosphatase over that found in homogenates of mucosal scrapings (Table 1). Sucrase enrichment was 18.0-fold for CaClz and 16.5fold for MgC12 precipitated vesicles, whereas alkaline phosTable

1. Microvillus Membrane Vesicle Enzyme Enrichment” and P-Face Intramembrane Particle Areal Density

Vesicle preparation CaCIZc CaCl,/KSCNd MgC1ze MgCl,/KSCN

Sucraseb 18.0 f 2.4 (7) 29.3 + 6.0 (6) 16.5 + 2.5 (7) 27.8 ? 2.9 (6)

Alkaline phosphatase 12.2 I? 2.6 (7) 19.1 + 2.5 (6) 11.3 + 4.0 (7) 19.7 t 1.6 (6)

IMP/km’ 2362 949 1033 710

(3) (3) (3) (4)

’ Enrichment of specific activity compared to homogenates of mucosal scrapings, mean + SD. b Numbers in parentheses indicate the number of preparations tested. ’ Prepared by a modification of the method of Kessler et al. (9). d Prepared by a modification of the method of Peerce and Wright (15). e Prepared by a modification of the method of Hauser et al. (10).

Figure 6. A. Replica of the P-face of the membrane of two microvilli from an isolated absorptive cell. The IMP density is considerably less than that seen in microvilli of isolated sheets (compare with Figure 3A). B. Replica of the P-face of the basolateral membrane of the same isolated absorptive cell. Intramembrane particle density appears comparable to that on the P-face of the microvillus membrane shown in A. Magnification x111,000.

phatase was enriched 12.2- and 11.3fold respectively (Table 1). Further treatment of CaClz and MgCIZ precipitated vesicles with KSCN increased sucrase enrichment to 29.3-fold and to 27.8-fold, and alkaline phosphatase enrichment to lQ.l-fold, and 19.7-fold, respectively (all p < 0.001 compared with vesicles prepared without KSCN). Electron microscopic evaluation of sectioned pellets of MVM prepared by the four different methods revealed dramatic differences in vesicle size, shape, and apparent cytoskeletal content. Profiles of CaCl, precipitated MVM vesicles (Figure 7A) were relatively small and more or less circular. Most contained substantial amounts of electron dense cytoskeletal material. Morphologic evidence of membrane discontinuity was unusual. Vesicles precipitated with MgClz were much larger and pleomorphic. Some vesicle profiles resembled elongated microvillus fragments with intact central actin filaments (Figure 7B). Many vesicles showed evidence of membrane discontinuity and contained less core material, suggesting that some MgClz precipitated vesicles were not sealed. Treatment of CaCl, precipitated MVMs with KSCN produced vesicles that varied in shape, appeared to contain less core cytoskeletal material, had more membrane discontinuities, and included more ellipsoid-shaped vesicles than vesicles not exposed to

December

1986

Figure

membrane vesicles prepared by CaCl, precipitation. Vesicles are generally spherical and contain abundant core cytoskeletal material. B. Microvillus membrane vesicles prepared by MgCl, precipitation. Vesicles vary dramatically in size and shape; some resemble microvillus segments with recognizable actin filament bundles (F). Apparent membrane discontinuities are common (arrows). C. Microvillus membrane vesicles prepared by CaCl, precipitation and then exposed to KSCN. Vesicle size and shape are more variable and retained cytoskeletal core material is decreased compared to the vesicles shown in A. D. Microvillus membrane vesicles prepared by MgCl, precipitation and then exposed to KSCN. There appears to be much less cytoskeletal core material than in the vesicles shown in B, which were not exposed to KSCN. Magnification X68,000.

ABSORPTIVE

CELL AND MVM ISOLATES

1407

7. A. Microvillus

KSCN (Figure 7C). Vesicles precipitated with MgClz and treated with KSCN also showed an apparent diminution of retained cytoskeletal material. They varied in size and shape, often appeared partially collapsed, and many appeared larger than CaClz precipitated vesicles treated with KSCN (Figure 7D). Occasional membrane discontinuities were observed.

