ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 198, No. 2, December, pp. 349-359, 1979
Isolation
and Characterization of Plamsa Membrane from Monolayer Cultures of Epithelial Type II Lung Cells
MUKUL K. BASU,’ GIUSEPPE COLACICCO, PAUL T. PICCIANO, M. ROSENBAUM AND MURRAY WITTNER Albert Einstein
College of Medicine 1300 Morris
Park Avenue, Bronx,
ROBERT
New York 10461
Received May 30, 1979; revised August 1, 1979 Pneumocyte type II produces a phospholipid, dipalmitoyl lecithin, which is stored in and secreted from the cell’s inclusion bodies and is indispensable for alveolar stability. Cloned rat lung type II cells were harvested at monolayer confluence and homogenized in swelling buffer. After sequential differential centrifugations, the crude membrane fraction was subjected to discontinuous sucrose density gradient centrifugation at 65,000 x g. Quality of the relevant fractions was monitored by enzyme activities and phase contrast and electron microscopy of two major bands at densities 1.16 and 1.18, respectively. The less dense band contained only small quantities of organelles, little cytochrome c oxidase, and some glucose 6-phosphatase, but had a significant (Na+, K+)-ATPase activity; this and ultrastructural evidence certified the product as a suitable plasma membrane preparation. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein pattern consisted of 11 major protein bands between 13,000 and 68,000 M, ranges, and several minor ones. The lipid pattern was studied by two-dimensional thin layer chromatograpy, followed by various group reactions (e.g., amine, unsaturation, phosphorus, sugars). In the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, palmitic acid was the least abundant of four major fatty acids, accounting for 14.20% in phosphatidyl choline and 5.70% in phosphatidyl ethanolamine, whereas the most abundant were stearic and palmitoleic with about 28% each in phosphatidyl choline, and palmitoleic (29.90%) and oleic (23.05%) in the ethanolamine phosphatide. Apparently, the palmitic acid containing phosphatidyl choline must be in the lamellar inclusion bodies of type II cells and not in their plasma membranes.
The isolation of plasma membrane from animal cells with the various criteria for identification and homogeneity has been the subject of several reviews (l-4). Preparations from in viva sources are more common, but preparations have also been made from cell cultures in vitro (5-8). Although some workers published the isolation of plasma membrane from rat lung tissue (9) there is no report concerning the isolation of plasma membrane from rat lung cell grown is tissue culture. 1 To whom correspondence should be sent at the present address: Hematology Division, St. Luke’s Hospital Center & Department of Medicine, College of Physicians and Surgeons, Columbia University, Amsterdam Avenue at 114th Street, New York, N. Y. 10025. 349
The primary objectives of the present work were (a) isolation of the plasma membrane from rat lung epithelial (type II) cells in culture, (b) identification of the membrane preparations by morphological as well as chemical methods, (c) membrane protein pattern by SDF-polyacrylamide gel electrophoresis, (d) membrane lipid pattern by two-dimensional thin layer chromatography, and (e) fatty acid compositions of these lipids with particular regard to phosphatidyl choline and phosphatidyl ethanolamine. * Abbreviations used: SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine; glc, gas-liquid chromatography; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; DOC, deoxycholate. 0003-9861/79/140349-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
350
BASU ET AL. MATERIALS
AND METHODS
Tbe rat lung cells (type II pneumocytes) obtained by the cloning method of Douglas and Kaighn (10) were grown in monolayers under 5% CO, and 95% air in petri dishes, using Dulbecco’s modified Eagle’s medium containing also 15% fetal calf serum, 100 pg/ml streptomycin, and 100units/ml penicillin. Transfer were made at 1 to 4 split. At the beginning of the seventh day when the cells had reached confluency, they were harvested by scraping with a rubber policeman, followed by lo-min centrifugation from 0.15 M NaCl at 300 g. The pellets were washed 2 x in 0.15 M NaCl and kept in ice bath. Phase contrast microscopy was used to monitor the effectiveness of cell disruption as well as of subsequent fractionation procedures. Isolation of plasma membrane. The packed cell pellets were resuspended in 6 vol of cold homogenizing medium containing 18 mM Tris (pH 8.0), 25 mM NaCl, and 0.5 mM CaCI,. All the homogenization steps were carried out in the presence of 1 mg/ml ribonuclease A (Sigma Chemical Co.) in order to remove possible ribosomal contaminants. Optimal rupturing of cells was produced by 35-40 manual strokes of a Dounce-type steel homogenizer with O.OOZ-in.clearance. After 20 up and 20 down strokes, the homogenized material was diluted with 2 vol of the same homogenizing medium and homogenized again after keeping the content at room temperature for 15 min. At this stage, 90% cells were ruptured. The contents were diluted again with 4 vol of the homogenizing medium and were subjected to differential centrifugation to obtain crude plasma membrane. The centrifugation steps were very similar to those in the method described by Forte et al. (5). The cell homogenate was centrifuged in a Sorvall refrigerated centrifuge (using SS 34 rotor) for 1 min at 200 g. The supernatant (I) was collected and the sediment was redispersed in homogenizing medium and centrifuged again for 1 min at 200 g. The supernatant (II) was collected and combined with supernatant I. The sediment consisted of nuclei and whole cells. The combined supernatant was centrifuged at 700 g for 10 min. The pellet contained plasma membranes and nuclei. The pellet was resuspended and washed twice in the homogenizing medium for preparation of final crude membrane pellet, which again was homogenized by means of 3 up and down strokes in a Dounce loose pestle homogenizer, in order to remove residual cytoplasmic components. The content was redispersed in 22 ml of I:1 mixture of resuspending medium and 60% sucrose. About 7.0 ml of this material was applied to a discontinuous density gradient, consisting of successive 6-ml layers of 60, 50, 45, and 40% sucrose (w/v), distributed equally in three 30-ml centrifuge tubes. The centrifugation was carried out in a SW 25 rotor at 25,000 rpm for 1 h. A pellet and four interfacial bands were noticed. The bands were collected by hypodermic needle, and the particulate
material therein was centrifuged in homogenizing medium for half an hour at 22,000 rpm using a SW 27 rotor and washed twice with this medium by the same centrifugation procedure. For further purification, the band containing plasma membrane (band II, 40-45% sucrose) was treated with 10 mM MgCl, (11) and redispersed in Tris buffer (pH 8.0), containing 18 mM Tris and 25 mM NaCI. The dispersion was homogenized again in a Dounce loose pestle homogenizer by 2 up and down strokes and subjected to a second discontinuous density gradient centrifugation using 8ml layers of 50, 45, and 40% sucrose (w/v). The membrane fraction was applied to the gradient in 7.0 ml of a resuspending medium (18 mM Tris and 25 mM NaCI, pH 8.0) and 60% sucrose; centrifugation, collection, and washing were done in the same manner as described above. Unless otherwise stated, all centrifugation steps were carried out at 4°C. Enzyme assays. Ouabain-sensitive (Na+, K+)ATPase was measured by the method of Jorgensen et al. (12). Since ATPase is tightly bound to the membrane, sodium deoxycholate was used to facilitate release of the enzyme. The membrane fractions and homogenates containing 150-350 pg protein were incubated in 1 ml solution containing 0.6 mg of sodium deoxycholate, 2 mM EDTA, 25 mM imidazole (pH 7.0) at 20°C. After 30 min, lOO-to 200~~1aliquots were incubated again in 1 ml medium containing 50 mM Tris-HC1 pH 7.4, 1 mM EDTA neutralized to pH 7.0, 3 mM Tris-ATP (Sigma Chemical Co.), 100 mM NaCI, 20 mM KCI, and 3 mM MgCl,. Ouabain octahydrate (Sigma Chemical Co.), 1 mM, was included. The mixtures were incubated for 10 min at 37”C, the reaction was terminated by addition of 25% trichloroacetic acid, and the reaction mixtures were left to stand for 15 min. The insoluble material was removed by centrifugation in rotor SS 34 at 2000 rpm for 20 min at 5°C. Aliquots of 1 ml supernatant were removed from each tube, and the released inorganic phosphorus was determined therein by the molybdate method of Martin and Doty as described by Lindberg and Ernster (13). Greater reproducibility was achieved when the samples were preincubated for 2 min at 37°C. The total ATPase activity (Na+, K+, Mg*+) was calculated from the absorbance at 730 nm in the absence of ouabain. The Mg’+-ATPase activity was calculated from the absorbance in the presence of ouabain. The difference between the two was taken to be the ouabain-sensitive (Na+, K+)-ATPase activity. Cytochrome c oxidase activity was measured by a method very similar to the one described by Cooperstein et al. (14). Cytochrom c (horse heart, Sigma Grade IV), 50 mg, was dissolved in 1 ml of Tris-HCl buffer (pH 7.4) containing 50 mM Tris and 154 mM NaCI. Tris-ascorbate (pH 7.4), 0.5 M, 1 ml, was added to reduce the cytochrome c. The solution was then layered on a Sephadex A-25 column prepared with the same Tris-HCI buffer, and the cytochrome c was
