THE PERMEABILITY OF NATURAL MEMBRANES b HUGH SAvSor and J. F. DANIELLICHAPTER VIIL PERMBABILITY TO NON-ELECTROLY Tp By J. F. Danrentr By non-electrolytes we refer to substances which, over th physiological pH range, exist in a non-ionic form only and Which also penetrate in an unionised form. Other substances (west acids and weak bass) penetrate in most cases mainly in the unonet form, but they present several special problems which maja’ preferable to deal with them separately. Within the group of non-lectrolytes a wide diversity op chemical and physical characteristics is still posible, Contdet the molecular series hexane, propyl alcohol, urea and glyecsn Hexane has an olive oil:water partition coefficient of about Soy" whereas that of glycerol is 0-00007. Propyl alcohol and urea bave intermediate values of about 0-1 and 0-00015, Thus there is = difference of ten million-fold between the relative “affinities” ot Hexane —Propyl alcohol ‘Urea Glycerol CH, cH, NE, CH,OH CH, du, db HOH du, dion Nu, a, du, 1H, H,OH hexane and glycerol for water and oil. It is not surprising that such enormous differences between molecules should lead to great differences in permeability, provided the membrane to be pene- trated can distinguish between molecules by virtue of these specific characteristics. Thus in water the dominant variable affecting the rate of diffusion on passing from one of the molecules to another is the molecular weight, so that the relative rates of Penetration of a water layer are as 1-0 ; 0:89 : 0-83 : 1-025 ie. there is very little difference between the rates. In diffusing through e.g, a collodion membrane between two water layers, the = __ — Escaneado con CamScannerpRMBABILITY TO NONELROTROLY TRS 81 r ating factor 18 molecular diameter, and again there mini little difference between these four molecules, beth two fifferentcollodion membranes the following jita une Neabilities for the last three of these substances: i .0, 1:0, 0-8; membrane (2) 1-0, 0-97, 0:21, On ind, with cell membranes the ratios would be, roughly, the other ea 1-0 : propyl alcohol 1-0 : urea 0-006 : elyeeral ext Bye discrimination now is between molecules of different }0005." seacter, the least polar penetrating most rapidly, the polar charac rapidly. Bearing in mind these very large oe which may exist between different non-clectrolyte we may now proceed to consider the permeability of different types of membranes. "rhe Permeability of the Exythrocyte. The earliest systematic studies of the permeability of erythrocytes to non-clectrolytes are those SrGriyns (1896) and of Hedin (1897), and it will be seen that, Sidhough their work was done so long ago, at a time when even the conception of the erythrocyte as a cell had first to be argued, their results are largely upheld by later work; in fact, it is worth pointing out that the work of Hedin was carried out under more {trictly physiological conditions than most of the later investiga- tions on non-electrolyte permeability. Quantitative work on the erythrocyte is comparatively recent, and is almost entirely based on the haemolysis technique. The quantitative analysis of times of haemolysis is almost entirely due to Jacobs and his colleagues. For many points we are still de- pendent of qualitative evidence. Griyns first showed, by chemical methods, that urea added to chicken blood is equally distributed between cells and serum. He then, using the haemolysis method, showed that the following non-electrolytes did not penetrate the cell at all: amino acids, slucose, mannose, inositol, disaccharides. The following substances were shown to penetrate the cell: MeOH, EtOH, glycerol, ethers, urea, biuret, pyridine. * Griyns was mainly interested in whether the substances pene: trated the erythrocyte or not, and paid little attention to the actual rates at which this process occurred. However, he was able to draw the important conclusion that “substances with the same * There are inthis serigg? °F COUR» Many cases where specific factors cause displacement bp 6 Escaneado con CamScanner82 PERMEABILITY TO NON-ELECTROLY py chemical groupings behave similarly”, which with modifi relating to the size of the molecules, and the distribu groups in them, may be upheld to-day. Hedin, using the method of freezing-point depressig blood, obtained the results summarised in Table VII. He ‘ti his results in relation to Overton’s theory of lipoid Solubility, the more rapidly penetrating substances being the more lipoia sola hie substances, with the exception of urea and ethylene glycol, a enunciates the rule that with polyhydric alcohols the rate : penetration decreases with the number of OH BrOUpS in the molecule. Thus already the principle of the importance of cae chemical groupingsin regard to permeability had been broughtor® Cation ON Of the My and og TABLE VII. Permeanturry oF ox eryrHRocyres (Henin, 1897) Dextrose Galactose Sita Cane sugar } Do not penetrate ‘Mannitol Adonitol Ethylene glycol) Penetrate in the order given, times for completion of the Glycerol } process being a few minutes for ethylene glycol, less thes Erythritol hours for glycerol and greater than 28 hoursfor erythritol Amides Urea Aldehydes Penetrate too rapidly for rates to be compared Ketones Ethers Penetrate only slightly within 24 hours Besides developing the above principle, the results of Kozawa (1914), using a haemolysis technique, led to the conception of real differences in permeability according to the species of erythrocyte studied. Thus with human erythrocytes he found the results shown in Table VIII. The erythrocytes of the ape (Macacus rhesus) behaved in the same way as human erythrocytes. It was TABLE VIII, PERMEABILITY OF HUMAN ERYTHROGYTES (Kozawa, 1914) Permeable to: Arabinose, xylose, galactose, mannose, sorbose, ' glucose, faevulose Slightly permeable 10: Adonitol Impermeable to; Cane sugar, maltose, lactose, glucoheptosts e, methylglucoside, galactoside, glycine, alanin mannitol, dulcitol “4 Escaneado con CamScanner- PERMEABILITY TO NON-ELEGTROLYTES 83 d, however, that the erythrocytes of the ox, pig, rabbit, founds ie, goat, horse, camel and cat were all impermeable to ances considered; dog cells were shown to be permeable the Mycoee ‘The important point was also made that isomeric wea ate at different rates, and has since been confirmed 38). 