Mean IMP area1 density in convex (putative Pface] profiles of freeze fracture replicas of vesicles prepared by the four methods are summarized in Table 1.Replicas of CaClz precipitated vesicles (Figure 8A) showed vesicles that varied in size but were generally spherical with a mean area1 IMP density of 2362 ? 85 IMPS/pm’. Intramembrane particles were generally distributed quite uniformly over the P-face

1408 BJORKMAN ET AL.

GASTROENTEROLOGY Vol. 91. No. 6

Figure 8. A. Replica of MVM vesicles prepared by CaCl, precipitation. On convex (presumably P-face] profiles, IMP density is relatively high and IMP distribution is quite uniform. Concave [presumably E-face] profiles have few IMPS. B. Replica of MVM vesicles prepared by MgCl, precipitation. The P-face IMPS are aggregated resulting in large IMP-free membrane domains (arrows). The P-face IMP density is relatively low. C. Replica of MVM vesicles prepared by CaCl, precipitation and then exposed to KSCN. The P-face IMPS are less abundant and more aggregated than in CaCl, precipitated vesicles not exposed to KSCN (A). D. Replica of MVM vesicles prepared by MgCl, precipitation and then exposed to KSCN. The P-face IMPS are sparse and, where present, are aggregated. Magnification ~68,990.

surface, although occasional vesicles showed aggregation of particles with focal areas devoid of IMPS. Concave (presumably E-face) fracture faces displayed few IMPS (Figure 8A). Convex fracture faces of MgClz precipitated vesicles had fewer IMPS than those of CaCl, precipitated vesicles (Figure 8B) with an average area1 density of 1033 2 47 IMPS/pm2 (p < 0.01). The distribution of IMPS differed dramatically

from that seen in CaClz precipitated vesicles and in intact microvilli (Figure 3A). There were broad expanses of IMP-free membrane adjacent to regions with densely aggregated IMPS (Figure 8B). The heterogeneity of vesicle size and shape was again evident. Vesicles precipitated with CaCl,, which were subsequently treated with KSCN, also showed a much lower P-face IMP area1 density that CaCL

ABSORPTIVE CELL AND MVM ISOLATES 1409

December 1986

Table

2. MicroviJJus Quasielastic

Vesicle preparations

Membrane Vesicle Size by Light Scattering’ Nb

CaCl,”

6

CaC1,/KSCNd MgC12” MeClqIKSCN

6

5 5

Vesicle diameter (Al 2448 2111 3420 2741

t + t ?

146 221 415 259

Sample variance (%I 26.3 15.1 46.3 29.6

L k 2 +

6.5 6.5 15.5 21.2

a Mean ? SD. b N, number of preparations tested. c Prepared by a modification of the method of Kessler et al. (9). d Prepared by a modification of the method of Peerce and Wright (15). ePrepared by a modification of the method of Hauser et al. (10).

precipitated

vesicles not exposed to KSCN (949 + 47 areas of particle aggregation and depletion similar to MgClz precipitated vesicles (Figure 8C). Vesicles precipitated with MgClz treated with KSCN, had a significantly lower P-face IMP area1 density than M&l, precipitated vesicles not treated with KSCN (710 * 51 IMPslpm2,

IMPslpm2,

p < O.Ol), with

Figure

p < 0.01). There was variation in vesicle size and shape and membrane domains with particle aggregation or depletion (Figure 80). Incubation of the

vesicle preparations at 24°C and 37°C for 40 min before fixation did not alter IMP aggregation. The size of MVMs isolated by the four preparative techniques differed when quantitated by QLS (Table 2). Vesicles precipitated with CaC12, treated with KSCN, were the smallest and most uniform in size, averaging 2111 A in diameter with a mean variance of 15.1% [Table 2). Vesicles prepared with CaC12 and without KSCN treatment were larger (2448 A, p < 0.02) with a 26.3% variance. Vesicles precipitated with Mg&, treated with KSCN, averaged 2741 pi in diameter with a 29.6% variance. The largest and least uniform in size were vesicles prepared with MgC12 and without KSCN, which were 3420 A in diameter with a variance of 46.3% (p < 0.002 compared with MgC12-precipitated, KSCN-treated vesicles). However, as mean QLS variances were ~50% in all preparations, it seems unlikely that any of the preparations consisted of two or more distinct populations of vesicles. Vesicle size was not affected by incubation for 1 h at 0°C or 24°C or by repeated thawing and refreezing prior to QLS evaluation. Evaluation of MVM proteins separated on SDSpolyacrylamide gels revealed that the protein band migrating with actin standards consistently stained less intensely in vesicles exposed to KSCN during their isolation (Figure 9). Reduction in the protein content of this band was confirmed by pyridine extraction, which consistently revealed less protein in MVMs treated with KSCN than in vesicles prepared from the same homogenates with CaCl, or MgCl, alone [Table 3). Several additional protein

9. Sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie Blue (left) and immunoblots prepared with antiactin and lZ51 protein A (right) of solubilized MVM vesicles. Lanes 1 and 1' are of CaCl, precipitated MVM vesicles, lanes 2 and 2’ of CaCl, precipitated MVM vesicles subsequently exposed to KSCN, lanes 3 and 3' of M&I, precipitated MVM vesicles, and lanes 4 and 4’ of M&I, precipitated MVM vesicles subsequently exposed to KSCN. The arrow indicates the position of the purified actin standard. Note that the intensity of the actin band is diminished considerably after exposure of MVM vesicles to KSCN. Note also that several other unidentified protein bands are reduced in the Coomassie Blue-stained gels of vesicles exposed to KSCN.

bands stained less intensely after gel electrophoresis of KSCN treated vesicles (Figure 9). However, immunoblot analysis of the gels with specific antiactin antibody showed labeling only of the band migrating with the actin standard (Figure 9). This suggests that higher molecular weight bands, which stain less intensely after KSCN treatment, are not actin polymers but are other cytoskeletal or membraneassociated proteins.

Table

3. Comparison of Protein Comigrating With Actin in MicroviJJus Membrane Vesicle Preparations Without

Preparation

and With KSCN Treatmenta

N”

Without KSCN” (%)

With KSCNd (%)

CaCl,

8

loo0

M&L

8

1001

50 +- 15.2" 52 ir 13.3'

a Protein determined by pyridine extraction after SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. b N, number of preparations tested. c Protein determination of each preparation without KSCN treatment was normalized to 100%. d Mean 2 SD of percentage found in companion preparations not treated with KSCN. ’ p < 0.001. ‘p < 0.001.

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Discussion Like other transporting epithelial cells, absorptive cells of the small intestine are highly polarized cells. This polarity is evident in the plasma membrane in which it has been shown that the MVM differs strikingly in its structure and composition from the BLM. For example, the area1 density of IMPS, which appear to represent intramembrane proteins (30,31), is substantially greater in the P-face of MVMs than in the P-face of BLMs (l), correlating with biochemical determinations that show a threefold greater protein/lipid ratio in isolated MVMs vs. enriched BLMs (32). Moreover, there is abundant evidence that many membrane-associated enzymes, receptor proteins, and transport proteins are localized to specific plasma membrane domains in absorptive cells. Disaccharidases, peptidases (33), the intrinsic factor-cobalamin receptor (34,35), and the Na+, e-glucose cotransporter (36) are localized to the MVM, whereas sodium-potassium-stimulated ATPase and adenyl cyclase (37) appear confined to the BLM. There is also evidence that tight junctions preserve epithelial membrane polarity by forming a barrier to the movement of integral membrane proteins; when tight junctions are disrupted by Ca+ + deprivation (19,38-40) or trypsinization (41), redistribution of membrane proteins occurs. Plasma membrane polarity becomes reestablished in MadinDarby canine kidney cell monolayers when tight junctions again develop in previously disbursed cells (41,42). In the present study, we demonstrated that sheets of intestinal epithelial cells in which intact tight junctions separate the MVM from the BLM domains maintain their polarity. A considerably greater IMP area1 density is evident on MVMs than on BLMs when such sheets are first isolated and is similar to that observed in absorptive cells of the small intestine of intact rats (17). This gradient between MVMs and BLMs persists in epithelial sheets incubated in oxygenated Fluosol for 30 min and morphologic preservation remains excellent. In contrast, no gradient between MVM and BLM IMP distribution was present in preparations of individual absorptive cells fixed as rapidly as possible after their isolation. Our observations suggest even more rapid redistribution of some membrane proteins than was observed by Ziomek et al. (19), whose studies employing isolated absorptive cells from mouse intestine suggested that complete redistribution of the MVM proteins, alkaline phosphatase, and leucine aminopeptidase in 50% of cells required 9-12 and 16-42 min, respectively, of in vitro incubation at 37°C. There is additional evidence that redistribution of all membrane proteins in isolated villus absorptive