PLASMA MEMBRANE
OF EPITHELIAL
eluted by monitoring the band visually. A 3-ml aliquot of the reduced cytochrome c solution was pipetted into a Beckman spectrophotometer cuvette and 0.02 or 0.04 ml of membrane fraction or cell homogenate was added. The reactants were mixed by inverting the cuvette and readings were taken every 30 s at 550 nm. At the end of 3 min a few grains of potassium ferricyanide were added (to oxidize completely the cytochrome c), and the optical extinction was redetermined. Glucose 6-phosphatase was assayed by the method of Swanson (15). The substrate consisted of 11 mM glucose 6-phosphate in 55 mM Tris, with 11 mM mercaptoethanol (pH 6.6). To 0.8 ml of substrate were added O&ml aliquots of the membrane fractions or cell homogenate (0.1-0.2 mg protein). The reaction was allowed to proceed for about 15 min and was then stopped by the addition of 1 ml of trichloroacetic acid (10%). Protein was removed by centrifugation, and 1 ml ofthe cleared supernatant was used for phosphate determination (16). Chemical analyses. Protein was determined by the method of Lowry et al. (1’7). Lipids were extracted from the membrane fractions as well as from the cell homogenate, by conventional procedures (18), in which 1 ml of the cell suspension or membrane dispersion was shaken with 3.75 ml of methanol:chloroform (2:1, v/v), followed by 4.75 ml of methanokchlorofornuwater (l&5:4, v/v). Lipid phosphorus was assayed by the technique of Marinetti et al. (19). Phase contrast and electron microscow. Light microscopy observations were made with a Nikon phase contrast microscope, equipped with camera. Membrane pellets were processed for ultramicrotomy and electron microscopy by standard procedures (20). The pellets were dispersed and fixed in 2.5% glutaraldehyde which was buffered with cacodylate (pH 7. l), and postfixed in similarly buffered 1% 0~0,. The pellet membranes were dehydrated with graded ethanol and embedded in epoxy resin. The sections were cut with a Sorvall MT-2 ultramicrotome at about 600 A and then stained with many1 acetate for 15 min, followed by lead acetate for 7 min, and then examined with a high performance electron microscope (Siemens, Elmiskop 101). SDS-polyacrylamide gel electrophvresis. Electrophoresis on slab gel was carried out to establish membrane proteins pattern. A discontinuous buffer system (21) was used with some modification (22); a 5-15% gradient gel was prepared with acrylamide containing methylenebisacrylamide (Polyscience), N,N,N’,N’-tetramethylethylenediamine (TEMED, Eastman), ammonium persulfate (Fisher, analytical grade), sucrose (Sigma), and SDS (Fisher) in the standard proportions. The plasma membrane was solubilized in 2% SDS and heated at 90°C for 90 s in the presence of Pmercaptoethanol (Eastman). The protein standards for molecular weight determination
(TYPE II) LUNG
CELLS
351
consisted of ribonuclease A (13.7 K, Sigma Chemical Co.), chymotrypsin (22 K, Sigma Chemical Co.), Rabbit muscle actin (43 K, Worthington Biochemicals), bovine serum albumin (68 K, Pentex Biochemical& and phosphorylase (Y(94 K, Sigma Chemical Co.). The gel was stained with 0.25% Coomassie brilliant blue (R-250, Mann) in methanol:water:acetic acid 50~437 for 1 h at 37°C and destained by keeping overnight in 30% methanol and 7% acetic acid at room temperature. Two-dimensional thin layer chromatography. The lipid pattern was studied by two-dimensional thin layer chromatograms (23), using chloroform:methanol: 28% aqueous ammonia 65255 in the first direction, and chloroform:acetone:methanol:acetic acid:water 6:8:2:2:1in the second direction. The lipids were shown on the plate by various group reactions e.g., 0.2% ethanolic ninhydrin for the primary amino groups of phosphatidyl serine, phosphatidyl ethanolamine, and amino sugars of glycolipids; iodine vapors for unsaturation and general lipid detection; lipid phosphorus by the molybdate spray; a-napthol reaction followed by charring with cont. H,SO, at 120°C for sugar and sialic acid of glycolipids. Apart from these group reactions directly on the thin layer plate, the lipid spots destined to quantitative analyses (for phosphorus and fatty acids) were evidenced by a light water spray, scraped off the plate and extracted with chloroform:methanol:diethyl ether mixtures 1:l:l. Gas-liquid chromatography (glc). The fatty acid analysis was restricted to the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. Upon hydrolysis of the phospholipid, the fatty acids were methylated according to the procedures described by Vance and Sweeley (24); to the dry lipid residue (about 500 pg) was added 2 ml methanol acidified with HCl gas to a final 0.75 N HCl concentration. The mixture was heated in sealed Pyrex tube for 22 h at 80°C. The methyl ester, in hexane, was then applied to the gas chromatograph. The glc analysis was carried out with a HewlettPackard 402 gas chromatograph (equipped with flame ionization detector), isothermally at 170°C with a 6-R U-shaped column of 3% OV-? coated on loo-120 Supelcoport (Supelco, Inc. Bellefonte, Pa.). Peak identifications were verified with a 6-ft column of 15% EGSS-X on 100-120 Gas Chrom P (Applied Science Laboratories, Inc., State College, Pa.). The fatty acid methyl ester standards were obtained from Applied Science Laboratories. Peak areas were measured with an Infotronics CRS-101 electronic integrator.
RESULTS AND DISCUSSION
The major centrifugation fractions are referred to in Table I. The plasma membrane material, collected as a white band (band II>
352
BASU ET AL. TABLE
I
COMPOSITION OF DIFFERENT FRACTIONS FROM EPITHELIAL Total protein bg) Homogenate Band I d = 1.14 Band II d = 1.16 (plasma membrane) Band III d = 1.18
at the 40-45% interface is shown by phase contrast micrograph in Fig. 1. Consistently with the electron microscopy and with the marker enzyme analysis this fraction seems to contain the plasma membrane and is clearly devoid of nuclei. Band I (30-40% interface) showed mitochondria-like particles and band III (45-50% interface) contained some plasma membrane aggregates along with partially ruptured enucleated cells. Band IV (50-60% interface) and the pellet consisted mostly of nuclei and some unidentified particles. The electron micrograph of the plasma membrane fraction is shown in Fig. 2, at magnifications of 20,000 in upper panel
42.4 0.13 0.14 0.17
k 2 2 2
4.1 0.02 0.02 0.04
TYPE II LUNG CELL mg phospholipidl mg protein 0.25 0.58 0.40 0.34
2 2 2 2
0.02 0.05 0.04 0.01
(before RNase treatment) and of 60,000 in lower panel (after RNase treatment). The larger units appear as typical membranous sacs. Within and around the plasma membrane texture a few particles resembled contamination by cytoplasmic organelles, which were not identified. As it was observed by other investigators, irrespective of the isolation procedure, it is virtually impossible to obtain a membrane fraction that is free of such contaminations (5, 8, 25-28). Among our attempts at improving the quality of the membrane preparation, treatment with ribonuclease (RNase) in the presence of Mg2+ afforded more satisfactory results (Fig. 2); obvious is the elimination of
FIG. 1. Phase contrast micrograph of plasma membrane (band II) from epithelial (type II) lung cells. Membranes appear as shrunken sacs. Magnification x520. Some “ghost-like” units are shown with arrows.
