[5]Isolation of plasma membranes from rat and mouse livers and hepatones

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I s o l a t i o n of P l a s m a M e m b r a n e s f r o m R a t a n d Mouse Livers and Hepatomas

By P. EMMELOT, C. J. BOS, R. P. VAN HOEVEN, and W. J. VAN BLITTERSWIJK

Erythrocyte ghosts have long since been isolated and studiedl but it is only more recently that plasma membranes have been prepared from ceils of solid mammalian tissues. The first achievement of this kind was the isolation of plasma membranes from rat liver, reported by Neville I in 1960. In subsequent years this method has beeh modified in various laboratories, and other methods have also been introduced (Table I). The original Neville procedure, as modified in our laboratory 2-e and found highly satisfactory for the isolation of plasma membranes from rat and mouse liver and various liver tumors, will be described here. The principle of the method is the homogenization of the tissue in water buffered with sodium bicarbonate, sedimentation of the plasma membranes followed by a number of washing cycles using low speed differential centrifugation in order to remove the bulk of contaminating materials, and a final flotation in a discontinuous sucrose gradient to remove remaining contaminants. Tissue homogenization forms a crucial step. Success depends on the generation of large plasma-membrane fragments (sheets) which by centrifugation settle upon nuclei and debris, and which can be separated from the bulk of the other organelles by the washings. Therefore, tissue homogenization should not disrupt too much (a) the plasma-membrane skeleton, and (b) the nuclear membrane. Otherwise, (a) small-sized plasma-membrane fragments may not be sedimented by the low speed centrifugation (while increasing speed may introduce too much unwanted material), and (b) a deoxynucleoprotein gel is formed that acts as a glue incorporating the plasma membranes and thus prevents any further effective separation. Different tissues may vary as to the extent to which these requirements can be met. Conditions satisfactory to rat and mouse liver are not necessarily so for liver tumors indigenous to the same hosts. Among the 1D. M. Neville, Jr., J. Biophys. Biochem. Cytol. 8, 413 (1960). 2p. Ernmelot and C. J. Bos, Biochim. Biophys. Acta 58, 374 (1962). P. Emmelot, C. J. Bos, E. L. Benedeni, and P. Riimke, Biochim. Biophys. Acta 90, 126 (1964). 4 p. Emmelot and C. J. Bos, Int. J. Cancer 4, 705 (1969). P. Emmelot and C. J. Bos, Int. J. Cancer 4, 723 (1969). ° P. Emmelot and E. L. Benedetti, in "Carcinogenesis, a Broad Critique," Syrup. Fundam. Cancer Res. ~,0, 471 (1967).

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TABLE I METHODS USED FOR ISOLATING PLASMA MEMBRANES FROM RAT LIVERa

Homogenization medium

Authors Neville'; Emmelot and Bose; Emmelot el a l / Neville e Pfleger et a l / Song el aly Evans h Ray ~ Newkirk and Waite i Takeuchi and T e r a y a m a k Coleman et al. ~ Stein el al. '~ B e r m a n et al. ~ Touster et al.°; Ashworth and Green~; Henning et al.~; House and Weidemann r E1-Aaser et al. ° Weaver and Boyle t Hinton et al. ~

Ratedependent centrifugation

Isopycniczonal centrifugation

b

d

SW 2

b b b b" b + 0.5 m M CaCI~ b (10 m M ) s W 0.5 m M CaCI~ s (0.3 M ) s s W 0.5 m M CaC12

r SW r ZB -r ZA d r ZB d, r SW d~ d d

SW 2 Z s ~/ A 7 SW ~/ SW 2 -SW 2 SW ~/ A 2 SW ~/, /

s s (0.08 M ) s (0.08 M ) s (0.08-0.25 M )

d (r ZA) ~ r ZB r ZA

SW 2' or ~/ Z A ~/ Z B ~/ SW 2

a I n all cases plasma membranes were isolated from the crude nuclear fraction, except for studies °,v,r in which plasma membranes were alsoo, r or exclusivelyp isolated from the microsomal fraction. Media: bicarbonate (b; 1 m M unless indicated otherwise) or sucrose (s, 0.25 M unless indicated otherwise). Centrifugation methods: rate-dependent centrifugation (d, differential centrifugation with fixed-angle head rotor; r, rate zonal centrifugation) and isopycnic-zonal centrifugation (~/ and 2 , respectively, sedimentation and flotation in sucrose gradient). Rotors: A, fixed-angle head rotor; SW, swing-out rotor; ZA and Z B, zonal rotors A and B. b-, Key to references : b D. M. Neville, Jr., J. Biophys. Biochem. Cytol. 8, 413 (1960). c p. Emmelot and C. J. Bos, Biochim. Biophys. Acta 58, 374 (1962). d p. Emmelot, C. J. Bos, E. L. Benedetti, and P. Rtimke, Biochim. Biophys. Acta 90, 126 (1964). D. M. Neville, Jr., Biochim. Biophys. Acta 154, 540 (1968). s R. C. Pfleger, N. G. Anderson, F. Snyder, Biochemistry 8, 2826 (1968). 0 C. S. Song, W. Rubin, A. B. Rifkind, and A. Kappas, J. Cell Biol. 41, 124 (1969). h W. H. Evans, Biochem. J. 116, 833 (1970). ~ T. K. Ray, Biochim. Biophys. Acla 196, 1 (1970). i J. D. Newkirk and M. Waite, Biochim. Biophys. Acta 225, 224 (1971). k M. Takeuchi and H. Terayama, Exp. Cell Res. 40, 32 (1965). ~R. Coleman, R. H. Michell, J. B. Finean, and J. N. Hawthorne, Biochim. Biophys. Acta 135, 573 (1967). '~ Y. Stein, C. Widnell, and 0. Stein, J. Cell Biol. 39, 185 (1968). = H. M. Berman, W. Gram, and M. A. Spirtes, Biochim. Biophys. Acta 183, 10 (1969). o O. Touster, N. N. Aronson, Jr., J. T. Dulaney, and H. Hendriekson, J. Cell Biol. 47, 604 (1970). p L. A. E. Ashworth and C. Green, Science 151, 210 (1966). q R. Henning, H. D. Kaulen, and W. Stoffel, Z. Physiol. Chem. 351, 1191 (1970). r P. D. R. House, and M. J. Weidemann, Biochem. Biophys. Res. Commun. 41, 541