0 Times OF HAEMOLYSIS IN ETHYLENE GLYCOL AND UTIONS FOR DIFFERENT sreciss (JAcons, 1931) ‘Time in seconds for 76% haemolysis eee LE IX: TAR anol #0 » (2) () =O) B=) species InNaGl_ In glycol In glycerol (1) TH aa 4-20 66 19 05 35 3:00 39 19 12-9 3-00 113 80 22 218 5-00 157 196 21 38-2 8:35 126 43 08 51 6:10 286 1548 37 253-0 265 18:3 1222 59 459-0 3:00 16-7 1024 46 340.0 380 36-1 2325 83 612-0 Sheep 1-90 241 1623, naz 850-0 Column (1) gives time of haemolysis in 002 M NaCl; column (2) that in ethylene Gheol4 002M NaCl; column (8) that in glycerol +0-02-M NaCl TABLE X. (Utrrcu, 1934) Species Substance Penetration Ox, pig, rabbit, rat Hexoses mannitol Not at all Mouse Hexoses Not at all Mouse Sorbitol, mannitol, dulcitol Rapid Guinea-pig Pentoses, glycine, alanine Penetrate Since Kozawa’s work appeared, a great many investigators have made more detailed studies of the variation in permeability with species. In Table IX some results of Jacobs on the relative rates of penetration of ethylene glycol and glycerol into the cells of ten species are shown. The results are given in times of hae- molysis, which are, very roughly, inversely proportional to permeability, The polyhydric alcohols and sugars have been studied from a species point of view by Ulrich (1934), whose results are shown in Table X. For the polyhydric alcohol, erythritol, the times in Table XI were found for 75 % haemolysis. * * It will be seen later, when Davson’s (1939) results on loss of potasslum in PiReletrolyte solutions are discussed, that times of haemolysis of more than ‘minutes are sometimes of doubtful value. 6-2 Escaneado con CamScannerILITY TO NON-ELEGTROLYTES st pRRMBAB TABLE XL. Haswonysts tn EBRYTHRITOL SOLUTIONS Species ‘Time for 75% haemolysis Ox 8 hours: Rat 6 hours R 38 how ‘ 5 hours oe 1-75 hours Mowe Few minutes Jacobs & Glassman in preliminary report (1937) give a brie yn species. characteristics, the most important of ed below. The permeabilities to glycerol, urea rythrocytes of 9 species of elasmobranch, 6 turtles, 4 snakes and 4 birds were com, neralisations may be made: account of results 0 which are summaris and thiourea of the ¢ 14 of teleosts, 2 frogs, pared. The following ge Fuss, Ethylene glycol penetrates fastest, Urea and thiourea highly variable from species to species. Thiourea usually faster than urea. Glycol and glycerol penetrate very rapidly at nearly equal rates. Thiourea much slower, urea slowest. Reriiss. Urea relatively rapid, glycol next; thiourea much slower. Permeability to glycerol only slight. Masotats. Urea very rapid; ethylene glycol much slower and thiourea still slower. Birps. The most interesting features of the work so far described on species characteristics are first, that species may be grouped in accordance with their permeability to a given substance, ¢. glycerol; second, that many species show a specially high permeability to a given substance which is out of all proportion to its permeability to other substances; e.g. human cells are permeable to glucose, whilst rat and mouse cells are not at all permeable to glucose, but are more permeable to glycerol than are human cells: further, mouse cells are permeable to the poly- hydric alcohols, sorbitol, mannitol and dulcitol, whilst human cells are not. _, So far enough has been said of species characteristics per s¢ for the reader to appreciate how real and significant they are; in the later parts of this section further species characteristics will be brought out in relation to other phenomena of non-clectrolyte penetration. “4 Escaneado con CamScannerPERMEABILITY TO NON-: eTROLYT, ah The question of the relation of the rate of i substance with a low lipoid solubility to its aera ‘ i been investigated by Mond & Hoffmann (1928) and Mond ie Gertz (1929). More recently Hober & Orskov (1038) hare ne a more extensive study from the same point of view, aint as dheie results agree with those of Mond & Hoffmann, it will he cufficinn to quote them alone, The method used was the haemolysis technique, and some of the results have been collected in Table XII. The figures in the table are values of t= (ly~t)/4,, where in 002MNaCl, and ty=time of haemolysis +0-02M NaCl. In this way differences in tl cells are to some extent discounted. as ime of haemolysis in non-clectrolyte he fragility of the TABLE XII. Revative times oF HAEMOLYSIS oF ERYTHROCYTES IN SOLUTIONS OF DIFFERENT NON-ELECTROL: OF DIF TES (SEE TEXT FOR DEFINITION oF ) (HéuER & Orsxov, 1933) Species AcetamidePropionamide Lactamide —Malonamide M.R. 149 19-5, 21 Rat 0-47 0-45 18-4 Man 0-90 1:20 315 Ox 1:30 1-70 720 Species Urea Methyl-urea—-Thiourea MR. 13-7 18:5 19-6 Rat Ol 19 31-7 Man 03 23 57 Ox OF 30 119 Species MeOH EtOH PrOH MR. 82 128 . Rat 0 O11 045 Man 0-125 03 03 Ox -019 = 0-05 0-03 Species Glycol Glycerol —_Exythritol MR. 4d 20-6 26-8 Rat 094 47 1,500 Man 17 60 10,750 Ox 16 650 Infinite Hober & Orskov conclude that the absolute value of the “Molecular Refraction”, i.e. the volume of the molecule, is only the determining factor when a given series of homologous sub- stances is considered. The basis for this is clear from the table; in each group of homologous or chemically similar substances the value of f increases as the molecular volume increases. On the Escaneado con CamScanner86 PERMEAMILITY TO NON-ELECTROLY ny, other hand, for a given species, there is no correlation 4 the rates of haemolysis and the molecular volumes 22° stances belonging to different homologous series ar, results of Hober & Orskov were extended to nine differences as striking as those described earlier were h Ween nen Sub. © Used, " Species ang Observed, TABLE XIII. (Jacons et al, 