PLASMA
MEMBRANE
OF EPITHELIAL
particles (possibly ribosomal) which were prevalent in the untreated preparation. Because of the small quantity of starting material (cell culture), we chose not to pursue further purifications by methods (isopycnic and rate-zonal centrifugations) in which material losses are prohibitive. Table I presents the composition of different fractions in terms of both total protein content and phospholipidlprotein ratio. This value is similar to that obtained for the Ehrlich ascites tumor cell membrane (5), but smaller than the one found for the newborn rat brain isolated cell membrane (25), indicating that the present membrane preparation has a relatively high protein content. The total protein content increases in going from band I to band III, whereas the phospholipidlprotein ratio decreases. The fraction with largest phospholipid/ protein ratio floats at the interface with lower density. This ratio in the membrane fraction is 1.6-fold greater than in the homogenate. If one considers that the plasma membrane represents only a small part of the total cell material, the recovery values in Table I are reasonable, and compare with those reported by other investigators for different cells (5, 25). The distribution of selected marker enzymes in different fractions is shown in Table II. (Na+, K+)-activated ATPase, one of the most significant markers of plasma membrane, has specific activity 7-fold greater in the membrane fraction than in the homogenate, while the band III fraction has a specific activity only just above 4-fold. Similar (Na+, K+)-ATPase activities of plasma membrane fractions isolated from different sources have been reported (25, 26). Glucose 6-phosphatase (a marker for endoplasmic reticulum) activity of this membrane fraction is less than one-fifth that in the homogenate; in spite of the efforts made in the isolation procedures, glucose 6-phosphatase activity in plasma membranes was also noticed by previous workers (5, 27, 28), and it is not clear whether that is due to contamination by endoplasmic reticulum (unbound and bound to the membrane) or to the presence of such an enzyme in the plasma membrane itself. The cytochrome c oxidase activities (a marker for
(TYPE
II) LUNG
CELLS
353
mitochondria) in bands II and III were respectively 76- and 19-fold smaller than in the homogenate, so minute indeed as to indicate that the membrane preparation described is practically free of mitochondria. Figure 3 shows the SDS-polyacrylamide gel electrophoresis pattern of the proteins isolated from the plasma membrane fraction. Eleven major protein bands are seen in the molecular weight range of 14,000 to 68,000. Five of them are seen in 14,000 to 30,000 range and six of them in 43,000 to 68,000 range. A few weak bands could be seen in the 68,000 to 94,000 range. The protein patterns were superimposable in the various membrane preparations obtained. Figure 4 shows the lipid patterns on two-dimensional thin layer chromatography plates. The standards were put on both sides of the plate and run along the unknown mixtures in two directions. The purpose of this was to facilitate identification of the unknown lipid (especially with similar migration pattern) at the intersection of the two lines drawn from the centers of the two corresponding lipid standard spots, one on each side of the plate (M. K. Basu, unpublished). Two plates are presented in Fig. 4, in order to illustrate the group reactions which had to be done on two separate plates, to make possible identification of certain lipids, which would otherwise mask one another under the simple iodine stain. This was the case of the cr-napthol reaction in the identification of the phosphorus-free cerebroside spot which on the basis of its migration could be mistaken for cardiolipin (diphosphatidyl glycerol). Similarly, spot 2 contains two lipids, phosphatidyl ethanolamine and phosphatidyl glycerol; the first, much more abundant than the other, was shown by a positive ninhydrin reaction, and the second was revealed by the Schiffperiodate (29). Spot Nos. 1 and ‘7 stained faintly with iodine but did not produce a positive phosphorus reaction; since both spots on a different plate gave a positive cy-napthol reaction, in line with the available data (30) it is suggested that spot 1 is a cerebroside and spot 7 is a ganglioside. For the quantitation of the phosphatidyl ethanolamine, separate plates were resolved only
354
BASUETAL.
FIG. 2. Electron micrograph of a thin section through the plasma membrane pellet (band II). The larger membranous units can best be described as typical membranous sacs. Within the domain of plasma membrane units, could be seen a few foreign particles, which may represent contamination by intracellular organelles. The upper panel (magnification x20,000) represents a typical picture of the band II material, before RNase treatment and the lower panel (magnification ~60,000) represents same material, after RNase treatment.
356
BASU ET AL. TABLE II DISTRIBUTION OF SELECTED MARKER ENZYMES IN DIFFERENT FRACTIONS FROM EPITHELIAL TYPE II LUNG CELL
Ouabain sensitive (Na+, K+)-ATPase”
Homogenate Band II (plasma membrane) Band III
With DOC
Without DOC
Glucose 6-phosphatase”
Cytochrome c oxidaseb
0.75
0.55
1.86
1.93
5.70 3.60
2.23 1.67
0.34 0.50
0.026 0.105
’ Micromoles of P,lmg protein/h. * Nanomoles/ml/min/mg of protein.
in the first direction, whereby phosphatidyl glycerol migrated slightly faster than phosphatidyl ethanolamine and could be separated out. The lipid pattern as monitored by the given group color reactions and quantitative phosphorus analysis was: phosphatidyl
choline + phosphatidyl ethanolamine > sphingomyelin > phosphatidyl serine = phosphatidyl inositol > cerebroside = ganglioside > phosphatidyl glycerol = lyso phosphatidyl choline. Except for the cerebroside and ganglioside which have not been reported for other membrane preparations, the lipid pattern resembles that obtained from rat liver plasma membrane (31). The fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) of the lipid extracted from plasma membrane fraction is presented in Table III. The fatty acid patterns were: stearic > palmitoleic > oleic > palmitic in PC and palmitoleic > oleic > stearic > arachidic > palmitic in PE.
FIG. 3. Coomassie blue-stained electrophoretic patterns of plasma membrane fraction (band II) and marker proteins, as described under Materials and Methods. Eleven major bands are seen between the molecular weight range of 14,000 to 68,000. A few bands (not so prominent) are also seen in the higher molecular weight range (68,000 to 94,000). The dye front is shown with an arrow.
Arachidic acid (C,,,,) was not found in PC. The unsaturated fatty acids, amounting to a total of less than lo%, were distributed evenly among three shallow and broad peaks, which defied comparison with the available standards. In comparing the fatty acid composition of PC of plasma membrane with those of interspecies lung lavage and lung tissue (32, 33), it is interesting to note that all of them have similar contents of oleic acid (C,,,,) around 14%; notably, the PC of lung lavage and lung tissue has insignificant amounts of stearic (C,,,,) and palmitoleic (C,,:,) acids, less than 7%, against a relatively high level of both, around 28%, in the PC of the plasma membrane presently derived from this type II
PLASMA MEMBRANE
OF EPITHELIAL
epithelial lung cell. On the other hand, palmitate (C,,,,), the essential constituent of pulmonary surfactant, dipalmitoyl phosphatidyl choline, is about 14.2% in PC of this plasma membrane as opposed to its large content in the PC of lung lavage (63%) and in PC of lung tissue (51%) (32, 33). It is apparent that most of the palmitic acidcontaining PC is in the lamellar bodies of
357
(TYPE II) LUNG CELLS TABLE III
FA~Y ACID COMPOSITION OF PHOSPHATIDYL CHOLINE AND PHOSPHATIDYL ETHANOLAMINE OF THE PLASMA MEMBRANE ISOLATED FROM EPITHELIAL (TYPE II) LUNG CELLS”
Fatty acid methyl ester
Phosphatidyl choline
Phosphatidyl ethanolamine
14:o 14:2 160 161 18:O 18:l 20:o Unidentified
2.55 3.10 14.20 28.05 28.15 14.55 Not detectable 9.40
3.90 3.05 5.70 29.90 14.35 23.05 10.65 8.40
(1Thedataareexpressedasapercentageofthetotal fatty acids.
type II cells and not in their plasma membranes. Biological
FIG. 4. Thin layer chromatograms of plasma membrane lipid (extracted from band II) developed in two dimensions. First solvent, chloroform:methanol: 28% aqueous ammonia 65~25~5;second solvent, chloroform:acetone:methanoI:acetic acid:water k&2:2:1. All lipid spots appear after staining with I, vapors, but they are better identified by specific group reactions as (1) cerebroside, (2) phosphatidyl ethanolamine and phosphatidyl glycerol, (3) phosphatidyl choline, (4) phosphatidyl serine and phosphatidyl inositol, (5) sphingomyelin, (6) lysophosphatidyl choline, (7) ganglioside, (8) cholesterol, triglycerides etc. Standard lipids: upper plate, (PE) phosphatidyl ethanolamine, (PC) phosphatidyl choline, (SP) sphingomyelin, (LY) lyso-phosphatidyl choline (100 pg each). Lower plate, (PG) phosphatidyl glycerol, (PS) phosphatidyl serine, (PI) phosphatidyl inositol (100 pg each).
