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ROLE OF THE CELL MEMBRANE IN THE METABOLISM OF INORGANICELECTROLYTES BY MICROORGANISMS'

ASER ROTHSTEINDepartments of Radiation Biology and of Pharmacology, University of Rochester School of Medicine and

Dentistry, Rochester, New York

CONTENTSI. Introduction 175

II. OsmoticBarrier. 177A. Intact Cells versusExtracts.177B. Retention of Internal Solutes and the Action ofAgents. 178C. Osmotic Behavior: The Penetration or Exclusion of Extracellular Electrolytes. 180D. Volume ofDistribution. 181E. Conclusions Based on Osmotic and DistributionStudies.183

III. Permeability to IndividualIons. 184IV. Active Transport ofElectrolytes. 186

A. Transport ofPhosphate.186B. Transport of MonovalentCations. 186C. Transport of BivalentCations. 190D. Mechanisms ofTransport. 191

V. Nature of the Membrane: Chemical and Cytological Observations.. 194VI. Extramembrane Factors in IonDistribution.195VII.Comment.. 196

VIII. References. 197

I. INTRODUCTION special emphasis on the role of the cell mem-brane.All cells face common problems with respect Phene n

to electrolytes. One of these is the somewhat henomensi ated since 1945,a.. . ~~have been intensively investigated since 1945, aparadoxical problem of maintainng a unique . . .itrndoxaleleroblte cofmpositin d c f primary stimulus being the availability of radio-inthrnat thelenvronmentcom,andt ,thetse time active isotopes. In the last few years numbers ofthat of the environment, and at the same timesypiandm ogphdelgwthheuballowing rapid exchanges of ions. One case su,the abil ,ofe a hh ect have appeared which provide useful generalpoint istn ability OI cent Iomamtam gahgh background material (1, 3, 36, 55, 73, 90, 110).internal potassium and low sodium while living E i h b d frinaenironenthigin odim an loin Existing information has been derived fromin an environment highmn sodium an lo in biological disciplines: from the animal* or * * ~~~~many boola ~cpms rmteaia

potaemonssium. Yt, bytmeans oi p it isey physiologists who work with red blood cells,to demonstrate that both Na+ and K+ can enter kiny inetie gati muoa muce nor leave the cell with some rapidity. A second kidey inetie gati muo uce n

orlave thconcell with some rapidity. Al sondp

frog skin; from the plant physiologist workingchase isconceeds withi thehighlevelas cofmpho primarily with cells of roots, storage organs, and

leaves; and from the microbiologist, with yeastwith the environment despite the rapid ex- r m m achanges of phosphate that can be demonstrated ro te reiw of Mitell an Moyler(67by experiments with 1Pt. In seeking explanations Frmtervesoice.adMye(7byrsue pherentsnwith most,In tseek aingexlantios 69) on bacteria and of Conway (7, 8) and Roth-forsuchphenmenamosbunotalli stein (91) on yeast, it is obvious that large gaps

gators have assigned a major role to the cellmembrane. It is the purpose of the present paperto review the metabolism of electrolytes, with microorgns. Fortunately, however, as in the

case of biochemical problems, cross fertilization1 This study is based on work performed under between disciplines can be most fruitful. Con-

contract with the U. S. Atomic Energy Coin- cepts derived from one type of cell can often bemission at the University of Rochester Atomic usefully applied to another. For this reason, anEnergy Project. attempt will be made to place the studies with

175

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176 ASER ROTHSTEIN [VOL. 23

microorganisms in the context of information centrations of solute, competition betweenderived from studies with all kinds of cells. For structurally similar solutes, and a high degree ofthe same reason it is necessary to discuss briefly specificity. The term facilitated diffusion hassome of the general concepts and terminology been applied to such systems (113).which have found usage among those studying Whether the mechanism of penetration of themembrane phenomena. membrane is "simple diffusion" or "facilitated