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liver tumors studied by the authors 4,~ there are rapidly growing, rather undifferentiated rat hepatomas, which contain large nuclei whose membranes are apparently easily disrupted by homogenization in the bicarbonate medium, however carefully performed. In such cases it has been found profitable to change the homogenization medium so as to afford protection to the nuclear membrane. Hardening of the nuclear membrane has been achieved either by replacing the bicarbonate medium by dilute citric acid *,7 or by fortifying it with calcium ions? ,s The citric acid method of homogenization has been found less useful as to yield and properties of the resulting plasma membranes. 4 This is presumably due to the extensive fragmentation of the plasma-membrane skeleton following removal of endogenous Ca 2÷ from the membranes by citric acid2 This fragmentation may result in a fractionation of heterogeneous plasma-membrane fragments during preparation. By contrast, the presence of Ca 2÷ during homogenization protects the plasma membranes against breakage as shown by the larger size of the isolated membrane sheets, but it also has certain disadvantages referred to below. P r e p a r a t i o n of P l a s m a M e m b r a n e s Reagents. Analytical grade reagents are used.

Sodium bicarbonate, 1 m M in bidistilled water, p H 7.5, freshly prepared, without or with calcium chloride, 2 m M Sucrose solutions in bidistilled water of 26.6 (d 1.10), 37.6 (d 1.14), 42.9 (d 1.16), 48.0 (d 1.18), 53.4 (d 1.20), and 81% (d 1.30) (% as w / v ; d42° in parentheses). P. Emmelot, E. L. Benedetti, and P. Riimke, in "From Molecule to Cell" (P. Buffa, ed.), p. 253. (Symp. on Electron Microscopy Modena). C.N.R., Rome, 1964. 8 p. Emmelot anti C. J. Bos, Biochim. Biophys. Acta 121, 434 (1966). 9E. L. Benedetti and P. Emmelot, in "The Membranes" (A. J. Dalton and F. Haguenau, eds.), p. ~3. Academic Press, New York, 1968. (1970). * A. A. E1-Aaser, J. T. R. Fitzsimons, R. H. Hinton, E. Reid, E. Klucis, and P. Alexander, Biochim. Biophys. Acta 127, 553 (1966). t R. A. Weaver and W. Boyle~ Biochim. Biophys. Acta 175, 377 (1969). ~ R. H. Hinton, M. Dobrota, J. T. R. Fitzsimons, and E. Reid, Eur. J. Biochem. 12, 349 (1970). " Subfractibnation of plasma membrane fragments generated by vigorous rehomogenization in 8% (w/v) sucrose-Tris buffer. 4, Vigorous rehomogenization of 1000 g pellet results in slower sedimentating plasma membrane fragments. Isolation carried out in a single step; plasma membranes reach equilibrium; particles with sedimentation constant smaller than that of plasma membranes do not.

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Tissue Sources. Liver of rats (3-month-old inbred strain R-Amsterdam, and hybrids with other strains) and mice (strain CBA, of various ages). Animals are not fasted prior to sacrifice by decapitation. Gallbladder of the mouse liver is removed. Hepatomas: Rat hepatoma-484, and its subline 484A, originally induced in a female rat of strain R by 4-dimethylaminoazobenzene, rather anaplastic and rapidly growing hepatoma of the liver-ceU type, transplanted intraperitoneally (i.p.), and harvested after 10 days of growth. The peritoneal cavity is opened, then ascites fluid is removed by blotting with paper towel, and tumor nodules are dissected free from adhering, e.g., fat, tissue. (As is frequently found with this type of tumor, subcutaneous (s.c.) transplants cannot be used. The subcutaneous tumors usually have a necrotic core covered by a shell of living tumor cells and many fibrotic elements. The consistency of this tissue is too firm to allow the gentle homogenization required for the purpose of isolating plasma membranes, and the homogenization necessary for breaking up sufficient cells leads to extensive gel formation even in the presence of Ca '-'~.) Mouse hepatoma-147042 and -143066 arose spontaneously in old CBA males and were transplanted s.c. on similar young animals. These are well differentiated and slowly growing tumors, containing little necrosis and few fibrotic elements, harvested after 2 months of growth, on the average. 5 The excised tumors are dissected free from their well defined fibrotic capsules. Plasma Membranes ]rom Liver All operations are carried out at 0-4 ° with prechilled materials. Cell Rupture. Rat liver corresponding to 30-40 g of wet weight of tissue (20 g in the case of mouse liver) is collected in a beaker containing 50 ml of bicarbonate medium and is finely cut with scissors. Portions corresponding to about 5 g of cut liver are each homogenized in 20 ml of bicarbonate medium using an all-glass homogenizer of the Potter-Elvehjem type with a pestle clearance of 0.5-0.6 mm and tube content of about 50 ml. The pestle is driven at about 1400 rpm and should be carefully centered during the 4-6 up-and-down movements of the tube, each of which takes 5 seconds on the average. The broken cell preparations are poured into a vessel containing 300 ml of bicarbonate medium, and the collected homogenate is diluted with similar medium to 500 ml, vigorously stirred for 2 minutes, and filtered twice through prewetted surgical gauze (18 threads/cm~-), first through one layer and then through a double layer. Low Speed Differential Centrffugations. Centrifugation is carried out in a cooled centrifuge capable of stable low speed. Accordingly, working quantities should be adapted to the volume capacity of the rotor.