Sign$cance
In order to establish the relevance of the present work to the real structure and function of the plasma membrane of the type II cell, one must first evaluate the criteria which are currently employed to identify type II cells. Such criteria are: (a) the presence of typical lamellar inclusion bodies of phospholipid surfactant, (b) the ability of the cell under certain conditions to secrete dipalmitoyl phosphatidyl choline, and (c)the ability of the cell to incorporate palmitate and synthesize dipalmitoyl phosphatidyl choline. Not all such criteria may be fully tested, primarily because little knowledge is available since very few type II cell lines have been made to grow successfully for extended periods of time in monolayer cultures, and when they grow they tend to transform (34, 35). The following observations, however, may serve as guidelines. Ultrastructural analysis was used routinely to assess the state of the inclusion bodies at various cell transfers. Quantity and integrity of the inclusion bodies tended to gradually diminish in the cloned cells after numerous transfers; this effect was more or less marked, depending on the animal species (341, and
BASU ET AL.
probably also on the chemistry of the culture medium. The cells used in the present study still had inclusion bodies. They also incorporated palmitate avidly (34). With regard to lipid secretion, which is the alleged function of type II cell in viva (36), the monolayer may not be the right model; although type II cells secrete huge quantities of lame&r lipid structures when they are grown from lung explants on spongy matrices in the form of alveolar-like cysts (35,37), the same cells secrete very little dipalmitoyl phosphatidyl choline when they are grown in monolayer-s (35, 38). Therefore with the monolayer model, quantitation of the lipid secreted into the culture medium would not bear useful or relevant information, Irrespective of which culture modelthe monolayer or the cyst-is more relevant to the physiology of type II cells, the most instructive finding of the present work is that under the given experimental conditions, dipalmitoyl phosphatidyl choline is not a major constituent of the membrane lipid of type II cell. Whether this is also true in vivo, and how in experimental and in physiological conditions this situation relates to the abundance of the phospholipid surfactant in the inclusion bodies, are a few of the questions open to investigation. ACKNOWLEDGMENTS This work was supported by NIH Grants HL 16137 and NOI-HR52952. Thanks are due to Dr. James Haley of the Neurology Department for help in the gas-liquid chromatography analysis. REFERENCES 1. DEPIERRE, J. W., AND KARNOVSKY, M. L. (1973) J. Cell Biol. 56, 275-303. 2. WARREN, L., AND GLIEK, M. C. (19’71) in Biomembranes (Manson, L. A., ed.), Vol. 1, pp. 257-288, Plenum, New York. 3. STEEK, T. L., AND WALLACH, D. F. H. (1970) in Methods in Cancer Research (Busch, H., ed.), Vol. 5, pp. 93-153, Academic Press, New York. 4. NEVILLE, D. M., JR. (1960) J. Biophys. Biochem. Cytol. 8, 413-422. 5. FORTE, J. G., FORTE, T. M., AND HEINZ, E. (1973) Biochim. Biophys. Acta 298, 827-841. 6. PERDUE, J. F., WARNER, D., AND MILLER, K. (1973) B&him. Biophys. Acta 298, 817-826.
7. ATKINSON, P. H. (1974) Methods in Cell Biology, Vol. XII, pp. 157-188, Academic Press, New York. 8. JOHNSEN, S., STOKKE, T., AND PRYDZ, H. (1974) J. Cell Biol. 63, 357-363. 9. RYAN, J. W., AND SMITH, U. (1971) Biochem. Biophys. Acta 249, 177-180. 10. DOUGLAS, W. H. J., AND KAIGHN, M. E. (1974) In Vitro 10, 230-237. 11. RAY, T. K., AND FORTE, J. G. (1974) Biochim. Biophys. Acta 320, 320-339. 12. JORGENSEN, P. L., SKOU, J. C., AND SOLOMON, L. P. (1971) Biochim. Biophys. Acta 233, 381-394. 13. LINDBERG, O., AND ERNSTER, L. (1963) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 3, pp. l-22, Interscience, New York. 14. COOPERSTEIN, S. J., AND LAZAROW, A. (1951) J. Biol. Chem. 189, 665-670. 15. SWANSON, M. A. (1956) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 541-543, Academic Press, New York. 16. NORTON, W. T., AND AUTILIO, L. A. (1966) J. Neumchem. 13, 213-222. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. KATES, M. (1972) in Techniques of Lipidology (Work, T. S., and Work, E., eds.), pp. 351352, American Elsevier, New York. 19. MARINETTI, G. B., ERBLAND, J., AND STOTZ, E. (1959) Biochim. Biophys. Acta 31, 251-252. 20. PEASE, D. C. (1964) in Histological Techniques for Electron Microscopy, 2nd ed., Academic Press, New York. 21. NEVILLE, D. M. (1971) J. Biol. Chem. 246, 6328-6334. 22. COHEN, R. S., BLOMBERG, F., BERZINS, K., AND SIEKEVITZ, P. (1977) J. Cell Biol. 74, 181-203. 23. ROUSER, G., FLEISCHER, S., AND YAMAMOTO, A. (1970) Lipids 5, 494-496. 24. VANCE, D. E., AND SWEELEY, C. C. (1967) J. Lipid Res. 8, 621-630. 25. HEMMINKI, K., AND SUOVANEIMI, 0. (1973) Biochim. Biophys. Acta 298, 75-83. 26. CONSTANTINO-CECCARINI, E., NOVIKOFF, P. M., ATKINSON, P. H., AND NOVIKOFF, A. B. (1978) J. Cell Biol. 77, 448-463. 27. NIGAM, V. N., MORAIS, R., AND KARASAKI, S. (1971) B&him. Biophys. Acta 249, 34-40. 28. RAY, T. K. (1970) Biochim. Biophys. Acta 196, l-9. 29. SHAW, N. (1968) Biochim. Biophys. Acta 164, 435-436.
PLASMA 30. SIAKOTOS, A.
N.,
MEMBRANE
AND ROUSER,
OF EPITHELIAL G.