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cells may not occur indiscriminately at the same rate. For example, enriched BLMs prepared from isolated absorptive cells contain substantial amounts of alkaline phosphatase but little sucrase (43,44). Moreover, some distinctive proteins and glycoproteins can be identified by SDS-polyacrylamide gel electrophoresis of isolated MVMs that are not evident by gel electrophoresis of BLMs prepared from rat isolated absorptive cells (43,45). Several factors might influence the rate at which specific membrane proteins redistribute from MVMs to BLMs and from BLMs to MVMs once tight junctions are disrupted. There is convincing evidence that at least some membrane proteins are attached either directly or by linking proteins to elements of the underlying cytoskeleton which would, in turn, influence their mobility within the membrane (46-48). It is also possible that the extent to which a given protein species is anchored in the hydrophobic core of the membrane may influence its redistribution; large, extensively glycosylated proteins such sucrase-isomaltase are generally anchored by a small hydrophobic tail, whereas proteins of smaller molecular weight such as alkaline phosphatase are more deeply embedded within the membrane (49). It has also been suggested that the hydrophilic components of membrane proteins that protrude beyond the membrane core may be subject to constraints that reduce their mobility within membranes (48). In any case, whether the composition of BLMs and MVMs prepared from isolated absorptive cells (4,43-45,50,51) represents that found in vivo must be questioned in view of the rapid redistribution of IMPS observed by us. Indeed, redistribution of proteins from plasma membrane domains normally maintained by intact tight junctions may explain the observation that brush border hydrolases are found in significant amounts in BLMs prepared from isolated absorptive cells. When possible, it would seem desirable to prepare MVMs and BLMs from fresh mucosal scrapings or isolated epithelial sheets with intact tight junctions. We cannot rule out the possibility that rapid selective reabsorption or release of membrane proteins from the microvillus domain may also contribute to the equalization of IMP area1 density observed in MVMs and BLMs prepared from isolated absorptive cells. In our studies, isolated cell preparations that were incubated in oxygenated Krebs-Ringer phosphate buffer (19) for 30 min consistently showed reduced trypan blue exclusion and increased ultrastructural evidence of cytoplasmic damage compared with freshly isolated cells. When oxygenated Fluosol replaced Krebs-Ringer phosphate buffer as the incubation medium, trypan blue exclusion and morphologic preservation were maintained, possibly reflecting enhanced oxygen delivery by Fluosol.

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However, incubation in Fluosol for 30 min resulted in a significant overall increase in IMP area1 density in both MVM and BLM domains of isolated cells and isolated sheets (Figure 4). Although the cause of the consistent increase in IMP density with Fluosol incubation is not known, internalization of IMP-poor membrane domains into the cytoplasm during pinocytosis of medium or extraction of membrane lipid components by Fluosol may have contributed to this change. Alternatively, rapid insertion of newly synthesized IMP-rich membrane into both MVM and BLM membrane domains during Fluosol incubation could account for these observations. Nutrients absorbed via the transcellular pathway from the intestinal lumen must cross the apical membrane of absorptive cells. Isolated MVM vesicles are particularly useful for examining the binding of nutrients to (34),their transport across (ll), and the chemical composition of (10,11,32) this strategically located membrane. There is no standardized, universally used technique for preparing MVMs; rather, a variety of isolation methods provide useful preparations (for review, see Reference 11). Available studies including determination of the area1 density of IMPS on concave and convex vesicle freeze fracture faces, accessibility of enzymes on the outer surface of vesicles to immunological probes, absence of stimulation of outer surface enzyme activity after detergent exposure, and absence of accessibility of the microvillus core protein, actin, to trypsin all indicate that at least 90% of vesicles are “right side out” regardless of the preparative method used (9,17,52,53). Indeed, there is no known method presently available for preparing “inside out” MVM vesicles from small intestine. On the other hand, there is evidence that vesicles prepared by the different widely used methods are not strictly comparable. Microvillus membranes prepared by different methods vary in their chemical composition (lo-12,14,15). For example, Hauser et al. (10) have shown that the Ca+ + precipitation method activates an intrinsic phospholipase A producing vesicles with high levels of lysophospholipids and free fatty acids, whereas chelating cytosolic Ca++ with ethyleneglycol-bis(~-aminoethylether)N,N’-tetraacetic acid and using Mg+ + for precipitation of unwanted contaminating membranes results in vesicles without extensive decomposition of phospholipids. Also, exposure of MVMs to high concentrations of SCN+ results in greater enrichment of MVM enzymes (sucrase and alkaline phosphatase) and the Na+, o-glucose cotransporter, presumably by removing residual cytoskeletal core proteins such as actin (l&15). Additionally, functional differences such as variation in ion permeability have been noted among MVMs prepared by

