A PRACTICAL MODEL OF THE ANIMAL CELL MEMBRANES’ HARRY N. HOLMES Sevsrance Chemical Laboratory, Obctlin College, Oberlin, Ohio Received Augwt 7 , 1080
Animal cell walls aa living membranes permit the passage of watersoluble or fat-soluble substances to nourish the cell. This perplexing, yet essential, behavior has been explained by a number of theories, but no Satisfactory working models have been offered to the biologist. Osterhout (4) in a condensed statement wrote, “It had been suggested by Quincke that the outer layer of the cell consists of a film of oil: Overton came to a similar conclusion, substituting for ‘oil’ the term lipoid’, which includes such substances as lecithin and cholesterin.” Kahlenberg (2) separated water-soluble substances from one another by dialysis through membranes of thin silk saturated with lanolin and through parchment bags impregnated Wth lanolin. He thought that the selective properties were due to the cholesterol in the lanolin. In his own words, “It would seem likely that sterols give living cells their wonderful selective osmotic properties.” Clowes (1) suggested that living membranes resembled emulsions so near the balance between oil-in-water and water-in-oil types as to be affected differently in respect to permeability by monovalent and divalent cations. Single permeability is well known with membranes of collodion formed by evaporation of the ether and removal of alcohol by washing with water or with toluene. The first is used in dialysis of water solutions, while the second is useful only in dialysis of toluene solutions (or “oils”), but neither membrane shows the double permeability of living animal cell membranes. It seemed that if some substance such aa lecithin, oleic acid, or rosin could be incorporated in common collodion solution, the resulting membranes might offer continuous, although irregular, paths of two types of material for migration of both water-soluble and fat-soluble substances. Lecithin dissolved readily in common collodion solution, but a 1 per cent solution was selected from a number of concentrations tried. After allowing the ether component to evaporate and washing out the alcohol Presented at the Sixteenth Colloid Symposium, held at Stanford University, California, July 6-8, 1939. 1161
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with water, the resulting bag (almost transparent) was filled with water containing about 1 per cent sodium chloride and was hung in a vessel of water containing a slight amount of silver nitrate as indicator. In 4 sec. an initial opalescence or cloudiness appeared in the outer solution. The membrane was therefore permeable to water solutions. Another collodion-lecithin bag filled with toluene (to represent lipid solvents) which had been dyed red with Sudan I11 was hung in a vessel of colorless toluene. I n 1.5 min. the red solution appeared in the outer liquid. Evidently the same membrane was permeable to water solutions and to toluene solutions. A 15 per cent solution of a fat (cottonseed oil) in toluene was dialyzed against pure toluene while samples of the dialysate were frequently withdrawn for fat spot tests on paper. I n 15 min. appreciable amounts of fat had gone through the collodion-lecithin membrane. Next, a similar bag containing an emulsion of fat (dyed red) in water, with sodium oleate as emulsifying agent, was hung in a beaker containing a layer of toluene resting on a layer of water. Within 3 sec. some sodium chloride in the water of the emulsion passed through the membrane and formed faint opalescence with a trace of silver nitrate in the outer water layer. At the same time red drops of fat passed through and dissolved in the colorless toluene layer. .Incredible as it seems, red drops also formed on the areas of the membrane immersed in water. These drops, seen after 5 sec., often coalesced and rose into the upper toluene layer. Evidently the emulsion film of sodium oleate broke on contact with the collodion-lecithin membrane. With gelatin as emulsifying agent no oil passed through. This emulsion procedure described above was reversed by using a water-in-oil emulsion prepared by dispersing water in a solution of gum dammar in toluene. The water was dyed with fluorescein and the toluene with Sudan 111. When a sac filled with this emulsion was hung in toluene, drops of colored water appeared on the outside of the sac. Other substances were used successfully in place of lecithin. Oleic acid, stearic acid, rosin, cholesterol, and a 5 : l mixture of lecithin and cholesterol (to simulate their natural occurrence in animal cells) gave collodion membranes double permeability, although there was some variation in the speed of diffusion. As a rule 1 per cent solutions were most satisfactory, although a 1.5 per cent solution of lecithin was used to advantage. It is noteworthy that soaking a collodion-oleic acid membrane in toluene for 18 hr. to replace the oleic acid with toluene decreases the penneability to water considerably, although permeability to toluene remains high.
MODEL OF ANIMAL CELL M E M B R A N E S
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Pervaporation (3) through an ordinary collodion sac containing 78 g. of water, heated only by summer sunshine, showed a loss of 15 per cent in 2 hr. A membrane prepared from collodion, containing 1 per cent of lecithin, permitted a 9 per cent loss of water. Pervaporation of toluene through a collodion membrane, prepared by washing out the alcohol with toluene, permitted a 35 per cent loss of toluene in 2 hr., while the usual collodion-lecithin membrane permitted a toluene loss of 28 per cent. Variations in intensity of sunshine doubtless caused some error in these results but do not change the fact of remarkable double permeability. Pervaporation can be used as a simple test for membrane quality. SUMMARY
1. A working model of animal cell membranes of double permeability can be prepared quickly and simply. 2. A membrane permeable to water-soluble and also to fat (or lipoid)soluble substances is made by dissolving lecithin (1 per cent) in common collodion solutions of cellulose nitrate, evaporating the ether as the fluid is swirled in a beaker or flask, and washing out the alcohol with water. 3. The addition to collodion solutions of 1 per cent of oleic acid, stearic acid, cholesterol, a 5 : l mixture of lecithin and cholesterol, rosin, and similar substances also permits formation of good membranes of double permeability. 4. When a collodion-lecithin sac containing an emulsion of toluene (dyed red with Sudan 111) in water containing sodium oleate as emulsifying agent is hung in a beaker of water, red drops of toluene form on the outside of the sac. 5. Collodion-lecithin sacs filled with water and hung in warm sunshine lose water rapidly by pervaporation. Similar sacs filled with toluene lose toluene rapidly, showing double permeability. 6. Collodion-lecithin membranes are more slowly permeable to solutions of fats in toluene.
Credit is due W. Alexander, J. Gofman, and D. Adams for laboratory assistance in this research. REFERENCES (1) CLOWES,G . H . A.: J. Phys. Chem. 29, 407 (1916).
(2) KAHLENBERG, L.: Phil. Mag. 1, 385 (1926). (3) KOBER,P. A , : J. Am. Chem. SOC.39, 944 (1917). (4) OSTERHOUT, W. J. V. : Some Fundamental Properties of Cellular Physiology. Yale University Press, New Haven (1927).