(1965)
J. Amer. Oil Chem. Sot. 42, 913-918. 31. BERGELSON, L. D., DYATU)VITSKAYA, E. V., TORKHOVSKAYA, T. I., SOROKINA, I. B., AND G~RKOVA, N. P. (1970) Biochim. Biophys. Acta 210,287-298. 32. COLACICCO, G., RAY, A. K., AND BUCKELEW, A. R., JR. (1977) Lipids 12, 879-888. 33. ROONEY, S. A., NARD~NE, L. L., SHAPIRO, D. L., MOMYAMA, E. K., GOBRAN, L., AND ZAEHRINGER, N. (1977) Lipids 12, 438-442. 34. COLACICCO, G., BASU, M. K., RAY, A. K.,
(TYPE
II) LUNG
CELLS
359
WITTNER, M., AND ROSENBAUM, R. M. (1979)
Z. Naturforsch 34C, 96-100. 35. ROSENBAUM, R. M., PICCIANO, P. T., KRESS, Y., AND WITFNER, M. (1977) Anat. Rec. 188, 241-262. 36. KIKKAWA, Y., KAIBARA, M., MOTOYAMA, E. K., ORZALESI, M. M., AND COOK, C. D. (1971) Amer. J. Pathol. 64, 423-442. 37. DOUGLAS, W. H. J., AND TEEL, R. W. (1976) Amer. Rev. Resp. Dis. 113, 17-23. 38. SMITH, B. T. (1977) Amer. Rev. Resp. Dis. 115, 285-293.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 198, No. 2, December, pp. 349-359, 1979
Isolation
and Characterization of Plamsa Membrane from Monolayer Cultures of Epithelial Type II Lung Cells
MUKUL K. BASU,’ GIUSEPPE COLACICCO, PAUL T. PICCIANO, M. ROSENBAUM AND MURRAY WITTNER Albert Einstein
College of Medicine 1300 Morris
Park Avenue, Bronx,
ROBERT
New York 10461
Received May 30, 1979; revised August 1, 1979 Pneumocyte type II produces a phospholipid, dipalmitoyl lecithin, which is stored in and secreted from the cell’s inclusion bodies and is indispensable for alveolar stability. Cloned rat lung type II cells were harvested at monolayer confluence and homogenized in swelling buffer. After sequential differential centrifugations, the crude membrane fraction was subjected to discontinuous sucrose density gradient centrifugation at 65,000 x g. Quality of the relevant fractions was monitored by enzyme activities and phase contrast and electron microscopy of two major bands at densities 1.16 and 1.18, respectively. The less dense band contained only small quantities of organelles, little cytochrome c oxidase, and some glucose 6-phosphatase, but had a significant (Na+, K+)-ATPase activity; this and ultrastructural evidence certified the product as a suitable plasma membrane preparation. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein pattern consisted of 11 major protein bands between 13,000 and 68,000 M, ranges, and several minor ones. The lipid pattern was studied by two-dimensional thin layer chromatograpy, followed by various group reactions (e.g., amine, unsaturation, phosphorus, sugars). In the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, palmitic acid was the least abundant of four major fatty acids, accounting for 14.20% in phosphatidyl choline and 5.70% in phosphatidyl ethanolamine, whereas the most abundant were stearic and palmitoleic with about 28% each in phosphatidyl choline, and palmitoleic (29.90%) and oleic (23.05%) in the ethanolamine phosphatide. Apparently, the palmitic acid containing phosphatidyl choline must be in the lamellar inclusion bodies of type II cells and not in their plasma membranes.
The isolation of plasma membrane from animal cells with the various criteria for identification and homogeneity has been the subject of several reviews (l-4). Preparations from in viva sources are more common, but preparations have also been made from cell cultures in vitro (5-8). Although some workers published the isolation of plasma membrane from rat lung tissue (9) there is no report concerning the isolation of plasma membrane from rat lung cell grown is tissue culture. 1 To whom correspondence should be sent at the present address: Hematology Division, St. Luke’s Hospital Center & Department of Medicine, College of Physicians and Surgeons, Columbia University, Amsterdam Avenue at 114th Street, New York, N. Y. 10025. 349
The primary objectives of the present work were (a) isolation of the plasma membrane from rat lung epithelial (type II) cells in culture, (b) identification of the membrane preparations by morphological as well as chemical methods, (c) membrane protein pattern by SDF-polyacrylamide gel electrophoresis, (d) membrane lipid pattern by two-dimensional thin layer chromatography, and (e) fatty acid compositions of these lipids with particular regard to phosphatidyl choline and phosphatidyl ethanolamine. * Abbreviations used: SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine; glc, gas-liquid chromatography; PC, phosphatidyl choline; PE, phosphatidyl ethanolamine; DOC, deoxycholate. 0003-9861/79/140349-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
350
BASU ET AL. MATERIALS
AND METHODS
Tbe rat lung cells (type II pneumocytes) obtained by the cloning method of Douglas and Kaighn (10) were grown in monolayers under 5% CO, and 95% air in petri dishes, using Dulbecco’s modified Eagle’s medium containing also 15% fetal calf serum, 100 pg/ml streptomycin, and 100units/ml penicillin. Transfer were made at 1 to 4 split. At the beginning of the seventh day when the cells had reached confluency, they were harvested by scraping with a rubber policeman, followed by lo-min centrifugation from 0.15 M NaCl at 300 g. The pellets were washed 2 x in 0.15 M NaCl and kept in ice bath. Phase contrast microscopy was used to monitor the effectiveness of cell disruption as well as of subsequent fractionation procedures. Isolation of plasma membrane. The packed cell pellets were resuspended in 6 vol of cold homogenizing medium containing 18 mM Tris (pH 8.0), 25 mM NaCl, and 0.5 mM CaCI,. All the homogenization steps were carried out in the presence of 1 mg/ml ribonuclease A (Sigma Chemical Co.) in order to remove possible ribosomal contaminants. Optimal rupturing of cells was produced by 35-40 manual strokes of a Dounce-type steel homogenizer with O.OOZ-in.clearance. After 20 up and 20 down strokes, the homogenized material was diluted with 2 vol of the same homogenizing medium and homogenized again after keeping the content at room temperature for 15 min. At this stage, 90% cells were ruptured. The contents were diluted again with 4 vol of the homogenizing medium and were subjected to differential centrifugation to obtain crude plasma membrane. The centrifugation steps were very similar to those in the method described by Forte et al. (5). The cell homogenate was centrifuged in a Sorvall refrigerated centrifuge (using SS 34 rotor) for 1 min at 200 g. The supernatant (I) was collected and the sediment was redispersed in homogenizing medium and centrifuged again for 1 min at 200 g. The supernatant (II) was collected and combined with supernatant I. The sediment consisted of nuclei and whole cells. The combined supernatant was centrifuged at 700 g for 10 min. The pellet contained plasma membranes and nuclei. The pellet was resuspended and washed twice in the homogenizing medium for preparation of final crude membrane pellet, which again was homogenized by means of 3 up and down strokes in a Dounce loose pestle homogenizer, in order to remove residual cytoplasmic components. The content was redispersed in 22 ml of I:1 mixture of resuspending medium and 60% sucrose. About 7.0 ml of this material was applied to a discontinuous density gradient, consisting of successive 6-ml layers of 60, 50, 45, and 40% sucrose (w/v), distributed equally in three 30-ml centrifuge tubes. The centrifugation was carried out in a SW 25 rotor at 25,000 rpm for 1 h. A pellet and four interfacial bands were noticed. The bands were collected by hypodermic needle, and the particulate
material therein was centrifuged in homogenizing medium for half an hour at 22,000 rpm using a SW 27 rotor and washed twice with this medium by the same centrifugation procedure. For further purification, the band containing plasma membrane (band II, 40-45% sucrose) was treated with 10 mM MgCl, (11) and redispersed in Tris buffer (pH 8.0), containing 18 mM Tris and 25 mM NaCI. The dispersion was homogenized again in a Dounce loose pestle homogenizer by 2 up and down strokes and subjected to a second discontinuous density gradient centrifugation using 8ml layers of 50, 45, and 40% sucrose (w/v). The membrane fraction was applied to the gradient in 7.0 ml of a resuspending medium (18 mM Tris and 25 mM NaCI, pH 8.0) and 60% sucrose; centrifugation, collection, and washing were done in the same manner as described above. Unless otherwise stated, all centrifugation steps were carried out at 4°C. Enzyme assays. Ouabain-sensitive (Na+, K+)ATPase was measured by the method of Jorgensen et al. (12). Since ATPase is tightly bound to the membrane, sodium deoxycholate was used to facilitate release of the enzyme. The membrane fractions and homogenates containing 150-350 pg protein were incubated in 1 ml solution containing 0.6 mg of sodium deoxycholate, 2 mM EDTA, 25 mM imidazole (pH 7.0) at 20°C. After 30 min, lOO-to 200~~1aliquots were incubated again in 1 ml medium containing 