1935) Time of haemolysiy Species Substance in seconde Ox Glycsrot 2169 Feiethylene glycat i" Diethylene glycol a ay Dioxypropane ie Ethylene glycol aa 2 Dioxypropane Propyl alcohol i Rabbit Glycerol Trlethylene glycol et Diethylene glycol 7 ay Dioxypropane a Einpene alyeol 17 af Dioxypropane 7 Propy! alcohol ia The results of Hober & Orskov made it quite clear that besides the molecular volume of the molecule and its lipoid solubility, there is at least one other factor of importance in determining the permeability of a membrane to a given substance. Hedin's finding that OH groups tend to slow the rate of penetration of a molecule has received ample confirmation in the study of the comperative rates of penetration of sugars and their analogous polyhydric alcohols (with the exception in this case of the mouse erythrocyte). Further evidence of the importance of specific chemical groupings is given by the work just quoted of Hober & Orskov, and Fleischmann (1928) has shown that although Shucose penetrates rapidly into the human erythrocyte, methyl glucoside docs not penetrate at all, thus emphasising the im- Portance of the groups in the sugar molecule. Quantitative work on the effect of introducing OH groups into the propane molecule, has been published by Jacobs ef al. (1935) and is shown in Table XIII, and it is seen that on introducing successive OH Eroups into this molecule the rate of penetration decreased with cach group introduced. In the dihydroxy compound itis evident that the position of the OH group is important, the compoun with its OH groups on adjacent («f) carbon atoms penetrating Escaneado con CamScannerPERMEABILITY TO NON-ELKOTROLY TRS 87 snore rapidly than that with the OH groups at the ends of the molecule (ay). . Before proceeding with the remaining points of importance in relation to non-electrolyte permeability it would be useful to recapitulate the principles derivable from the work so far de- feribed, as follows: apparently important variables are (a) Species characteristics, (b) Lipoid solubility, (0) Molecular volume, (d) Specific chemical groupings, (e) Position of the groups on the molecule. All of these factors, with one possible exception, are of funda- mental importance in determining the rate of penetration of a substance. The exception is molecular volume, The results we have given above show without doubt that, in any one homologous series, as molecular volume increases rate of penetration decreases. But many other properties of an homologous series vary in the same way as molecular volume, so that whilst it is of interest to observe this general parallelism between rate of penetration and molecular volume, a quantitative treatment is necessary before we can be sure whether this correlation is fortuitous or not. Conclusions on the Structu e of Red Cell Membranes, Many results are available to show that if a series of cells, such as (a) rat, (6) human, (¢) ox erythrocytes, is considered, substances are found to penetrate the cells in different orders, For example, Héber & Orskov (1933) found that with acetamide the order of speeds is rat >man > ox, whereas with propyl alcohol the order is ox> man >rat. According to Jacobs et al. (1935) glycerol penetrates in the order rabbit > guinea-pig > rat > man > cat > ox; butwith thiourea the order is cat > rat> rabbit > man > guinea-pig> ox. According to some authors the differences between such series are such that they can only be accounted for by a mosaic structure of the plasma membrane of at least some species; i.e. the membranes of these cells must consist of areas of different chemical properties. To the present writers this view does not seem to be well founded, since it is based on qualitative rather than on quantitative arguments. In Table XIV are given values of the permeability, temperature coefficient and of PM? Q'Z4000 for ox red cells. The experimental data were taken from Jacobs et al. (1936) and the values of P calculated by the method of Jacobs (1934). For the substances Escaneado con CamScannerae PERMEABILITY TO NON-ELECTROLYTpy . jiethylene glycol, triethy] 1, zy dioxypropane, diethy! fool, 'ylene gl ayer eit cis 0% should be approximately constany, got for the other molecules, if the membrane is a homogeneous jipgey Tayer'® It will be seen that this s the ease, 20 that the memo tie red cells behaves, to a first approximation, as a homogense® geneous lipoid layer. tt TABLE XIV. DATA FOR OX RED CELLS AT 20°C, Substance P PMIQt Hone Propy! alcohol Jos x10-% Lbeto-s , . : 5x 10-1 Thiourea 0-019 xi i Glycol 0-209 x I 20x Ios ap Dioxypropane 0-405. x 10-"* 82 Glycerol 0-017 x 1 17 ay Dioxypropane —0°105 x 10-"* ous Diethylene glycol 0-075. x 10-48 16 Triethylene glycol 0-083. x 10-1" 028 On the other hand, in the case of red cells of man, rat and rabbit, it can be shown that a small part of the surface of the cell is specially adapted to allow passage of glycerol. Results of ‘Jacobs ef al. (1935) show that for these cells the Qyyis about 1-2 for glycerol, and about 1-4 for urea. Consequently, if the membrane were homogeneous, glycerol should penetrate these cells more rapidly than urea.