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The filtered suspension is equally divided among the centrifuge tubes (e.g., 6 of 100 ml), and subjected to 1500 gl0 for 10 minutes. After reaching standstill, the fatty layer (mainly triglycerides) floating at the surface of the supernatant is sucked off with a pipette equipped with a suction bulb, while the tubes are left standing in the rotor so as not to disturb the floating layer. The supernatant is either decanted or sucked off by a pipette with water-pump aspiration, but, because of the loose packing of the sediment, care should be taken to leave a small layer of the supernatant above the undisturbed sediment. Any fatty material sticking to the wall of the tube is removed with the aid of filter paper. Bicarbonate medium, 5-10 ml, is added to each centrifuge tube, and the precipitate is suspended in toto by stirring with a glass rod. Each suspension is transferred, rinsing the tubes with bicarbonate medium, to a 75-ml smooth-walled tube equipped with a loosely fitting smooth Perspex pestle, and made up to a final volume of 35 ml with bicarbonate medium. Further suspension is carried out with three very gentle strokes of the pestle by hand. The resulting six suspensions are transferred to 35-ml centrifuge tubes (glass or other translucent material). The centrifuge is slowly accelerated by first spinning for 5 minutes at 100 g and then 10 minutes at 1000 g. The centrifuge is also decelerated slowly to avoid disturbance of the precipitate. The precipitated material consists of two easily distinguishable portions: an upper layer of loosely packed membranes, pale tan in appearance, covering a large more consistent bottom layer of dark-red nuclear material and other debris. The bulk of the supernatant is drawn off with a pipette connected with a water pump; the remainder of the supernatant is drawn off without such suction in order not to interfere with the precipitate. Bicarbonate medium, 3-5 ml, is gently layered over the precipitate. The fluffy upper layer is suspended with the aid of a glass rod bent and flattened at one end, while leaving the bottom layer intact. The suspended membranes of two tubes each are carefully transferred with a Pasteur pipette to the hand-driven homogenizer, made up to a final volume of 35 ml of bicarbonate medium and further suspended by three gentle strokes. The three suspensions are recentrifuged for 10 minutes at 1000 g. The supernatant, which still contains many mitochondria, is removed, leaving behind a sediment consisting of a fluffy layer of plasma membranes above a small amount of more tightly packed, mainly nuclear material that sticks to the bottom of the tube. As before, the fluffy layer is carefully suspended, further suspended in the hand-driven homogenizer, and centrifuged (now in 2 tubes) for 10 minutes at 1000 g. The washing procedure is repeated. After the last centrifugation, the supernatant is no longer 1°The centrifugal fields refer to the bottom of the tubes (fixed-angle rotor, 36.5°).

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turbid. The precipitate consists of plasma membranes, mitochondria, and some nuclei. This material is used in the flotation step. Flotation. After removal of the supernatant, the two precipitates are separately suspended and transferred using 3 ml of bicarbonate medium, to a 10-ml tube equipped with a loose Perspex pestle and calibrated at 3.6 ml. Bicarbonate medium is added to a final volume of 3.6 ml, and resuspension is carried out by three gentle strokes by hand. The two suspensions are transferred with a pipette to two translucent tubes fitting the SW 25-1 swinging-bucket rotor of a Beckman L ultracentrifuge. Ten milliliters of a sucrose solution of d 1.30 is added drop by drop to both tubes with vigorous shaking in order to prevent membrane agglutination (and subsequent contamination of the membranes). This brings the density of the solution to 1.22. This discontinuous sucrose gradient is built up by carefully adding (along the wall of the tube and using a Pasteur pipette with a tip bent at 90 °) 4.5 ml of a sucrose solution of d 1.20, 8 ml of d 1.18, and 4.5 ml of d 1.16. The two tubes containing the gradients and a third tube containing sucrose solution are tared, capped, and subjected to 70,000g for 90 minutes. Plasma membranes gather at the d 1.16/1.18 interface as a compact band. At the d 1.20/1.22 and d 1.18/1.20 interfaces, hazy bands of mitochondria are present. The latter interface also contains some plasma membranes, but these together with the few plasma membranes which are part of the bottom pellet, are discarded. The plasma membranes are harvested from the d 1.16/1.18 interface using a Pasteur pipette with a tip bent over 90 °. The collected membranes are resuspended in the large hand-driven homogenizer by 3 strokes, transferred, and centrifuged in 35 ml of bicarbonate medium for 10 minutes at 2500 g. Routinely two such washings are applied, but for particular experiments (e.g., hexose determination) up to 5 washings have to be carried out. The final membrane precipitate is suspended in bicarbonate medium (routinely) or in any other medium desired for future use. Comments 1. The method is suitable for the isolation of plasma membranes from rat and mouse ~,6 liver. For routine isolation 1 mM NaHCO.~ solution without CaC12 is used for homogenization and throughout all washing steps. 2. The membranes are gradually concentrated in the various washing cycles. For the last two washings and the flotation, the crude membrane fractions stemming from 30-40 g wet weight of rat liver are divided over two centrifuge tubes, instead of being collected in one. This has been found to improve yield and purity of the membranes, mitochondrial contamination being counteracted. Overloading of the gradient should be avoided, otherwise plasma membranes are trapped in layers of higher