In the classical studies which were initiated diffusion" the driving force is the same, theas early as 1855 and culminated in the work of difference in concentration of solute on the twoOverton at the end of the 19th century, the sides of the membrane (in strict chemical terms,concept of a semipermeable membrane was estab- the difference in activity). At equilibrium thelished, relatively permeable to water but re- concentrations will be equal, and no net transfersisting the diffusion of solutes to varying de- will occur, because the rates of movementgrees. The resistance to diffusion was expressed (fluxes) are equal in both directions. With somequantitatively in terms of a permeability con- penetrating solutes, however, the concentrationstant in modifications of the Fick equation for inside the cell may be considerably higher thandiffusion. According to this concept, driing that outside. In other words, accumulation occursforce for solute transfer is the concentration against the apparent concentration gradient. Indifference between the inside and the outside of some cases, accumulation results from trappingthe cell. The membrane plays a passive role, but of the solute within the cell in some form notis regulatory in the sense that it is discrimina- distinguishable from the original by the particulartory, offering high resistance to diffusion of some method used in the experiment. Trapping may besolutes and lower resistance to others. The im- due to such reactions as (a) binding by cellularportant factors thought to determine the re- constituents, (b) chemical alteration, or (c) dis-sistance of the membrane to solute penetration sociation of molecular to ionic form in the casewere physical properties of the solute, its lipid of weak electrolytes due to differences in pH.solubility, and its molecular size. From the per- In other cases, accumulation cannot be explainedmeability studies a number of theories of mem- by trapping effects, and the concept of activebrane structure were developed, usually involv- transport has evolved, in which the solute noting some kind of lipid structure (21). only passes through the membrane in combina-Most of the substances used in the earlier tion with specific membrane receptors or car-

permeability studies were not physiologically riers, but is moved against its activity gradientimportant. They were, however, useful because by a mechanism deriving energy from metabolicof the information they provided concerning some reactions. Here, the driving force is not the con-of the properties of the membrane. In recent centration gradient, but is some kind of molecu-years, studies of the entry and exit of physio- lar or ion "pump."logically important substances into and out of The foregoing discussion applies to bothcells have revealed many deviations from the nonelectrolytes and to electrolytes. In the caserelatively simple model of diffusion through a of the electrolytes, the situation is complicatedselectively permeable lipid membrane. These to a considerable degree by the electrical chargesdeviations are of two kinds, one concerned with of the ions. Molecules of nonelectrolytes behavethe mechanism of penetration through the mem- as independent particles with respect to move-brane, and the other concerned with the driving ment through a membrane, whereas ions areforce for solute movement. In the simple model, electrically coupled. If a cation moves through athe entry process, although it may involve parti- membrane, it must be electrically balanced bytion into a lipid phase, is essentially a diffusion movements of an equivalent number of cationsprocess. In contrast, many physiologically in the opposite direction, or of an equivalentimportant substances seem to pass through the number of anions in the same direction. In othermembrane by a combination with specific re- words, the movement of one ion through theceptors or carriers. The kinetics of entry in this membrane sets up an electrical potential whichcase do not fit the diffusion model. Instead, they acts on other ions, either repelling or attractinghave many of the properties of enzyme reactions, them through the membrane. For this reason,such as saturation of the receptor by high con- when considering the forces acting on ions it is