* But in fact urea penetrates thirty-five times or more faster than glycerol. This can only be accounted for if 3% or less of the surface of these cells is specially differentiated to permit passage of glycerol at a high rate.tt The Influence of the Medium on Permeability of Cells to Non-Electro- bytes. So far as we are aware, no extensive systematic study of this aspect of non-electrolyte permeability has been described and most of what is known arises out of accidental discoveries. The principal work in this field is that of Jacobs and of Davson. Jacobs et al. (1935) mentioned that “acidity” of the medium causes, with a group of species, a marked retardation of the rate of haemolysis in glycerol solutions; the effect seems to be confined to this molecule. These authors do not define the pH at which the effect of acidity shows itself. In the description of the equilibrium conditions of the erythrocyte in Chapter m it was * See Chay . sSeations attendiO€ these sar it and Appendix A for the evidence and qualifications a Escaneado con CamScannerPERMEABILITY TO NONRLECTROLYTES 89 shown that the non-electrolyte suspension medium is acid and sigy have a pH of 6 or even less, 0 that the effect of acidity Teectibed by Jacobs ¢t al. must show itself at even more acid ‘eactions. Davson (10304) has investigated the matter further and Tome representative results on the rabbit erythrocyte, using a phosphate buifer, are shown in Table XV, and it seems clear that The retarding influence of acidity begins at a pH of from 4:7 to 5; however, in view of the importance of the ionic strength of the medium in these almost completely clectrolyte-free suspensions Gferythrocytes, it is questionable whether the pH of the medium Sione is of much significance. The fact that the effect of acidity may be revealed by the “incautious breathing” ofthe investigator faves come idea of the difficulties encountered in a great deal of the work on penetration of non-clectrolytes. TABLE XV. Tue Errect oF THE pH oF THE MEDIUM ON THE RATE TA NAEMOLYSIS OF RABBIT RED CELLS IN ISOTONIC GLYCEROL sOLU- trons (Davson, 19304) Suspension medium ‘Time of haemolysis 0-32 Glycerol 30 see. 0-32 Glycerol buffer, pH 48 sec. 0-32 Glycerol buffer, pH 41 sec. 0-32 Glycerol buffer, pH 39 sec. 0-32 Glycerol _ buffer, pH 5: 34 sec. 0-32 Glycerol buffer, pH 5: 30 sec. 0:32 Glycerol buffer, pH 4:75 53 sec. 0:32 Glycerol buffer, pH 45 320 sec. Another influence of the medium on permeability is described by Jacobs & Corson (1934) in a short note. These authors state that minute traces of copper in the solution of glycerol causes a pronounced retardation in the rate of haemolysis of certain species of erythrocytes, attributable presumably to a slowing of the rate of entry of glycerol. The effect shown is with those species which show the effect of acidity: man, rat, mouse, guinea-pig, rabbit. It is inhibited by NaHCO,. Davson (19394), in a general study of the action of heavy metals, has shown that the effect of copper can be greatly increased bywashing thecellswith saline, thisaction being due to thewashing out of the bicarbonate and removal of the serum proteins from the system, Addition of NaCl or any alkaline buffer inhibits the action of copper, the pH at which this action begins being as acid as 45. ‘These facts are shown in Tables XVI and XVII. Escaneado con CamScanner00 PERMEABILITY TO NONELROTROLY gp, Jacobs had noticed that the time course of fie glycerol in the presence of copper is peculiar in ¢hae "oly ig marked auto-acceleration towards the end ofthe proce "Sis has found an explanation for this, attributing this efter in alkalinity of the suspension medium, due to the "tie itself, As alkalinity inhibits the action of copper, it will th lysis accelerate the rate of penetration of glycerol, The alka et® following haemolysis is due to the breakdown of the equity distribution of anions, which fora cel ina non-electrolyts mem will be such as to cause the cell to be alkaline in respecy su suspension medium. Probably adsorption of copper ns haemoglobin released by haemolysis is aiso an important aa in accelerating haemolysis. or TABLE XVI. Errzcr or wasiine o exvrunocyres oy », INUIT TION OF PENETRATION OF CLYCEROL BY CoPvan’(Shyrtt 1939, : nl Suspension medium ‘Time of hemos Unwashed cells 032M Glycerol Fe arope Unwashed cells 0-32 M Glycerol 1x 10-§M Cu Once washed cells 0°32 M Glycerol 1 x 10-*f Cu Twice washed cells 0-32. M Glycerol 1 x 10-*M Cu TABLE XVII. Inrivence OF REACTION OF THE MEDIUM ON THE INHIBITING ACTION OF COPPER ON GLYCEROL PENETRATION, aE SHOWN BY CHANGES I TIME OF HAEMOLYSIS (IN SECONDS) (Davson, 19394) The time of haemolysis in glycerol alone was 30 sec. Glycerol + buffer pH Glycerol+ buffer +10-§M Gut* 6-75 41 “4 475 52 53 45 320 600 Jacobs et al. (1937) have investigated the effect of small quantities of salts on the rate of haemolysis of ox cells in glycerol solutions and find a marked acceleration, which, however, they attribute chiefly to a change in the fragility of the cells due to anionic shifts. It is possible that other factors may be involved. A possible influence of salts and plasma proteins on penetration is revealed by Somogyi’s (1928) results on the distribution of Slucose between human plasma and cells; Somogyi’s claims, that the passage of glucose from the cells to the suspension medium takes place within a minute or two, would give a permeability Escaneado con CamScannerPERMEABILITY TO NONKLY stant of the same order as that of eo conien are stable {n pure glucces Manan Yet erythrocytes temperature, As most of the work on noneelectrol ister has been done with the haemolysis technique, whicl ees Pilty enormous reduction of the salt content of the medion qe olves a the cell, it seems that the thorough investigation of the of environment on permeability to non-electrolytesw aa siicance of results obtained by the haemolysis technique will In concluding this description, it shoul . centage hal be ne ot ha non-clectrolyte permeability in this brief account, so that a certain amount of selection has been necessary; thus the voluminous literature on the vexed question of the distribution of glucose and amino acids between cells and serum has not been touched upon. Plant Cells. A very large amount of work has been done iy the permeability of plant cells (including yeasts), but of this extremely little is published in a form suitable for quantitative treatment. The most outstanding work is that of the Finnish school of Collander & Barlund. Fig. 26 shows the permeability of cells of Chara ceratophylia plotted against oil-water partition coefficient— a form of graph originally due to Collander & Barlund (1933). The larger the oil-water partition coefficient, and the smaller the molecular volume, the more rapidly a molecule penetrates. This work is based entirely.on