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different methods (20,21). In this report we have shown that certain structural features of MVMs isolated from rat small intestine prepared by four established methods differ dramatically from one another. Differences in vesicle size, shape, core structure, and membrane structure were demonstrated. We also confirm differences in enzyme enrichment and document differences in actin content between the preparations. Size differences among vesicle preparations were evident in transmission electron micrographs of sectioned pellets, and freeze fracture replicas and were quantitated by QLS. Sizing of artificial vesicles with QLS, sepharose gel filtration, and electron microscopy have been shown recently to provide similar results (54) validating QLS as a useful method for estimating vesicle size. The average diameter of Ca++ precipitated rat MVM determined by QLS in our studies was similar to that observed for Ca+ + precipitated rabbit MVM by flow cytometry (55). Our finding that MVMs prepared by the Mgf + precipitation method are substantially larger than MVMs prepared by Ca+ + precipitation has implications regarding the comparability of some studies of transport kinetics using these preparations. If one assumes that most vesicles in both preparations are spherical, MVMs prepared by Ca+ + precipitation 40% more membrane would present approximately surface to the medium per unit of intravesicular volume than those prepared with Mg+ + precipitation. As electron microscopy showed that Mg+ + precipitated MVMs contained a substantial number of oblong profiles, the actual difference in surface to volume ratio may be somewhat less, although still substantial, especially because oblong vesicles may become spherical during transport experiments. Although results using initial rate kinetics would be unaffected by such size differences, studies relying on equilibrium kinetics would be influenced by vesicle surface to volume ratios. The consistent reduction in vesicle size observed by us after KSCN exposure may reflect collapse of some vesicles following extraction of core cytoskeletal components. The P-face IMP distribution and area1 density in replicas of MVMs prepared with Ca+ + and without KSCN exposure resembled that seen in replicas of intact microvilli, confirming the observations of Haase et al. (17). Microvillus membranes prepared with Mg+ + with or without KSCN and Ca++ with KSCN showed P-faces with striking aggregation of IMPS and large membrane domains devoid of IMPS. The P-face IMP area1 density in these preparations with aggregated IMPS was only 30%~43% of that observed in Ca+ + prepared MVMs not exposed to KSCN. It has been demonstrated that brush border cytoskeletal organization and cytoskeletal-mem-