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densities containing mitochondria, and some mitochondria may be carried over by the membranes to the position where the latter arrive. 3. To judge the progress of separation, the entire procedure can be followed by phase contrast microscopy. In the final membrane sediment obtained by low speed centrifugation mitochondria are still present. Only if too many mitochondria are present (to be judged from experience) is it advisable to repeat washing. A useful criterion is the turbidity of the supernatant. It has been found that if the wash supernatant is no longer turbid, the mitochondria still present in the membrane sediment can easily be removed in the following flotation step provided the sucrose gradient is composed as indicated. Also the small amounts of nuclei and debris found in the membrane sediment at this stage are easily removed in the flotation step. 4. If, for comparative reasons, liver plasma membranes should be isolated in the manner required for the plasma membranes of certain hepatomas (next section), 2 mM CaCI., is added to the homogenization medium. Dilution of the homogenate and washings are, however, carried out with the unsupplemented bicarbonate medium. The number of washings should be increased (usually with another two washings) since the calcium ions promote the adherence of mitochondria to the plasma membranes. Under these conditions the first washings remove fewer mitochondria than do later ones, as shown by the turbidity of the supernatant and phase contrast microscopy of both supernatant and membranes. The use of calcium ions does not infrequently lead to the presence of some mitochondria in the final preparations (without CaCI~ none are present), and introduces some cytoplasmic RNA in the membranes.

Plasma Membranes from Hepatomas Mouse Hepatomas. Conditions for isolation of plasma membranes from slowly growing and well-differentiated hepatomas ~,6 are the same as those described for rat liver. Since a small part of the hepatoma plasma membranes may show a lower buoyant density, 3 ml of a sucrose layer of d 1.14 may be added on top of the gradient, at the expense of an equivalent part of all other layers, if this material gathering now at the d 1.14/d 1.16 interface, is wanted separately. Rat Hepatomas. Basically, the same procedure is followed except for the following changes. From 4-5 animals 50-60 g of wet weight of tumor tissue is collected in a beaker containing 100 ml of bicarbonate medium fortified with 2 mM CaC12. After swirling the beaker, the tissue is allowed to settle, and most of the fluid is decanted. The tissue is finely cut and homogenized in 5-g portions each with 20 ml of bidistilled water containing 1 mM sodium bicarbonate and 2 mM CaC12. Homogenization is carried

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out very gently, by moving the homogenization tube very slowly up and down, each stroke taking about 8-10 seconds. The collected homogenate is diluted with bicarbonate medium (without CaCI~ in this and the following steps) to 750 ml and stirred for about 2 minutes. For the first centrifugation of the homogenate in translucent tubes, the centrifuge is set at 100 g for 5 minutes, then slowly accelerated and kept for 10 minutes at 2500 g. Much more floating lipid material accumulates at the surface than in the case of rat liver; this and any floating lipid arising later in the procedure is removed as indicated. After the supernatant has been sucked off, the upper fluffy layer of the sediment is harvested as described for liver. Separation between the fluffy membranes and bottom layer is less distinct than in the case of liver because (a) the two layers differ less in color, both appearing slightly tan, and (b) the upper layer sticks somewhat to the bottom one. This first separation should be considered as approximate, visibly carrying over also some of the nuclear material. After resuspension by homogenization by hand, another centrifugation is carried out as before. Separation between membranes and small nuclear sediment is now more pronounced. The membranes, in two portions, are subjected to 2-3 washhag cycles with bicarbonate medium during which centrifugation is carried out at 1500 g for 10 minutes. Supernatant containing mitochondria and the sticky bottom pellet are discarded each time. The number of washings is decided by the same criteria as applied to liver. The two membrane preparations are transferred to 2 tubes of the swinging-bucket rotor, as described for liver. Ten milliliters of sucrose solution of d 1.30 is added, followed by 3 ml of d 1.20, 4 ml of d 1.18, 4 ml of d 1.16, 3 ml of d 1.14, and finally 3 ml of d 1.10. Centrifugation is carried out as described. Most of the hepatoma membranes gather at the d 1.14/1.16 interface, and a few at the d 1.10/1.14 interface. At d 1.16/1.18 some membranes heavily contaminated with mitochondria are present; these are discarded. The membranes are collected and washed three times with bicarbonate medium; they are somewhat paler than the liver plasma membranes. Comments