chemical determination of the amount of penetrating substance which actually enters the cell sap, a fact which makes the work most reliable. From this diagram Collander (1937) concludes that “There is clearly a somewhat close concordance between the oil-water solubility of substances on the one hand, and their permeability constants on the other; this is not merely a general concordance, but, at least approximately, a direct proportionality. ...On the other hand, the smallest mole- cules obviously permeate faster than would be expected on account of their oil solubility alone....It seems therefore natural to conclude that the plasma membranes of the Chara cells contain lipoids, the solvent power of which is on the whole similar to that of olive oil. But, while the medium-sized and large molecules penetrate the plasma membrane only when dissolved in the lipoids, the smallest molecules can also penetrate in some other OLYT RS Escaneado con CamScannerfF a 92 PERMEABILITY TO NON-ELEGTROLY ops way. Thus, the plasma membrane seems to act solvent and as a molecular sieve. ‘This view of Collander’s is based on qualitative ay Daniel (Appendix A), from a theoretical study of ire both as a Selectiyg ents only, ails of det; 19 ‘Boeing acohet, #207 sete ‘Urethylan « sane as Ania Sree GOO The cy Propiomunite 9 vSarime pectoris Formamide oP: eyel os © Ween Pn aia eco me ee = ol Matyea Lscttmide @ Diethyl mabonamide Permeability (mol.fem*,/hour/mol. per litre) oor 07 Partition coefficient Pia. 25. “Permeability (ordinates) of Chara to non-electrolytes (cm. pet hr.) Plotted against olive oil-water partition coefficients (Collander, 1997). Arrows = om ‘0 permeability values which Collander & Barlund consider to the mechanism of diffusion across the plasma membrane, con- cludes that there is no justification for plotting P against partition Coefficients, so that the conclusions which Collander draws from Fig. 25 are not valid. Instead, one should plot* PM} (in the case of rapic 25002 JosPiy Penetrating molecules), and PM¥¢ RI" (in the case of slowly penetrating molecules), against partition coeflicients. Then, * See p. 76 or p. 336 for explanation of these formulae. = Escaneado con CamScannerPERMEAMILITY TO Non for a homogeneous lipid layer, the p Jines distant by a factor of 6 from the from Figs. 26 and 27 the reaulis of Co within these limits, and there inno differen ttlund fall molecules of diferent molecular volume. Heart at” between that the plasma membrane of Chara ellis pony yond lipoid layer, to a first approximation, ‘Thus, within the molecular species studied, molecular volume ig not {ause, of affecting the rate of penetration, and the reason win onatie & Bishund found degres of coreation berwery Colandet volume and rate of penetration is that molecular soln cat to run parallel to the quantities Mt and Mii? mod is aso a fortuitous correlation between P and moleesia see? Collander also gives data for fifteen other plant eels sion, the Helsingfors laboratory, shown on 28, The cells are arranged in order of increasing permeabil ity to erythritol. Cells of flowering plants, mosses, green algae, diatoms, brown algae, red algae, blue-green algaeand bacteria are represented, Collanic, (1937) interprets all these results in term is of the “lipoid-sieve”” theory. He remarks “ (1) Spirogyra and Chara are seen from Fig. 28 to agree very closely as to their permeability, except that the permeability of Chara cells is about three to ten ti imes greater than that of Spirogyra cells. Such a difference can, at least theoretically, be explained on the assumption that the membrane of Spiragra is correspondingly thicker than that of Chara. (2) The epidermal cells of Rhoco have an exceptionally low permeability to all amides... .This can easily be explained along lines first put forth by Hober and his school*,..we have only to assume that the plasma membrane lipoids of most cells are acidic in character, while those of Rhoeo are approximately neutral. (3) The root cells of Lemna differ from most other plant cells in being more permeable to urea than to the more lipoid soluble methyl urea, Perhaps this can be explained on the assumption that the plasma membrane of the cells in question contains a considerable number of pores of such a diameter as to be just penctrable by the urea molecules but not by the somewhat greater molecules of methyl urea. (4) The two diatoms so far studied, viz. Melosira and Licmophora, are both characterised by their remarkably high permeability to * Hober (1930), Wilbrandt (1931). MLBOT ROL y4 TRS ! 7) tween twa ints should e het rvernge line. As can be seen f Collander & cous Escaneado con CamScanner&@~> o PERMBADILITY TO NON-ELROTROL, YTKy 10 1 Bythritol Methyloluren 4. Urea 4, Glycerol 5. Malonamide 6. Urotropin Methylurea Dicyandiamide 9. Ethylene glyco! 10F 10, Lactamide 1, Acetamide ' oF 1 Partition coefficient o wer Fro. 28, PMle*F for non-electrolytes penetrating Chara, pl : olive oilowater partition coefficients, The points are not more' diver, wt factor of 5 from the straight line, showing the membrane to be homaveay 1 first approximation, earl) 107 : Partition coefficient 1G. 27, PMI for non-electrol i AE!) ; ytes penetrating Chara, plotted ag B fromaPattition coefficients. The points are not more distant than a factor of approxi ne ine, showing that the membrane is homogeneous to & an Batlnd conde to ea atached to permeability values which Collander — Escaneado con CamScannersihritol and sucrose, ic, To ipoid solubility and mats to the occurrence of plasma memtn Path in these cells. (5) A great abunda plasma membrane pores may be assumed lar volume. This rane pores of an extreme nee of somewhat smaller in the case of Oscillatoria, Propionamitle — Exhylene glycol — Methylurea—¢ Urea————— Malonamide Glycerol Enythritol cal 123456789 01213141516 Fic. 28, Permeability (cm. per hr.) of plant cells to non-electrolytes: (1) leaf cells of Plagiothecium denticulatum, (2) Oedogonium sp., (8) root cells of Lemna