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brane interactions are highly sensitive to changes in Ca++ concentration. This includes the interaction of calmodulin with the 110,000molecular weight protein which may be crucial for the anchoring of the core actin bundle to the MVM (56). This may account, in part, for the decreased density and the aggregation of P-face IMPS in MVMs prepared with ethyleneglycol-bis(p-aminoethylether)-N,N’-tetraacetic acid and Mg+ + precipitation. Difficulty in resolving individual particles in IMP aggregates may also have contributed to the reduction in IMP area1 density in preparations with aggregated IMPS. Rigler et al. (18)recently reported either aggregation or absence of IMPS on the majority of membrane faces of MVM vesicles prepared from pig jejunum with Mg+ + aggregation. They concluded that Mg+ + precipitation produced clustering of membrane proteins. However, other factors must also play a role because, as we have demonstrated, IMPS of Ca++ precipitated vesicles exposed to KSCN were also consistently aggregated. It is of interest that KSCN exposed MVMs which have the lowest density of IMPS (Figure 8) show the greatest enrichment of the membrane proteins sucrase and alkaline phosphatase (Table 1). Selective loss of membrane proteins other than these markers possibly may contribute in concert with loss of cytoskeletal core proteins to this phenomenon. Whereas SDS-polyacrylamide gel electrophoresis and immunoblotting confirm the suggestion that KSCN treatment reduces the actin content of MVMs (l4), several protein bands other than actin also stained less intensely with Coomassie Blue on gels of KSCN treated vesicles than on gels of companion MVM vesicle preparations not exposed to KSCN (Figure 9). The functional implications of the differences in IMP density observed in Ca’ + vs. vesicles are difficult to assess. Mg++ precipitated Whether or not decreased IMP density represents depletion of membrane proteins in Mgf + precipitated vesicles requires further study. Whereas sucrase and alkaline phosphatase specific activity are comparable in both preparations, these enzymes represent only a small fraction of total MVM protein. Again, differences in the intensity of several protein bands are evident after SDS-polyacrylamide gel electrophoresis of these preparations (compare lanes 1 and 3 in Figure 9). Thus, there may be selective loss of certain membrane proteins but not of others when the Mg+ + precipitation technique is employed. Careful comparative functional studies will be needed to define the functional correlates of the morphological differences reported here between the various MVM preparations. Production of protein-depleted lipid microdomains in MVMs solely by phase transition (57) or high cation concentrations appear to be unlikely

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explanations for the observed aggregation of IMPS; MVMs prepared with Ca+ + and not exposed to KSCN showed little IMP aggregation, and incubation of isolated MVMs at 24 or 37% for 40 min before fixation did not result in IMP redistribution. Definition of the factors responsible for the differences in P-face IMP area1 density and IMP aggregation observed in MVMs prepared by the various isolation methods used here requires further study. In conclusion, the findings reported here demonstrate that absorptive cells in epithelial cell sheets with intact tight junctions isolated from the rat small intestine closely resemble, by morphological criteria, absorptive cells in freshly fixed intact rat intestinal mucosa. Moveover, the structural features of such sheets remain stable following in vitro incubation for at least 30 min. In contrast, absorptive cells isolated as individual units lose their polarity within minutes of isolation; hence, whether in vitro study of binding, uptake, and metabolism of nutrients with such preparations accurately reflects what occurs in vivo is questionable. Microvillus membrane vesicles isolated by four preparative methods show striking differences in their size, shape, IMP content and distribution, and residual core cytoskeletal content. Further studies designed to clarify the functional implications of these structural differences are needed.

References 1. Trier JS, Madara JL. Functional

morphology of the mucosa of the small intestine. In: Johnson LR, ed. Physiology of the gastrointestinal tract. New York: Raven, 1961:925-61. 2. Bjerknes M, Cheng H. Methods for the isolation of intact epithelium from the mouse intestine. Anat Ret 1981; 199:565-74. 3. Stern BK. Some biochemical

properties of suspensions of intestinal epithelial cells. Gastroenterology 1966;51:655-67. 4. Weiser MM. Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J Biol Chem 1973;248:2536-41. of isolated 5. Carter JH, Poretz RD, Eichholz A. Separation hamster intestinal epithelial cells by velocity sedimentation on Ficoll gradients. J Cell Physiol 1962;111:66-76. 6. Hegazy E, Lopez de1 Pino V, Schwenk M. Isolated intestinal mucosa cells of high viability from guinea pig. Eur J Cell Biol 1983;30:132-6. 7. Eichholz A, Crane RK. Studies

on the organization of the brush border in intestinal epithelial cells. I. Tris disruption of isolated hamster brush borders and density gradient separation of fractions. J Cell Biol 1965;26:667-91. a. Schmitz J, Preiser H, Maestracci D, Ghosh BK, Cerda JJ, Crane RK. Purification of the human intestinal brush border membrane. Biochim Biophys Acta 1973;323:96-112. 9. Kessler M, Acute 0, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of o-glucose and choline transport systems. Biochim Biophys Acta 1976;506:136-54.