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1959] CELL MEMBRANE IN METABOLISM OF ELECTROLYTES 177

necessary to take into account not only the con- tendency toward an equilibrium distribution ofcentration gradient, but also the electrical gradi- electrolytes with no electrochemical gradient, isent. The over-all driving force is the electrochemical balanced against transport mechanisms derivinggradient. Electrical potentials on the membranein- energy from metabolic reactions which work influence the movement of ions, and the movement the opposite direction, away from diffusion equi-of ions through the membrane in turn induces librium. The membrane factors of importance inpotentials in an intimate relationship that has the system are (a) the permeability of the mem-been beautifully elucidated in certain animal brane to each ion species (resistance to diffusion),cells and tissues (41, 116). In microorganisms, (b) the electrical potential of the membrane, (c)the membrane potential cannot be readily the active transport systems, and (d) the cou-measured. Nevertheless, in any system in which pling between ion movements. The cytoplasmicions move across a membrane, or in which un- factors include (a) Donnan equilibrium, due toequal ion distributions exist across a membrane, nondiffusible ions in the cytoplasm, (b) trappingelectrical potentials constitute an important of various kinds, (c) metabolic production orparameter of the system, imposing certain distribution of ions, and (d) compartmentaliza-limitations on the ion movements. tion within the interior of the cell by other

Because of the problem of electroneutrality, membranes such as the vacuolar membrane orions must move in pairs. It is only with some mitochondrial membrane.difficulty that it can be determined which ion One technical matter should be raised at thisof the pair is moved by some primary driving time. Because ion movements are linked andforce such as active transport, and which ion is because they are occurring in both directionsmoving to satisfy the electrical gradient created through the membrane at the same time, elucida-by the movement of the first ion. In some cases, tion of membrane mechanisms requires knowl-the pairs of ions are specifically linked. For ex- edge of all of the ion movements. Chemicalample, the inward movement of potassium may analyses in themselves are not enough, for theybe coupled in an obligatory manner to the out- indicate only the net changes. Isotope measure-ward movement of sodium. Another phenomenon ments alone give the rates of movement in ais exchange diffusion in which the movement of given direction (unidirectional fluxes) but arean ion in one direction is coupled to the move- not a direct measure of net changes. Judiciousment of the same species of ion in the opposite use of both the chemical and isotope techniquesdirection (115). In such a system, exchanges of is usually required for a complete characteriza-an ion can occur rapidly with no net movement. tion of the system.Electrical coupling is also important within thecytoplasm because of the high concentration of Il. OSMOTIC BARRIERnondiffusible ions, giving rise to the well known Much of the recent work with bacteria hasDonnan effect. been concerned with establishing whether they

It is sometimes technically difficult to sepa- do or do not possess a semipermeable membranerate the various factors in electrolyte metabolism which resists to some degree the diffusion ofand to assign specific roles to the membrane. An solutes (including electrolytes) but which allowsover-all picture can be drawn, however, which is the rapid entry or exit of water. The studies cangenerally accepted, although some of the details be fitted into four categories: (a) comparisons ofare the subject of considerable controversy. Cells the action of electrolytes on enzymatic activitiescan, within certain limits, maintain an internal of intact cells versus those of broken cells orelectrolyte composition considerably different extracts, (b) leakage of cellular electrolytes, (c)from that of the environment. Under conditions osmotic behavior, and (d) volumes of distribu-of minimal metabolism, a tendency toward uni- tion of external electrolytes.form distribution of solutes is found, whereasunder conditions of maxmal metabolism, cer- A. Int(ct Cells vermss Extradstain ions are accumulated. The ion distribution In a number of cases, it has been demon-between the cells and the environment can be strated that electrolytes influence enzymaticdescribed as a steady-state situation in which activities of broken cells or cellular extracts to atwo opposing tendencies are operative. The marked degree, but have lesser or little effect on


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178 ASER ROTHSTEIN [voL. 23

the activity of the same enzyme in the intact reversible osmotic shrinking of the cells. If, oncell. the other hand, the concentration is reduced, the