50 mM Tris-HC1 pH 7.4, 1 mM EDTA neutralized to pH 7.0, 3 mM Tris-ATP (Sigma Chemical Co.), 100 mM NaCI, 20 mM KCI, and 3 mM MgCl,. Ouabain octahydrate (Sigma Chemical Co.), 1 mM, was included. The mixtures were incubated for 10 min at 37”C, the reaction was terminated by addition of 25% trichloroacetic acid, and the reaction mixtures were left to stand for 15 min. The insoluble material was removed by centrifugation in rotor SS 34 at 2000 rpm for 20 min at 5°C. Aliquots of 1 ml supernatant were removed from each tube, and the released inorganic phosphorus was determined therein by the molybdate method of Martin and Doty as described by Lindberg and Ernster (13). Greater reproducibility was achieved when the samples were preincubated for 2 min at 37°C. The total ATPase activity (Na+, K+, Mg*+) was calculated from the absorbance at 730 nm in the absence of ouabain. The Mg’+-ATPase activity was calculated from the absorbance in the presence of ouabain. The difference between the two was taken to be the ouabain-sensitive (Na+, K+)-ATPase activity. Cytochrome c oxidase activity was measured by a method very similar to the one described by Cooperstein et al. (14). Cytochrom c (horse heart, Sigma Grade IV), 50 mg, was dissolved in 1 ml of Tris-HCl buffer (pH 7.4) containing 50 mM Tris and 154 mM NaCI. Tris-ascorbate (pH 7.4), 0.5 M, 1 ml, was added to reduce the cytochrome c. The solution was then layered on a Sephadex A-25 column prepared with the same Tris-HCI buffer, and the cytochrome c was
PLASMA MEMBRANE
OF EPITHELIAL
eluted by monitoring the band visually. A 3-ml aliquot of the reduced cytochrome c solution was pipetted into a Beckman spectrophotometer cuvette and 0.02 or 0.04 ml of membrane fraction or cell homogenate was added. The reactants were mixed by inverting the cuvette and readings were taken every 30 s at 550 nm. At the end of 3 min a few grains of potassium ferricyanide were added (to oxidize completely the cytochrome c), and the optical extinction was redetermined. Glucose 6-phosphatase was assayed by the method of Swanson (15). The substrate consisted of 11 mM glucose 6-phosphate in 55 mM Tris, with 11 mM mercaptoethanol (pH 6.6). To 0.8 ml of substrate were added O&ml aliquots of the membrane fractions or cell homogenate (0.1-0.2 mg protein). The reaction was allowed to proceed for about 15 min and was then stopped by the addition of 1 ml of trichloroacetic acid (10%). Protein was removed by centrifugation, and 1 ml ofthe cleared supernatant was used for phosphate determination (16). Chemical analyses. Protein was determined by the method of Lowry et al. (1’7). Lipids were extracted from the membrane fractions as well as from the cell homogenate, by conventional procedures (18), in which 1 ml of the cell suspension or membrane dispersion was shaken with 3.75 ml of methanol:chloroform (2:1, v/v), followed by 4.75 ml of methanokchlorofornuwater (l&5:4, v/v). Lipid phosphorus was assayed by the technique of Marinetti et al. (19). Phase contrast and electron microscow. Light microscopy observations were made with a Nikon phase contrast microscope, equipped with camera. Membrane pellets were processed for ultramicrotomy and electron microscopy by standard procedures (20). The pellets were dispersed and fixed in 2.5% glutaraldehyde which was buffered with cacodylate (pH 7. l), and postfixed in similarly buffered 1% 0~0,. The pellet membranes were dehydrated with graded ethanol and embedded in epoxy resin. The sections were cut with a Sorvall MT-2 ultramicrotome at about 600 A and then stained with many1 acetate for 15 min, followed by lead acetate for 7 min, and then examined with a high performance electron microscope (Siemens, Elmiskop 101). SDS-polyacrylamide gel electrophvresis. Electrophoresis on slab gel was carried out to establish membrane proteins pattern. A discontinuous buffer system (21) was used with some modification (22); a 5-15% gradient gel was prepared with acrylamide containing methylenebisacrylamide (Polyscience), N,N,N’,N’-tetramethylethylenediamine (TEMED, Eastman), ammonium persulfate (Fisher, analytical grade), sucrose (Sigma), and SDS (Fisher) in the standard proportions. The plasma membrane was solubilized in 2% SDS and heated at 90°C for 90 s in the presence of Pmercaptoethanol (Eastman). The protein standards for molecular weight determination
(TYPE II) LUNG
CELLS
351
consisted of ribonuclease A (13.7 K, Sigma Chemical Co.), chymotrypsin (22 K, Sigma Chemical Co.), Rabbit muscle actin (43 K, Worthington Biochemicals), bovine serum albumin (68 K, Pentex Biochemical& and phosphorylase (Y(94 K, Sigma Chemical Co.). The gel was stained with 0.25% Coomassie brilliant blue (R-250, Mann) in methanol:water:acetic acid 50~437 for 1 h at 37°C and destained by keeping overnight in 30% methanol and 7% acetic acid at room temperature. Two-dimensional thin layer chromatography. The lipid pattern was studied by two-dimensional thin layer chromatograms (23), using chloroform:methanol: 28% aqueous ammonia 65255 in the first direction, and chloroform:acetone:methanol:acetic acid:water 6:8:2:2:1in the second direction. The lipids were shown on the plate by various group reactions e.g., 0.2% ethanolic ninhydrin for the primary amino groups of phosphatidyl serine, phosphatidyl ethanolamine, and amino sugars of glycolipids; iodine vapors for unsaturation and general lipid detection; lipid phosphorus by the molybdate spray; a-napthol reaction followed by charring with cont. H,SO, at 120°C for sugar and sialic acid of glycolipids. Apart from these group reactions directly on the thin layer plate, the lipid spots destined to quantitative analyses (for phosphorus and fatty acids) were evidenced by a light water spray, scraped off the plate and extracted with chloroform:methanol:diethyl ether mixtures 1:l:l. Gas-liquid chromatography (glc). The fatty acid analysis was restricted to the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine. Upon hydrolysis of the phospholipid, the fatty acids were methylated according to the procedures described by Vance and Sweeley (24); to the dry lipid residue (about 500 pg) was added 2 ml methanol acidified with HCl gas to a final 0.75 N HCl concentration. The mixture was heated in sealed Pyrex tube for 22 h at 80°C. The methyl ester, in hexane, was then applied to the gas chromatograph. The glc analysis was carried out with a HewlettPackard 402 gas chromatograph (equipped with flame ionization detector), isothermally at 170°C with a 6-R U-shaped column of 3% OV-? coated on loo-120 Supelcoport (Supelco, Inc. Bellefonte, Pa.). Peak identifications were verified with a 6-ft column of 15% EGSS-X on 100-120 Gas Chrom P (Applied Science Laboratories, Inc., State College, Pa.). The fatty acid methyl ester standards were obtained from Applied Science Laboratories. Peak areas were measured with an Infotronics CRS-101 electronic integrator.
RESULTS AND DISCUSSION
The major centrifugation fractions are referred to in Table I. The plasma membrane material, collected as a white band (band II>
352
BASU ET AL. TABLE
I
COMPOSITION OF DIFFERENT FRACTIONS FROM EPITHELIAL Total protein bg) Homogenate Band I d = 1.14 Band II d = 1.16 (plasma membrane) Band III d = 1.18
at the 40-45% interface is shown by phase contrast micrograph in Fig. 1. Consistently with the electron microscopy and with the marker enzyme analysis this fraction seems to contain the plasma membrane and is clearly devoid of nuclei. Band I (30-40% interface) showed mitochondria-like particles and band III (45-50% interface) contained some plasma membrane aggregates along with partially ruptured enucleated cells. Band IV (50-60% interface) and the pellet consisted mostly of nuclei and some unidentified particles. The electron micrograph of the plasma membrane fraction is shown in Fig. 2, at magnifications of 20,000 in upper panel
42.4 0.13 0.14 0.17
k 2 2 2
4.1 0.02 0.02 0.04
TYPE II LUNG CELL mg phospholipidl mg protein 0.25 0.58 0.40 0.34
2 2 2 2
0.02 0.05 0.04 0.01
(before RNase treatment) and of 60,000 in lower panel (after RNase treatment). The larger units appear as typical membranous sacs. Within and around the plasma membrane texture a few particles resembled contamination by cytoplasmic organelles, which were not identified. As it was observed by other investigators, irrespective of the isolation procedure, it is virtually impossible to obtain a membrane fraction that is free of such contaminations (5, 8, 25-28). Among our attempts at improving the quality of the membrane preparation, treatment with ribonuclease (RNase) in the presence of Mg2+ afforded more satisfactory results (Fig. 2); obvious is the elimination of
FIG. 1. Phase contrast micrograph of plasma membrane (band II) from epithelial (type II) lung cells. Membranes appear as shrunken sacs. Magnification x520. Some “ghost-like” units are shown with arrows.