rinor, (4) Pylaiella litoralis, (6) Zygnema cyanosporum, (6) subepidermal cells of Carcuma rubricaulis, (71) Spirogyra sp., (8) leaf cells of Elodea densa, (9) epidermal cxlls of Rhoeo discolor, (10) epidermal cells of Taraxacum pectinatiforme, (11) “leaf” cells of Chara ceratophylla, (12) internodal cells of Ceramium diaphanum, (13) Bac- terium paracoli, (14) Oscillatoria princeps, (15) Melosira sp., (16) Licmophora sp. (after Collander, 1937). which differs from most other cells in that the sieve principle is more dominant and the effect of the lipoid solubility less so than. in the other cases.”” However, as was the case with Chara, these arguments are entirely qualitative, and unfortunately the results available for these various cells are hardly sufficient for any positive conclusion to be obtained from quantitative examination. It may, however, be said that with one possible exception, if the same quantitative tests are applied to the results given in Fig, 28 as to those for Escaneado con CamScanner96 PERMEABILITY TO NON-ELROTR Chara, e.g. if PM* is Plotted against points for all these cells fall within 8 homogeneous lipotd membrane, evidence of any pore size factor bei the results for Melosira plotted o: plant cells the slope of the avera, Otyrns Partitio, : the limits to sortie, y, and that there jg .°P* ng involved. Seq n Fig. 29. For ait the siytls coe BC straight line, Passing qqadied the values of PM*e RI" for slowly Penetrating molg, against partition coefficient, is Greater than the Slope of p, MA rapidly penetrating molecules, plotted against the Party, coefficient, as is predicted theoretically (Daniell, Appendis a 10” Partition coefficient Fic. 29. PM! for non-electrol cells of the diatom Melosra, Plotted against olive oil-water partition coefficients, 10 iytes penetrating Permeability of Bacteria. Collander (1937) quotes a few values of P for Bacterium paracoli which are wor greatly dissimilar from those of cells known to have Table XXII), a definite lipoid membrane (see A very large amount of work has been done on the sulphur bacterium Beggiatoa mirabilis, particularly by Ruhland & Hoffmann (1926) and by Schénfelder (1981). The work of the first two qualitative, and therefore do not : clusion being made. Marklund ant Crone Values of P, which are quoted in Table XVII together with values of P ‘M*. The PM} values are constant within Escaneado con CamScannerPERMEABILITY TO NOW-ELECTROLYTES n imental error with the exception of th capmequently it may be said that the Proper oe azharone. Commbrane are those of a non-lipoidal molecular sieve, diffusion meng place through watersilled pores, many of which are Spout the same diameter as saccharose molecules. There eolot evidence of any correlation with partition coefficients. When the Ctiyes of Pare compared with those for other cells (Table XXII), will be seen, however, that Beggiaioa has a permeability of ihe same order of magnitude as other cells, and as a layer of 50 A TABLE XVIII. Vatues or PMI ror Beggiatoa mirabilis Substance P pn Saccharose 0-135 x 10-* Z )n16 ritol 0-838 10-18 sat Glycerol 1-060 10-1 102% 10 Urea 1-580 10-18 12-2 10 Methylurea 1-170 10" 1o1xio Glycol 1-390x 10-1" 11-0 10-18 of olive oil, but which is 10° times less than that of a water layer of the same thickness. Consequently, if the membrane is a non- lipoidal molecular sieve of the same thickness as other cell membranes, with penetration occurring through pores only, these pores must occupy an extremely small fraction of the membrane farea—about one part in 10°. Now the total surface area of a single cell of Beggiatoa is about 5x 10-* sq. cm. Hence the total cross-sectional area of the pores is 6 x 105+ 10°=5 x 10- cm. or 500 A. A pore large enough to admit saccharose has an area of about 30-40 A.2; hence there can only be about 10-20 pores in the membrane of a Beggiatoa cell. If, therefore, we accept the molecular sieve hypothesis, either the greater part of the mem- brane of Beggiatoa must have a permeability equal to or less than that of plant and animal cells, with a very small number of wide water-filled pores occupying one part in a thousand million of the total membrane area: or else the membrane has more pores but is proportionately thicker. tt . This seems a little peculiar, and it is perhaps worth pointing out that if we abandon the idea that the penetration of these cells is necessarily a simple diffusion process only, at least two possi- bilities are revealed which could explain their permeability: (1) pores having a diameter usually rather less than that of pp 7 Escaneado con CamScanner98 PERMEABILITY TO NON-RLEGTROLY 7p, saccharose appear periodically in the membrane, whig \ wise highly impermeable; (2) that Beggiatoa has two Taba an outer membrane with an average pore diameter m2, than that of saccharose and an inner highly imperm, leas, membrane, in which from time to time such » structar tle contractile vacuole opens, sucking material through fee & & porous membrane.tt Thus much further work is root iter any final conclusion can be reached on the membrane of Beggiatoa. A farther alternative explanation of the bel bacteria is that the resistance to penetration due is not'so large that the resistance to diffusion contents may be ignored. In this case we ar calculating the number of pores by the simpl on page 102, A similar calculation for Bacterium paracoli is stil more in. teresting. Its membrane has an area of 8:4%10-¥em, and” permeability of about 10- of that of a similar molecules such as urea. If molecules were to waterfilled pores, the area occupied by the pores must te 34X10 x 10“ cm.?=0-0084.A.