1. The differential centrifugations are carried out at higher speed than in the case of liver. The lower speed used for the latter is not fast enough to pack the majority of the tumor plasma membranes. 2. The two-step manner of the first and second centrifugation promotes stratification of the sediments, the nuclear material sedimenting faster so that its trapping of plasma membranes is counteracted. This is especially important for the process of hepatoma membrane separation because of the tendency of the nuclear material to gel formation. 3. For the same reason the initial separation of the plasma membranes

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from the bulk of the nuclear material is performed already after the first centrifugation, so that during the subsequent resuspension gel formation and mutual exposure is reduced to a minimum. 4. The sucrose layer of d 1.10 is introduced to facilitate collection of the plasma membranes from the gradient. If the d 1.14 layer is the top one, part of the membranes is diffusely floating in this layer and hard to collect. With the d 1.10 layer present, these membranes are packed at the d 1.10/ 1.14 interface. Note that not all hepatomas should necessarily show these differences in buoyant densities of their plasma membranes. Properties of Isolated Plasma Membranes Rat-liver plasma membranes will be principally dealt with. In general, the mouse-liver plasma membrane¢ ,G,9 resemble those of the rat; salient differences will be indicated. The properties of hepatoma plasma membranes are markedly tumor-strain specific, and common characteristics distinguishing these membranes from normal are very scarce. 4-9,11-14 The presentation is limited to findings made in the authors' institute. Yields. The isolation procedure aims not at quantity nor at rapidity, but at purity. Homogenization of the tissue is not complete in order not to fragment the membrane sheets too much, membranes stick to and are present in the nuclear pellets, membrane fragments may remain in the wash supernatant, and some 10% of the final membranes are lost in the flotation step. The average yields of plasma membranes in milligrams of protein (biuret) per l0 g, fresh weight, of tissue are 3.5 for rat liver and 6.4 for mouse liver homogenized in bicarbonate medium, 1.8 for rat liver homogenized in the presence of 2 mM CaCl_~, and 1.2 for rat hepatomas and about 3.0 for mouse hepatomas. The higher yield of the mouse liver membranes is due to the softness of this tissue which allows more complete homogenization. Lower yield obtained following homogenization in the presence of Ca -~÷,is due to an increased number of washings, and in the case of the rat hepatoma also by gel formation of the nuclear material trapping plasma membranes. A rough estimate, based on work of Weibel et al. 1~ shows the yield of rat-liver plasma membranes (minus the protein that is saline soluble, compare below) to be some 15 % of the theoretical value. Apart from the aforementioned factors contributing to this restricted yield, the question arises 1~p. 1"~P. 13p. 14R. I~E.

Emmelot and C. J. Bos, Biochim. Biophys. Acta 211, 169 (1970). Emmelot and C. J. Bos, Biochim. Biophys. Acta 249, 285 (1971). Emmelot and C. J. Bos, J. Membrane Biol. 9, 83 (1972). P. van Hoeven and P. Emmelot, I. Membrane Biol. 9, 105 (1972). R. Weibel, W. Sttiubli, H. R. Gn~igi, and F. A. Hess, J. Cell Biol. 42, 68 (1969).

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as to the extent to which the isolated liver plasma membranes represent the liver cell surface in situ. Loosely bound surface materials, such as part of the glycocalyx, could be lost during preparation. Furthermore, the cell surface is composed of heterogeneous elements, for example, membranes lining Disse and bile canalicular spaces, apposed membranes, and structurally differentiated intercellular contacts and contact zones. Disruption by homogenization might create small fragments more easily from the first mentioned than from the other membrane areas, since the former are of relatively long extension and lack structural supports. Such small fragments (vesicles) could behave during the differential centrifugations as mitochondria and microsomes, and subsequently be lost. Plasma membranes have been isolated from the liver microsomal fraction (see Table I). Buoyant Densities. Liver plasma membranes gather at the d 1.16/d 1.18 interface of the sucrose gradient. If another layer of d 1.17 is included in the gradient, the membranes are approximately equally divided over the two interfaces d 1.16/d 1.17/d 1.18. Thus 1.17 can be considered as the average buoyant density of these membranes. The buoyant density is a relative value dependent on the composition of the medium. The average buoyant density of rat-liver plasma membranes equilibrated in Ficoll, Urografin-16 and glycerol-water gradients amounts to 1.08, 1.14, and 1.21, respectively, and in corresponding gradients containing 2H.,O instead of H20 to 1.14, 1.19, and 1.22, respectively (and 1.21 for a sucrose-'-'H_~O gradient). The specific density of the waterless membrane material, calculated from the distance traveled by a drop of membrane-detergent (sodium deoxycholate, 1%, or dodecyl sulfate, 0.8%) solution against that of a drop of detergent solution in a continuous kerosene-bromobenzene density gradient, 17 amounts to 1.33 for fresh rat-liver plasma membranes, and to 1.29 for the saline-insoluble membrane portion. Purity of Isolated Plasma Membranes. Rat-liver plasma membranes, isolated from plain bicarbonate homogenates, are free from any mitochondrial contamination as shown by electron microscopy3,9 and the absence of mitochondrial enzymes'~ and cardiolipin. ~ Some ( 5 - 1 0 % ) mierosomal contamination is present on the basis of glucose-6-phosphatase measurements, but the plasma membrane activity could very well stem from an aspecific phosphatase activity.11 No smooth or rough microsomal membranes are present in the preparations ~.9.1'~ and certain drug-metabolizing N,N'-Diacetyl-3,5-diamino-2,4,6-triiodobenzoate,product of Schering A. G., Berlin, Germany. 17W. S. Bont, P. Emmelot, and H. Vaz Dias, Biochim. Biophys. ,4cta 173, 389 (1969). ~8p. Emmelot and E. L. Benedetti, in "Protides of the Biological Fluids" (H. Peeters, ed.), Vol. 15, p. 315. Elsevier, Amsterdam, 1968.