December 1986

10. Hauser H, Howell K, Dawson RMC, Bowyer DE. Rabbit small

intestinal brush border membrane preparation and lipid composition. Biochim Biophys Acta 1980;602:567-77. 11. Semenza G, Kessler M, Hosang M, Weber J, Schmidt U. Biochemistry of the Na+, n-glucose cotransporter of the smallintestinal brush-border membrane. The state of the art in 1984. Biochim Biophys Acta 1984;779:343-79. 12. Steiger B, Murer H. Heterogeneity of brush-border-membrane vesicles from rat small intestine prepared by a precipitation method using Mg/EGTA. Eur J Biochem 1983;135:95-101. 13. Carlsen J, Christiansen K, Bro B. Purification of microvillus membrane vesicles from pig small intestine by immunoadsorbent chromatography. Biochim Biophys Acta 1982; 689:12-20. 14. Hopfer U, Crowe TD, Tandler B. Purification of brush border membrane by thiocyanate treatment. Anal Biochem 1983; 131:447-52. 15. Peerce BE, Wright EM. Conformational changes in the intestinal brush border sodium-glucose contransporter labeled with fluorescein isothiocyanate. Proc Nat1 Acad Sci USA 1984; 81:2223-6. of intestinal sucrase16. Nishi Y, Takesue Y. Localization isomaltase complex on the microvillus membrane by electron microscopy using nonlabeled antibodies. J Cell Biol 1978; 79:516-25. 17. Haase W, Schafer A, Murer H, Kinne R. Studies on the orientation of brush-border membrane vesicles. Biochem J 1978;172:57-62. 18. Rigler WW, Ferreira GC, Patton JS. Intramembranous particles are clustered on microvillus membrane vesicles. Biochim Biophys Acta 1985;816:131-41. 19. Ziomek CA, Schulman S, Edidin M. Redistribution of membrane proteins in isolated mouse intestinal epithelial cells. J Cell Biol 1980;86:849-57. 20. Mandel KG, Harms V, Stevens BR, Schell RE, Wright EM. Effect of calcium on the transport properties of intestinal brush border vesicles. In: Bronner F, Peterlik M, eds. Epithelial calcium and phosphate transport: molecular and cellular aspects. New York: Alan R. Liss, 1984;273-9. 21. Sabolic I, Burckhardt G. Effect of the preparation method on Na+H+ exchange and ion permeabilities in rat renal brushborder membranes. Biochim Biophys Acta 1984;772:140-8. 22. Lowry OH, Rosebrough JJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75. 23. Messer M, Dahlqvist A. A one-step ultramicro method for the assay of intestinal disaccharidases. Anal Biochem 1966;14: 376-92. 24. Grand RJ, Chong DA, Isselbacher KJ. Intracellular processing of disaccharidases: the effect of actinomycin D. Biochim Biophys Acta 1972;261:341-52. 25. Bessey OA, Lowry OH, Brock MJ. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J Biol Chem 1946;164:321-9. 26. Laemmli UK. Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 1970;227:680-5. 27. Fenner C, Traut RR, Mason DT, W&man-Coffelt J. Quantification of Coomassie blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal Biochem 1975; 63:595-602. 28. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 1979;76:43504. 29. Kiehart DP, Kaiser DA, Pollard TD. Monoclonal antibodies