*: i.e. an area less than a mole. cular area, so small that even if the whole of this area were in ons Part it would be too small for any molecule to Penetrate. Its mem- brane consequently cannot be an inert molecular sieve, enormously thicker than most plasma membranes.t+ fn structure gy haviour of theye to the membrane through the cell © Not justified in © Procedure given water layer, to Penetrate through unless it is TABLE XIX. Suowino THE FRACTION OF THE TOTAL VOLUME oF A BODY TAKEN UP BY A PLASMA MEMBRANE OF NORMAL STRUCTURE, eel Smallest % of volume due Cell or virus diameter to plasma membrane Beagiatoa mirabilis By " . prodigiosus 750 m, B. coli 250, me 3 ‘Sewage micro-organism 160 mp. 19 Newcastle disease 100 mp 1 St Louis encephalitis 25 mp 75 Foot and mouth disease 10 mp 100 Quite apart from experimental work, considerations of the Space available inside a really small cell show that there is hardly Sufficient space for a plasma membrane of the type found with large cells. Table XIX shows the percentage of “cell” volume taken up by a plasma membrane 60 A. thick in the case o Escaneado con CamScanner~~ pEAMEADILITY TO NON-ELECTROLYTES 99 » of various volumes. It will be seen that as toon as a size weal” Of"%t which a plasma membrane of normal thickness is reached ge part of the cell—in fact an expensive Iuxury— pecomes & oer found, but only virus particles. t calls ate MP gs. The fist important study of the permeability of Marit eis that of R.S. Lille (1916, 1917, 1918), but his work marine cE confined to permeability to water. Work on non- as Mais solutes is more recent, and is mainly due to Jacobs, ceo McCutchcon and their colleagues. Table XX gives Luck mainly taken from Jacobs & Stewart (1936) on un- ised “Arbacia eggs. Qyo Values are available for butyramide, fertilise jaide and ethylene glycol, and for these three substances, propisiue of PM*Q{z*0° is constant within experimental error. Iefallows that the Arbacia egg membrane is possibly homogeneous, too few results are available to be certain of this. Tt will be btn from the table that the more lipoid-soluble substances tend to penetrate the faster, and that the more polar molecules penetrate more slowly. TABLE XX. Permeasiuity oF Arbacia EGG TO NON-ELECTROLYTES ‘Substance Butyramide Propionamide Acetamide Propylene glycol Ethylene glycol ay Dioxypropane Diethylene glycol PMigigtion9 2-95 x 10 1-05 x 10 2.75% 10° Glycerol 0-0083 x 10- Lucké et al. (1939) have compared Chaetopterus and Cumingia eggs with Arbacia eggs, finding that the former cells are con- siderably more permeable to glycol and glycerol than are Arbacia eggs. The permeability of Arbacia egys to ethylene glycol and ay dioxypropane is approximately doubled on fertilisation, as is‘also case with water. But whereas the Na*, K* and Ca** con- centrations have a very marked effect on the permeability to water, the electrolyte content of the medium has little effect on Permeability to glycol. From this Jacobs & Stewart (1936) conelude that the effect of electrolytes on the membrane is of a ferent nature from that of fertilisation. 18 > Escaneado con CamScanner100 PERMEABILITY TO NON-ELEGTROLY Tey sility of the Intestine, etc. There are many tissu . meres show a selective activity based on anvith to mechanism, but which to ater subway, even OF the sant ical group, are quite inert. Studies on the intestine’; Me eernerecing: ample of the methods whieh sar distinguish between the two groups of substances, Two pe methods have been used: (1) the effect of concentration of gua on the rate of absorption, (2) the effect of specific poisony. 2” ts rate of absorption. i TABLE XXI. Txs errzcr oF witiat concentnarton oy AMOUNT OF SUDSTANGE ABSORBED FROM THE tNrgerige’ THE Initial Millimoles absorbed molarity Initial concentration Urea (after 15 min.) 0-08 0-83 0-09 0-76 oz 0-69 0-18 0-80 0-24 on Erythritol (after 15 min.) 0-06 21 0-09 26 oz 25 018 22 0-24 22 Valine (after 20 min,} 0-056 25 0-084 15 O-115 Ld 0-167 06 0-293 O4 Table XXI shows some results obtained by Héber & Hober (1987) on the effect of concentration on the amount of a substance absorbed by rat intestine. Urea and erythritol have values of Amount absorbed 5 Initial concentration which, within experimental error, do not vary when the initial concentration is varied. This shows that for these substances the amount absorbed is directly proportional to the concentration in the intestine. But for valine the ratio falls off rapidly as concentration is increased, to an extent which Suggests that, above a threshold, the amount of valine absorbed 's independent of the concentration in the intestine. Hober Escaneado con CamScannertC MH PERMEABILITY TO NON-ELEGTROLY THs lor at in general the polyhydric alcoh: . gat nt inn a ri oe a = ‘herefore probably penetrate by simple diffusion, but pron and imino acids absorption is “complicated by the presence of with Mjerating facror, the effect of which becomes more visible an acrever than with higher concentrations”. In the case. of with Marie alcohols absorption resembles diffusion through a polyhyxe membrane, the maximum pore diameter of which is seve pat of mannitol, so that mannitol (C,) is searcely absorbed apa, but adonitol (C,) and erythritol (C,) are comparatively rapidly absorbed. But with the amides the effect of lipoid solubility is of marked importance. Thus butyramide, with a jnolecular volume of 113, should penetrate a sieve more slowly yan acetamide (molecular volume 68), but in fact butyramide netrates rather more rapidly than acetamide, ‘The use of poisons is well illustrated by the work of Wilbrandt & Laszt (1933) on the absorption of sugars. These workers measured the rate of absorption of sugars (a) without iodoacetate, (8) with iodoacetate, and found, in a typical case, that the ratios of (a): (b) were: galactose 2-1, glucose 3-0, fructose 1-2, mannose 13, xylose 1-0, arabinose 1-0. It was concluded from this that an active mechanism, possibly phosphorylation, was involved in the transport of at least the sugars galactose and glucose. This theory will, of course, require much further experimental examina- tion before it can be regarded as correct. The intestine is such a complex tissue, and so complicated by active processes, that it does not seem profitable to make further comment upon it at the present time. The Capillary