is

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enzymes 18 and cytochromes 13 which are characteristic for liver microsomes (see under Enzymatic Composition below) are absent. Some 25% of the membrane protein is soluble in physiological saline (0.15 M NaC1), and this protein contains several enzymes and antigens of the liver cell cytoplasm? -9,~3,18 The saline-soluble protein originates mainly, if not exclusively, from the soluble fraction of the homogenate. These proteins which are predominantly positively charged, interact with negatively charged plasma membranes proper under the hypotonic conditions prevailing throughout the isolation procedure. Associated with the saline-soluble protein is hemoglobin derived from lysed erythrocytes. This gives the isolated plasma membranes their faint reddish color. The saline-insoluble membrane portion, which represents the clean plasma membranes, is colorless. Prior perfusion of the liver, e.g., with physiological saline, yields uncolored membranes. Morphology (as Studied by Electron Microscopy). GENERAL APPEARANCE. The preparations consist of large sheets of membranes interconnected by the various types of junctional complexes found in liver sections, i.e., the gap junction (nexus, formerly called tight junction), desmosome (macula adhaerens), intermediate junction (zonula adhaerens), next to many bile spacelike structures and some vesicles. The preparations are free from any other recognizable organelles. Appropriate illustrations can be found in various publications? ,5,r,9,~8 These give the strong impression that of the liver plasma-membrane skeleton most is preserved, except perhaps some of the blood front lining. FINE STRUCTURES AND LOCAL SPECIALIZATION OF THE PLASMA MEM-

The isolated membranes reveaP the "classical" triple-layered membrane element of an overall width of about 80 A. Bile spacelike elements exhibit 9,1s an average membrane width of 95-100 A. As shown by colloidal iron hydroxide staining, sialic acid is located in a rather regular spacing at the extracellular side of the membrane, and is absent from the inner side of the membrane leaflet? 9 Sialic acid in desmosomes and intermediary junctions is occluded by some Ca~-+-dependent mechanism, since it can be unmasked and demonstrated at the internal plates of these junctions after treatment of the membranes with EDTA. By these criteria, gap junctions lack sialic acid. In negatively stained membrane preparations, globular knobs (50-60 A diameter) are seen projecting from the membrane surface of certain membrane sheets or areas. 9,'8 These knobs are released by exposing the isolated membranes to papain, but not to trypsin. The isolated knobs exhibit aU the BRANES.

19E. L. Benedetti and P. Emmelot, I. Cell Sci. 2, 499 (1967).

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Co~+-activated aminopeptidase activities displayed by fresh membranes, z°,21 With leucyl-/3-naphthylamide as substrate, histochemically demonstrable amino-peptidase activity is restricted to the plasma membranes lining the bile spaces. This activity can be considered as a marker enzyme for the globular knobs which coat the bile space-lining plasma membranes. In negative stain a hexagonal subunit (90 A) pattern of restricted location is outlined. 6,9,2~ This lattice is demonstrated by the gap junctions. ~3,-~4These junctions survive sodium deoxycholate ( 1 % ) treatment of the isolated membranes and are thus obtainable by centrifugation, but in an as yet impure form. 23,~' Only undifferentiated rapidly growing hepatomas may lack both the globular knobs (although not the enzymes contained therein) and the gap junctions, e Structural continuity between a few ribosome-dotted microsomal vesicles and the isolated plasma membranes has been observed for the various hepatomas examined, but not for liver. 6 Chemical Composition (Table II). The main components of hepatic plasma membranes are proteins and lipids, amounting to 66 and 30.5%, respectively, of the dry membrane weight in the case of rat liver. 9,13,14 Hepatic plasma membranes are characterized by a high content of cholesterol and protein-bound sialic acid, 9,19 and a particular phospholipid class profile in which the high contents of sphingomyelin and phosphatidylserine are noteworthy. 1~ These components may be considered as chemical "markers" which distinguish the liver plasma membranes from intracellular membrane species, except lysosomal membranes. 23 The chemical composition may help to provide criteria for judging the purity of liver plasma membranes preparations (e.g., the absence of cardiolipin). The various types of hepatoma plasma membranes contained significantly more cholesterol per micromole of phospholipid than did the liver plasma membranes, a4 The former's RNA content was also increased due to ribosomal RNA of the few rough microsomal vesicles structurally connected with the hepatoma plasma membranes. ~3 Liver plasma membranes contain about 1% RNA on a protein basis. The presence of calcium ions during homogenization increases this amount by 50%, and this addi~°P. Emmelot, A. Visser, and E. L. Benedetti, Biochim. Biophys. A c t a 150, 364 (1968). 2~p. Emmelot and A. Visser, Biochim. Biophys. Acta 241, 273 (1971). ~E. L. Benedetti and P. Emmelot, 1. Cell Biol. 26, 299 (1965). hE. L. Benedetti and P. Emmelot, J. Cell Biol. 38, 15 (1968). up . Emmelot, C. A. Feltkamp, and H. Vaz Dias, Biochim. Biophys. Acta 211, 43 (1970). Secondary lysosomes are partly derived from the plasma membranes.