ABSORPTIVE CELL AND MVM ISOLATES

1413

demonstrate limited structural homology between myosin isozymes from Acanthamoeba. J Cell Biol 1984;99:1002-14. 30. Edwards HH, Mueller JJ, Morrison M. Distribution of transmembrane polypeptides in freeze fracture. Science 1979;203:1343-6. 31. Verkleij AJ, Ververgaert PHJTh. The nature of the intramembraneous particle. Acta Histochem 1981;23(suppl):137-43. 32. Brasitus TA, Schachter D. Lipid dynamics and lipid-protein interactions in rat enterocyte basolateral and microvillus membranes. Biochemistry 1980;19:2763-9. 33. Crane RK. A digestive-absorptive surface as illustrated by the intestinal cell brush-border. Trans Am Microsc Sot 1975; 94:529-44. 34. Donaldson RM Jr, Mackenzie IL, Trier JS. Intrinsic factormediated attachment of vitamin Blz to brush borders and microvillus membranes of hamster intestine. J Clin Invest 1967;46:1215-28. 35. Levine JS, Allen RH, Alpers DH, Seetharam B. Immunocytochemical localization of the intrinsic factor-cobalamin receptor during cell maturation. J Cell Biol 1984;98:1111-8. 36. Hopfer U. Isolated membrane vesicles as tools for analysis for epithelial transport. Am J Physiol 1977;233:E445-9, 37. Wright EM, Mircheff AK, Hanna SD, et al. The dark side of the intestinal epithelium: the isolation and characterization of basolateral membranes. In: Binder HJ, ed. Mechanisms of intestinal secretion. New York: Alan R. Liss, 1979:117-30. 38. Hoi Sang U, Saier MH Jr, Ellisman MH. Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia. Exp Cell Res 1979;122:384-91. of surface macromole39. Pisam M, Ripoche P. Redistribution cules in dissociated epithelial cells. J Cell Biol 1976; 71:907-20. 40. Meldolesi J, Castiglioni G, Parma R, Nassivera N, De Camilli P. Ca’ +-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs. J Cell Biol 1978;79:156-72. EJ, et al. Bio41. Sabatini DD, Griepp EB, Rodriguez-Boulan genesis of epithelial cell polarity. Mod Cell Biol 1983;2: 419-50. 42. Herzliner DA, Ojabian GK. Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies. J Cell Biol 1984;98:1777-87. 43. Quaroni A, Kirsch K, Weiser MM. Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Biochem J 1979;182:203-12. 44. Van Corven EJJM, Roche C, van OS CH. Distribution of Ca*+-ATPase, ATP-dependent Ca’+-transport, calmodulin and vitamin D-dependent Ca*+-binding protein along the villus-crypt axis in rat duodenum. Biochim Biophys Acta 1985;820:274-82. 45. Quaroni A, Kirsch K, Herscovics A, Isselbacher KJ. Surfacemembrane biogenesis in rat intestinal epithelial cells at different stages of maturation. Biochem J 1980;192:13344. of mem46. Coudrier E, Reggio H, Louvard D. Characterization brane glycoproteins involved in attachment of microfilaments to the microvillar membrane. In: Porter R, Collins GM, eds. Brush border membranes, Ciba Foundation Symposium 95. London: Pittman Books, 1983:216-32. 47. Carraway KL, Carraway CAC. Plasma membrane-microfilament interaction in animals cells. BioEssays 1984;1:55-8. 48 McCloskey M, Poo M. Protein diffusion in cell membranes: some biological implications. Int Rev Cytol 1984;87:19-80. 49 Sigrist-Nelson K, Sigrist H, Bercovici T, Gitler C. Intrinsic proteins of the intestinal microvillus membrane. Iodonaphthylazide labeling studies. Biochim Biophys Acta 1977; 468:163-76.

1414

BJORKMAN ET AL.

50. Batt RM, Peters TJ. Analytical subcellular fraction studies on enterocytes from the jejunum and ileum of the rat and some properties of brush-border alkaline phosphatase. Clin Sci Mol Med 1978;55:157-65. 51. Colas B, Maroux S. Simultaneous isolation of brush border and basolateral membrane from rabbit enterocytes. Presence of brush border hydrolases in the basolateral membrane of rabbit enterocytes. Biochim Biophys Acta 1980;600:406-20. 52. Klip A, Grinstein S, Semenza G. Transmembrane disposition of the phlorizin binding protein of intestinal brush borders. FEBS Lett 1979;99:91-10% 53. Gains N, Hauser H. Detergent-induced proteolysis of rabbit intestinal brush border vesicles. Biochim Biophys Acta 1981;646:211-7.

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54. Schurtenberger P, Hauser H. Characterization of the size distribution of unilamellar vesicles by gel filtration, quasielastic light scattering and electron microscopy. Biochim Biophys Acta 1984;778:470-80. 55. Gorvel J, Mawas C, Maroux S, Mishal Z. Flow cytometry is a new method for the characterization of intestinal plasma membrane. Biochem J 1984;221:453-7. 56. Glenney JR Jr, Glenney P. Comparison of Ca++-regulated events in the intestinal brush border. J Cell Biol 1985;lOO: 754-63. 57. Armond PA, Staehelin LA. Lateral and vertical displacement of integral membrane proteins during lipid phase transition in Anacystis nidulans. Proc Nat1 Acad Sci USA 1979;76: 1901-5.