PLASMA
MEMBRANE
OF EPITHELIAL
particles (possibly ribosomal) which were prevalent in the untreated preparation. Because of the small quantity of starting material (cell culture), we chose not to pursue further purifications by methods (isopycnic and rate-zonal centrifugations) in which material losses are prohibitive. Table I presents the composition of different fractions in terms of both total protein content and phospholipidlprotein ratio. This value is similar to that obtained for the Ehrlich ascites tumor cell membrane (5), but smaller than the one found for the newborn rat brain isolated cell membrane (25), indicating that the present membrane preparation has a relatively high protein content. The total protein content increases in going from band I to band III, whereas the phospholipidlprotein ratio decreases. The fraction with largest phospholipid/ protein ratio floats at the interface with lower density. This ratio in the membrane fraction is 1.6-fold greater than in the homogenate. If one considers that the plasma membrane represents only a small part of the total cell material, the recovery values in Table I are reasonable, and compare with those reported by other investigators for different cells (5, 25). The distribution of selected marker enzymes in different fractions is shown in Table II. (Na+, K+)-activated ATPase, one of the most significant markers of plasma membrane, has specific activity 7-fold greater in the membrane fraction than in the homogenate, while the band III fraction has a specific activity only just above 4-fold. Similar (Na+, K+)-ATPase activities of plasma membrane fractions isolated from different sources have been reported (25, 26). Glucose 6-phosphatase (a marker for endoplasmic reticulum) activity of this membrane fraction is less than one-fifth that in the homogenate; in spite of the efforts made in the isolation procedures, glucose 6-phosphatase activity in plasma membranes was also noticed by previous workers (5, 27, 28), and it is not clear whether that is due to contamination by endoplasmic reticulum (unbound and bound to the membrane) or to the presence of such an enzyme in the plasma membrane itself. The cytochrome c oxidase activities (a marker for
(TYPE
II) LUNG
CELLS
353
mitochondria) in bands II and III were respectively 76- and 19-fold smaller than in the homogenate, so minute indeed as to indicate that the membrane preparation described is practically free of mitochondria. Figure 3 shows the SDS-polyacrylamide gel electrophoresis pattern of the proteins isolated from the plasma membrane fraction. Eleven major protein bands are seen in the molecular weight range of 14,000 to 68,000. Five of them are seen in 14,000 to 30,000 range and six of them in 43,000 to 68,000 range. A few weak bands could be seen in the 68,000 to 94,000 range. The protein patterns were superimposable in the various membrane preparations obtained. Figure 4 shows the lipid patterns on two-dimensional thin layer chromatography plates. The standards were put on both sides of the plate and run along the unknown mixtures in two directions. The purpose of this was to facilitate identification of the unknown lipid (especially with similar migration pattern) at the intersection of the two lines drawn from the centers of the two corresponding lipid standard spots, one on each side of the plate (M. K. Basu, unpublished). Two plates are presented in Fig. 4, in order to illustrate the group reactions which had to be done on two separate plates, to make possible identification of certain lipids, which would otherwise mask one another under the simple iodine stain. This was the case of the cr-napthol reaction in the identification of the phosphorus-free cerebroside spot which on the basis of its migration could be mistaken for cardiolipin (diphosphatidyl glycerol). Similarly, spot 2 contains two lipids, phosphatidyl ethanolamine and phosphatidyl glycerol; the first, much more abundant than the other, was shown by a positive ninhydrin reaction, and the second was revealed by the Schiffperiodate (29). Spot Nos. 1 and ‘7 stained faintly with iodine but did not produce a positive phosphorus reaction; since both spots on a different plate gave a positive cy-napthol reaction, in line with the available data (30) it is suggested that spot 1 is a cerebroside and spot 7 is a ganglioside. For the quantitation of the phosphatidyl ethanolamine, separate plates were resolved only
354
BASUETAL.
FIG. 2. Electron micrograph of a thin section through the plasma membrane pellet (band II). The larger membranous units can best be described as typical membranous sacs. Within the domain of plasma membrane units, could be seen a few foreign particles, which may represent contamination by intracellular organelles. The upper panel (magnification x20,000) represents a typical picture of the band II material, before RNase treatment and the lower panel (magnification ~60,000) represents same material, after RNase treatment.
356
BASU ET AL. TABLE II DISTRIBUTION OF SELECTED MARKER ENZYMES IN DIFFERENT FRACTIONS FROM EPITHELIAL TYPE II LUNG CELL
Ouabain sensitive (Na+, K+)-ATPase”
Homogenate Band II (plasma membrane) Band III
With DOC
Without DOC
Glucose 6-phosphatase”
Cytochrome c oxidaseb
0.75
0.55
1.86
1.93
5.70 3.60
2.23 1.67
0.34 0.50
0.026 0.105
’ Micromoles of P,lmg protein/h. * Nanomoles/ml/min/mg of protein.
in the first direction, whereby phosphatidyl glycerol migrated slightly faster than phosphatidyl ethanolamine and could be separated out. The lipid pattern as monitored by the given group color reactions and quantitative phosphorus analysis was: phosphatidyl
choline + phosphatidyl ethanolamine > sphingomyelin > phosphatidyl serine = phosphatidyl inositol > cerebroside = ganglioside > phosphatidyl glycerol = lyso phosphatidyl choline. Except for the cerebroside and ganglioside which have not been reported for other membrane preparations, the lipid pattern resembles that obtained from rat liver plasma membrane (31). The fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) of the lipid extracted from plasma membrane fraction is presented in Table III. The fatty acid patterns were: stearic > palmitoleic > oleic > palmitic in PC and palmitoleic > oleic > stearic > arachidic > palmitic in PE.
FIG. 3. Coomassie blue-stained electrophoretic patterns of plasma membrane fraction (band II) and marker proteins, as described under Materials and Methods. Eleven major bands are seen between the molecular weight range of 14,000 to 68,000. A few bands (not so prominent) are also seen in the higher molecular weight range (68,000 to 94,000). The dye front is shown with an arrow.
Arachidic acid (C,,,,) was not found in PC. The unsaturated fatty acids, amounting to a total of less than lo%, were distributed evenly among three shallow and broad peaks, which defied comparison with the available standards. In comparing the fatty acid composition of PC of plasma membrane with those of interspecies lung lavage and lung tissue (32, 33), it is interesting to note that all of them have similar contents of oleic acid (C,,,,) around 14%; notably, the PC of lung lavage and lung tissue has insignificant amounts of stearic (C,,,,) and palmitoleic (C,,:,) acids, less than 7%, against a relatively high level of both, around 28%, in the PC of the plasma membrane presently derived from this type II
PLASMA MEMBRANE
OF EPITHELIAL
epithelial lung cell. On the other hand, palmitate (C,,,,), the essential constituent of pulmonary surfactant, dipalmitoyl phosphatidyl choline, is about 14.2% in PC of this plasma membrane as opposed to its large content in the PC of lung lavage (63%) and in PC of lung tissue (51%) (32, 33). It is apparent that most of the palmitic acidcontaining PC is in the lamellar bodies of
357
(TYPE II) LUNG CELLS TABLE III
FA~Y ACID COMPOSITION OF PHOSPHATIDYL CHOLINE AND PHOSPHATIDYL ETHANOLAMINE OF THE PLASMA MEMBRANE ISOLATED FROM EPITHELIAL (TYPE II) LUNG CELLS”
Fatty acid methyl ester
Phosphatidyl choline
Phosphatidyl ethanolamine
14:o 14:2 160 161 18:O 18:l 20:o Unidentified
2.55 3.10 14.20 28.05 28.15 14.55 Not detectable 9.40
3.90 3.05 5.70 29.90 14.35 23.05 10.65 8.40
(1Thedataareexpressedasapercentageofthetotal fatty acids.
type II cells and not in their plasma membranes. Biological
FIG. 4. Thin layer chromatograms of plasma membrane lipid (extracted from band II) developed in two dimensions. First solvent, chloroform:methanol: 28% aqueous ammonia 65~25~5;second solvent, chloroform:acetone:methanoI:acetic acid:water k&2:2:1. All lipid spots appear after staining with I, vapors, but they are better identified by specific group reactions as (1) cerebroside, (2) phosphatidyl ethanolamine and phosphatidyl glycerol, (3) phosphatidyl choline, (4) phosphatidyl serine and phosphatidyl inositol, (5) sphingomyelin, (6) lysophosphatidyl choline, (7) ganglioside, (8) cholesterol, triglycerides etc. Standard lipids: upper plate, (PE) phosphatidyl ethanolamine, (PC) phosphatidyl choline, (SP) sphingomyelin, (LY) lyso-phosphatidyl choline (100 pg each). Lower plate, (PG) phosphatidyl glycerol, (PS) phosphatidyl serine, (PI) phosphatidyl inositol (100 pg each).