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TABLE II CHEMICAL COMPOSITION OF RAT LIVER PLASMA MEMBRANES

Protein Percent of dry weight (total membranes) Percent of dry weight a (saline-insoluble membranes) Soluble in 0.15 M NaC1 (% of total protein) Total protein per gmole phospholipid-P (mg) Lipids Percent of dry weight (total membranes) Total lipids per mg protein (nag) Cholesterol/phospholipid-P (molar ratio) Cholesteryl esters (mole-% of total cholesterol) Free fatty acids (ttmoles per mg protein) Triglycerides (ttmo]es per mg protein) Phospholipids (% of total lipids) Phospholipid composition (% of lipid-P) Sphingomyelinb Phosphatidylcholine Phosphatidylethanolamine~ Phosphatidylserine Phosphatidylinositol Phosphatidic acid Lysophosphatidylcholine Lysophosphatidylserine Cardiolipin Nucleic acids Percent of dry weight (total membranes) RNA (ttg per mg protein) DNA (ttg per mg protein)

66 + 2 (61-69) 58 25 _+ 4 (18-33) 2.83 + 0.12 30.5 _ 0.7 0.46 _+ 0.Ol 0.65 + 0.06 1-1.5 0.O88._+ 0.039 0.035 + 0.002 59.9 + 3.9 23.2_+2.1 30.0_+2.0 19.3_+1.3 15.2±0.8 6.0+0.5 2.3+1.1 3.5+1.1 0.5(0-1.6) 0.0 0.6 8.1_+0.4 1.4_+0.1

Bound carbohydrates Percent of dry weight (total membranes) Sialic acidsd (nmoles per mg protein) Percentage neuraminidase-resistant sialic acid Hexoses (nmoles glucose per mg protein) Hexosamines (nmoles per mg protein)

2.2 33 69 65 61

+2 +3 +_2 _+4

a Dry weight corrected for saline-soluble protein. b Containing some 1% lysophosphatidylethanolamine. c 17.5% as plazmalogen and 82.5% as diacyl. d Mainly N-acetylneuraminic acid; at least 95% of the sialic acid is protein bound. tional R N A ( v e r y p r o b a b l y transfer R N A ) is fully r e m o v e d b y 0.15 M NaCI. D N A is p r e s e n t in trace a m o u n t s ( r a t liver) o r b e l o w the level of detection ( m o u s e l i v e r ) . Its increased presence in h e p a t o m a m e m b r a n e s is due to a d d i t i o n a l c o n t a m i n a t i o n . 13

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TABLE III SPECIFIC ENZYME ACTIVITIES OF ]ISOLATED RAT-LIVER PLASMA MEMBRANES

Enzyme Mg 2+ (or Ca2+)-ATPase *a (Na+-K+)ATPase* 5'-Mononucleotidase* Glycerolphosphatase Alkaline* 9 Acid p-Nitrophenylphosphatase Alkaline* O Acid*

C

K+-nitrophenylphosphatase* Alkaline 9 Acetylphosphatase* K+-acetylphosphatase* ADPase*, Mg ~+ Ca2 ÷

IDPase*, Mg 2+ NAD pyrophosphatase* Inorganic pyrophosphatase*, b (Pi) Co ~+-Aminopeptidase(s)* Leucinamide Triglycine Leucylglycylglycine Leucylglycine Leucyl-B-naphthylamide Adenylcyclase* (nmoles cyclic AMP) Basal NaF Glucagon Epinephrine Lipase ( ~ triolein) Phosphodiesterase (bis- (p-nitrophenyl)phosphate) Alkaline* Acidc Ribonuclease (A~ ~m) Alkaline~ Acid* Glucose-6-phosphataseY PPi-glucose transferase

gMoles of substrate converted or product formed/mg protein/hour 46.4±7.8 11.7±2.3 51.0±6.7 3.8±0.4 1.0±0.2 0.4±0.1 3.31±0.2 1.97±0.04 9.1±1.3 7.3±0.33 1.6 1.6 11.4 10.8 20.3 42.2 30.1 5.72 4.0

±0.3 ±0.5 ±2.2 ±1.9 ±1.2 +_.3.9 ±3.2 ±0.2 ±0.3

6.5 8.4 11.3 1.4 3.9

±0.7 ±0.9 ±1.2 ± 0.16 ±0.2

2.3 25.3 44.2 11.1 267

±1.0 ±4.2 ±8.3 ±2.8

3.6 i 0 . 4 0.7 ± 0 . 1 2.2 _ 0.50 0.54 ± 0.24

1.0 ± 0 . 3 0.18 ± 0.01

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TABLE I I I (Continued)