Membrane. Despite its importance, extremely little is known about the permeability of the capillary membrane; it is probable that it will be found to resemble the glomerular membrane in being a molecular sieve of rather large pore size. According to Keys. (1937) the available evidence shows that substances penetrate the capillary membrane in the order H,O > urea > glucose > sucrose. Chitin Membranes. An investigation of lobster chitin membranes by C. M. Yonge (1936) shows that the membrane is much more Permeable to weak acids than to strong acids, and that for the Weak acids the relationship, PM* = a constant, is obeyed within “perimental error (Table XXI a). | — Escaneado con CamScanner102 PRRMBAMLITY TO NON-ELECTROLY rpg TABLE XX1a, Parweamiity o” Curry M ay Hydrochloric acid 36 24 in J Formic aci 45 : Acetic acid 60 1s igre Propionic acid 16 4 122 Butyric acid 89 13 B There is no tendency for the rate of permeation to fllyy citwater partition coeflsiens of the molecules, The sy.o" te to the relationship PAf*=constant, together with tke nee impermenbility to HCL shows that the weak acids prot? penetrate mainly in the undissociated form, that the peneyrat! takes place through waterflled pores which are eons? greater in diameter than is butyric acid, and thatthe pore we probably carry an electrical charge sufficient Breatly to reduce the rate of penetration of ionic substances, Relative Permeability of Various Cells. Table XXI1 gives value of the permeability P of fiteen different cells to non-elestays compared with the “permeability” of layers of water and dln oil of about the same thickness as the plasma membrane, ‘The water layer is always much more permeable than the cell men, brane in the cases cited. This, however, does not mean that there are not some substances which may diffuse just as rapidly as through water. Such may be the case with hydrocarbons, ‘But accurate permeability constants can at present only be determined for substances which do diffuse much more slowly through the cell membrane. What is of much more importance is that whereas in water all the substances concerned diffuse at about the same speed, through the cell membrane the rates differ by a factor of 10%, and that to the most rapidly penetrating of these molecules the membrane permeability is still 108 timer less than that of a water layer of equivalent thickness. The values given for the permeability of an olive-oil layer are calculated from the theory of Danielli (Appendix A), and are rough values only. Nevertheless, the values closely parallel those found for the cells, both in magnitude and in variation from molecule to molecule. The permeability of the oil layer may be varied, for some molecules at least, by a factor of probably more than 100, if different oils are considered. It will be seen, however, that in certain cases, e.g. urea and glycerol, there is a variation Escaneado con CamScannerto pERMEABILITY TO NON-ELEGTROLYTES 103, 000-fold in the permeability of diff Jeast 3 , ty of different cells ¢ of : molecule. Whether this large difference can be catch same jyed for by variation in the chemical character of the ofl eet cannot as yet be stated. a XXL. PERMEABILITY OF DIFFERENT CELLS, ETC. 1M woLS prFBRENOE Fatt . mi ales have BEER multiplied by 10%, except the olive oil: water partition A pices . 5 te oH FJ gu ill, dy Ee ho G 2 - & gb ga5B 5B GSP ab ON speimethylettrate + : - 67 on z 5 ; Doe oe pepronamide a 36 0-20 Peceamide 5 0-067 Urowppin oot «07st Teun Saeahylurea : : : + G19 oon fe wy O11 00038 UrSioxypropane O-d ; . sp oionypropane O-LL — 0-67 Malonamide . ; _ . faiyienc glycol 0-015 0-43 ; Glycerol 0.0017 0-005 1-0 i Erythritol . : : : Saccharose : : ‘ ere 1 32 : : i : ui # 44 oS ‘Trimethyl citrate . . . . . Antipyrin : . : : Propionamide 1-4 O48 01628) 30 Acctamide : ' 0a Urotropin . i : : a etal Glycol 0-097 oll oll O17 0-83 0-375 Methylurea 0-031 0-0089 0-028 0-0084 «= 0-097 O12 oe 0-083 0-0015 0-018 + 0-0028 0-083 (0-042 ty Dioxypropane ’ ‘ * ‘ + é. Malonamide 0-019 0-00036 0-00160-00022 0-025 0-018 Diethylene glycol . : ; : ; Ghreerol 0-0024 0-0018 0-033 0-011 0-021 0-033 ythrital 0-00012 0-00031 0-00042 0-0005 0-0021 0-013, Saccharose 0-000006 0-00008 00058 (ni Escaneado con CamScanner> _ {LiTY TO NON-ELECTROLYTEs pERMEAB 104 TABLE XXII (continued) a ie Ys @ g 3,f)47aga; t BS PaS be2ds peed § gFhs Soess Eee . 0-047 170) y 0-032 {is 9.80% 10 0.0038 30 Lagi 0-00083 0-83.79 1 = Goole 087 0.00049 0758 taxi 7 00044 0-058 1.0151 0-25 0-00015 0-018 taxi. 132 0.0057 26 = 1-60x 10» hatude 0-028 . . 0-00008 0-0021 1-38x 10° ethylene glycol = : 1 0-005 0485 1-45 x 10° o0gs 1-06 «00180-00007 O-0051, 1-44x 10» Sto 0005 (084 + 0:00003 000007 1-24 10+ Saccharose . OL 2 0.00008. (0 75x10" When comparing different molecules diffusing through the same membrane it is preferable to compare values of PA}, not Of P, since in this way the purely mechanical factor of variation fa molecular weight is eliminated. For example, values of P for tthylene glycol and ay dioxypropane usually differ significantly, but PM! values are often identical within experimental error. Finally, one experimental defect in practically all of these studies must be pointed out. It is usually assumed that the structure of the membrane is not significantly affected by the concentration of non-electrolyte used in permeability studies, and that therefore the permeability constant obtained in all cases refers to the same independent membrane. This is an assumption which is probably true in most, but not in all, cases. Investigation is needed of the variation in P: (1) with different concentrations of the same non-electrolyte; (2) when one non-clectrolyte pene trates in the presence of another. Collander (1949) has made a valuable new study of the perme \ ability of plant protoplasts to small molecules (Piysiol. Plan’ & 300), Wilbrandt has published a collatiori of permeability values (Tab. Biol. 19, part 2). 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