Sign$cance
In order to establish the relevance of the present work to the real structure and function of the plasma membrane of the type II cell, one must first evaluate the criteria which are currently employed to identify type II cells. Such criteria are: (a) the presence of typical lamellar inclusion bodies of phospholipid surfactant, (b) the ability of the cell under certain conditions to secrete dipalmitoyl phosphatidyl choline, and (c)the ability of the cell to incorporate palmitate and synthesize dipalmitoyl phosphatidyl choline. Not all such criteria may be fully tested, primarily because little knowledge is available since very few type II cell lines have been made to grow successfully for extended periods of time in monolayer cultures, and when they grow they tend to transform (34, 35). The following observations, however, may serve as guidelines. Ultrastructural analysis was used routinely to assess the state of the inclusion bodies at various cell transfers. Quantity and integrity of the inclusion bodies tended to gradually diminish in the cloned cells after numerous transfers; this effect was more or less marked, depending on the animal species (341, and
BASU ET AL.
probably also on the chemistry of the culture medium. The cells used in the present study still had inclusion bodies. They also incorporated palmitate avidly (34). With regard to lipid secretion, which is the alleged function of type II cell in viva (36), the monolayer may not be the right model; although type II cells secrete huge quantities of lame&r lipid structures when they are grown from lung explants on spongy matrices in the form of alveolar-like cysts (35,37), the same cells secrete very little dipalmitoyl phosphatidyl choline when they are grown in monolayer-s (35, 38). Therefore with the monolayer model, quantitation of the lipid secreted into the culture medium would not bear useful or relevant information, Irrespective of which culture modelthe monolayer or the cyst-is more relevant to the physiology of type II cells, the most instructive finding of the present work is that under the given experimental conditions, dipalmitoyl phosphatidyl choline is not a major constituent of the membrane lipid of type II cell. Whether this is also true in vivo, and how in experimental and in physiological conditions this situation relates to the abundance of the phospholipid surfactant in the inclusion bodies, are a few of the questions open to investigation. ACKNOWLEDGMENTS This work was supported by NIH Grants HL 16137 and NOI-HR52952. Thanks are due to Dr. James Haley of the Neurology Department for help in the gas-liquid chromatography analysis. REFERENCES 1. DEPIERRE, J. W., AND KARNOVSKY, M. L. (1973) J. Cell Biol. 56, 275-303. 2. WARREN, L., AND GLIEK, M. C. (19’71) in Biomembranes (Manson, L. A., ed.), Vol. 1, pp. 257-288, Plenum, New York. 3. STEEK, T. L., AND WALLACH, D. F. H. (1970) in Methods in Cancer Research (Busch, H., ed.), Vol. 5, pp. 93-153, Academic Press, New York. 4. NEVILLE, D. M., JR. (1960) J. Biophys. Biochem. Cytol. 8, 413-422. 5. FORTE, J. G., FORTE, T. M., AND HEINZ, E. (1973) Biochim. Biophys. Acta 298, 827-841. 6. PERDUE, J. F., WARNER, D., AND MILLER, K. (1973) B&him. Biophys. Acta 298, 817-826.
7. ATKINSON, P. H. (1974) Methods in Cell Biology, Vol. XII, pp. 157-188, Academic Press, New York. 8. JOHNSEN, S., STOKKE, T., AND PRYDZ, H. (1974) J. Cell Biol. 63, 357-363. 9. RYAN, J. W., AND SMITH, U. (1971) Biochem. Biophys. Acta 249, 177-180. 10. DOUGLAS, W. H. J., AND KAIGHN, M. E. (1974) In Vitro 10, 230-237. 11. RAY, T. K., AND FORTE, J. G. (1974) Biochim. Biophys. Acta 320, 320-339. 12. JORGENSEN, P. L., SKOU, J. C., AND SOLOMON, L. P. (1971) Biochim. Biophys. Acta 233, 381-394. 13. LINDBERG, O., AND ERNSTER, L. (1963) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 3, pp. l-22, Interscience, New York. 14. COOPERSTEIN, S. J., AND LAZAROW, A. (1951) J. Biol. Chem. 189, 665-670. 15. SWANSON, M. A. (1956) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, pp. 541-543, Academic Press, New York. 16. NORTON, W. T., AND AUTILIO, L. A. (1966) J. Neumchem. 13, 213-222. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. KATES, M. (1972) in Techniques of Lipidology (Work, T. S., and Work, E., eds.), pp. 351352, American Elsevier, New York. 19. MARINETTI, G. B., ERBLAND, J., AND STOTZ, E. (1959) Biochim. Biophys. Acta 31, 251-252. 20. PEASE, D. C. (1964) in Histological Techniques for Electron Microscopy, 2nd ed., Academic Press, New York. 21. NEVILLE, D. M. (1971) J. Biol. Chem. 246, 6328-6334. 22. COHEN, R. S., BLOMBERG, F., BERZINS, K., AND SIEKEVITZ, P. (1977) J. Cell Biol. 74, 181-203. 23. ROUSER, G., FLEISCHER, S., AND YAMAMOTO, A. (1970) Lipids 5, 494-496. 24. VANCE, D. E., AND SWEELEY, C. C. (1967) J. Lipid Res. 8, 621-630. 25. HEMMINKI, K., AND SUOVANEIMI, 0. (1973) Biochim. Biophys. Acta 298, 75-83. 26. CONSTANTINO-CECCARINI, E., NOVIKOFF, P. M., ATKINSON, P. H., AND NOVIKOFF, A. B. (1978) J. Cell Biol. 77, 448-463. 27. NIGAM, V. N., MORAIS, R., AND KARASAKI, S. (1971) B&him. Biophys. Acta 249, 34-40. 28. RAY, T. K. (1970) Biochim. Biophys. Acta 196, l-9. 29. SHAW, N. (1968) Biochim. Biophys. Acta 164, 435-436.
PLASMA 30. SIAKOTOS, A.
N.,
MEMBRANE
AND ROUSER,
OF EPITHELIAL G.
(1965)
J. Amer. Oil Chem. Sot. 42, 913-918. 31. BERGELSON, L. D., DYATU)VITSKAYA, E. V., TORKHOVSKAYA, T. I., SOROKINA, I. B., AND G~RKOVA, N. P. (1970) Biochim. Biophys. Acta 210,287-298. 32. COLACICCO, G., RAY, A. K., AND BUCKELEW, A. R., JR. (1977) Lipids 12, 879-888. 33. ROONEY, S. A., NARD~NE, L. L., SHAPIRO, D. L., MOMYAMA, E. K., GOBRAN, L., AND ZAEHRINGER, N. (1977) Lipids 12, 438-442. 34. COLACICCO, G., BASU, M. K., RAY, A. K.,
(TYPE
II) LUNG
CELLS
359
WITTNER, M., AND ROSENBAUM, R. M. (1979)
Z. Naturforsch 34C, 96-100. 35. ROSENBAUM, R. M., PICCIANO, P. T., KRESS, Y., AND WITFNER, M. (1977) Anat. Rec. 188, 241-262. 36. KIKKAWA, Y., KAIBARA, M., MOTOYAMA, E. K., ORZALESI, M. M., AND COOK, C. D. (1971) Amer. J. Pathol. 64, 423-442. 37. DOUGLAS, W. H. J., AND TEEL, R. W. (1976) Amer. Rev. Resp. Dis. 113, 17-23. 38. SMITH, B. T. (1977) Amer. Rev. Resp. Dis. 115, 285-293.