Enzyme Esterase, nonspecific a-Naphthyl laurate q-Naphthyl capryla~e p-Nitrophenylacetate Triose-3-P dehydrogenaseg Aspartate aminotransferaseh NADPH-cytochrome c reductase NAD nucleosidase

aMoles of substrate converted or product formed/rag protein/hour

0.46 -+ 0.02 16.1 _+ 2.3 2.5 + 0.5 2.04 -+ 0.3 128 -+ 15 7.68 _+ 0.5 0.5_+0.1

" Enzymes marked with an asterisk have been shown, or can reasonably be considered to be intrinsic, though not necessarily exclusive, components of plasma membranes. Distinct from the microsomal enzyme/ c This enzyme appears to be a genuine component of rat hepatoma membranes/; some 30% of the liver membrane activity is released by 0.15 M NaCI. d Very probably a genuine plasma-membrane enzyme,k • Half of the activity is soluble in 0.15 M NaC1. ~ s Not due to microsomal contamination in the case of rat-hepatoma plasma membranes.~ o Completely removable by 0.15 M NaC1.m,~ h Nearly completely removable by 0.15 M NaC1, specific activity of saline-insoluble membrane fractions amounting to 4.2 __ 0.6 +tmoles." i p. Emmelot and C. J. Bos, Biochim. Biophys. Acta 211, 169 (1970). i p. Emmelot and C. J. Bos, Int. J. Cancer 4, 705 (1969). k K. A. Norris, M. L. E. Burge, and R. H. Hinton, Biochem. J. 122, 53 1) (1971). z p. Emmelot and E. L. Benedetti, in "Protides of the Biological Fluids" (H. Peeters, ed.), Vol. 15, p. 315. Elsevier, Amsterdam, 1968. " P. Emmelot and C. J. Bos, Biochim. Biophys. Acta 121, 434 (1966). "P. Emmelot and C. J. Bos, J. Membrane Biol. 9, 83 (1972).

Enzymatic Composition (see Table 11I, and references cited in footnotes 2-9, 11-13, 18, 20, 21, 2 6 - 2 9 ) . T h e enzymes m a r k e d with a n asterisk in T a b l e I I I either have b e e n shown, or m a y r e a s o n a b l y be considered, to be intrinsic, though n o t necessarily exclusive, c o m p o n e n t s of the p l a s m a m e m b r a n e s . Some of the enzymes m a y p r o v i s i o n a l l y - - u n t i l m o r e is k n o w n of possible i s o z y m e s - - b e used as m a r k e r enzymes for hepatic plasma m e m b r a n e s , e.g., 5'-nucleotidase (at least in the case of rat liver; the specific activity of the e n z y m e in mouse-liver p l a s m a mereP. P. ~P. ~P.

Emmelot and C. J. Bos, Biochim. Biophys. Acta 121, 375 (1966). Emmelot and C. J. Bos, Biochim. Biophys. Acta 120, 369 (1966). Emmelot and C. J. Bos, Biochim. Biophys. Acta 150, 341 (1968). Emmelot and C. J. Bos, Biochim. Biophys. Acta 249, 293 (1971).

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branes is one-fourth that of the rat), Co2÷-activated aminopeptidase(s), fluoride- and glucagon-stimulated adenylcyclase, NAD-pyrophosphatase. Enzymes which are also present in the isolated plasma membranes, but whose authenticity is not settled or may stem from (microsomal) contamination, are also included in Table III. Enzymes below the level of detection of the analytic methods used are: hexokinase and glucokinase (hexokinase is present in and a genuine component of rat-hepatoma plasma membranes), 3'-mononuclbotidase, monoamine oxidase, succinate-cytochrome c reductase system, cytochrome c oxidase, phosphoprotein phosphatase, various proteolytic enzymes, microsomal NADPH-oxidase and N-demethylase (substrate: dimethylaminoantipyrine). Cytochromes of liver, including cytochrome c and the microsomal P450 and b~, could not be detected. Organ-Specific or Differentiation Antigens Present in Rat-Liver Plasma Membranes. By using a heterologous serum (rabbit) directed against isolated rat-liver plasma membranes freed from their saline-soluble protein, and suitable absorption, three classes of insoluble liver-specific components, protein in nature, have been detected in the liver cell surface and in isolated plasma membranes. 3° One of these is located in the bile space-lining membranes and found to reside in the 50-60 A globular knobs coating these membranes, thus providing independent evidence for the location of these knobs. Another is present at other regions of the plasma membrane skeleton. The third type of liver-specific component is normally masked, but can be uncovered by very mild trypsin digestion) I S°K. A. Norris, M. L. E. Burge, and R. H. Hinton, Biochem. 1. 122, 53 P (1971). 3~j. B. Sheffield and P. Emmelot, Exp. Cell Res. 71, 97 (1972).

[6] I s o l a t i o n of R a t L i v e r P l a s m a M e m b r a n e F r a g m e n t s in I s o t o n i c S u c r o s e

By NATHAN N. ARONSON, JR. and OSCAR TOUSTER During the differential centrifugation of liver homogenates, certain enzymes are noted to exhibit a bimodal distribution between the nuclear and microsomal fractions. It has been shown that many such enzymes are in actuality components of the plasma membrane. 1,2 Indeed, a good first assumption to be made is that an enzyme is localized on the plasma membrane of rat liver ceils if it exhibits such a nuclear-microsomal bimodal 1 O. Touster, N. N. Aronson, Jr., J. T. Dulaney, and H. Hendrickson, 1. Cell Biol. 47, 604 (1970). ~C. de Duve, 1. Cell Biol. 50, 20D (1971).