For example, the (3-galactosidase activity of light intensity fades continuously, representingintact Escherichia coli is relatively independent lysis of more and more cells. A value of 22.4of extracellular pH and of the presence of Rb+ atm. is equivalent to a 1 M sucrose solution. Otherand Nat, but the galactosidase activity of ex- organisms including Bacillus meaterium, Baciustracts of the same cells is very susceptible to pH subtilis, E. coli, Sarcina lutea, and Saccharomycesand is inhibited by Rb+ and Nat (54). Other cereviia have a somewhat lower osmotic pres-comparisons of intact and broken cells give sure, equivalent to 0.2 and 0.6 M sucrose (5 tosimilar results. The ions tested include H+, Nat, 13 atm.), based on plasmolysis thresholds orKE, Rb+, Cst, and NH4+. The enzymes and or- stability of protoplasts (25, 63, 122).ganisms include aspartic acid deaminase in The high osmotic pressure is retained for longProteus (114), glycolysis in Lactobacilus (4), periods of time in intact cells, or even in proto-glycolysis in yeast (105), oxidation of various plasts, when they are suspended in media freesubstrates by a marine bacterium identified as of electrolytes, such as sucrose. Thus the cellularPseumonas or Spirillum (61), and catalase in electrolytes which contribute to the osmoticMicrococcu lysodeiticu (28). In another type of pressure, do not readily leak out. The exactexperiment, titrations with hydrochloric acid contribution of inorganic salts to cytoplasmicwere made using intact cells of M. lysodeit8 osmotic pressure has not been directly assessed.and on butanol-treated cells. Large differences Mitchell and Moyle (69) calculate that most ofin titration values indicated that the interior of the trichloroacetic acid-extractable material ofthe cell was shielded from H+ by a barrier which S. aureus and of E. coli must be involved. Inis disrupted by butanol (30). yeast, with an osmotic pressure equivalent toThe studies cited above suggest that in the 0.55 m sugar (25) the potassium of the cell (0.25

intact cell, the enzyme centers are protected m), extractable phosphate (0.04 x), bicarbonatefrom extracellular electrolytes by some kind of (0.05 M), and organic acids (0.02 and 0.04 m) (91)barrier. The study with protoplasts (28) in- must contribute in large measure, because thedicates that the barrier is not the cell wall. nondiffusible ions are relatively inert from an

B. Retention of Internal Solutes and theosmotic point of view.B.ActntionofAgdernal Soutes and theBecause of the high internal osmotic pressure,

Action of Agents and because the internal solutes are retained by

All cells, including microorganisms, are rich a relatively impermeable membrane, the bac-in potassium and in phosphate. In microor- terial cell tends to absorb water. This tendencyganisms this is particularly true after periods of is balanced by the mechanical resistance of theactive metabolism or growth during which elec- cell wall. As in the case of plant cells, microbialtrolyte accumulation occurs. Most of the cellular cells are in a state of turgor with the walls underelectrolyte is free within the cytoplasm to con- outward pressure. For this reason, any weakeningtribute to the osmotic pressure of the cell, which of the cell wall by the use of enzymes such asis very high in some microorganisms. Osmotic lysozyme, or by agents which interfere with cellpressures have been determined in different wall synthesis leads to a rapid osmotic burstingways. In Staphylococcus aureus, vapor pressure (43, 62, 107, 123). On the other hand, if theequilibria (71) and studies of stability of proto- extracellular osmotic pressure is kept above thatplasts (70) indicate a value of over 20 atm. A of the cytoplasm with a nonpenetrating solute,similar osmotic pressure is found in M. lyso- then the cell remains intact as a protoplastdeikticus also by studies with protoplasts (68) even after the cell wall is completely removedand in luminescent bacteria based on the quench- (122).ing of luminescence after osmotic lysis (45). Some direct measurements of the retention ofFigure 1 represents a typical experiment with cellular potassium and phosphate have beenluminescent bacteria (5). The light intensity made. Cells vary considerably in leakiness. Forremains normal at 0.8 M sucrose. If the concen- example, the yeast cell has a "tight" membrane.tration of solute is increased, the light intensity It leaks cellular phosphate at a very slow rateis stable but somewhat reduced, representing a (39, 47) even though the phosphate content of


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19591 CELL MEMBRANE IN METABOLISM OF ELECTROLYTES 179

00s/2/2mQ2M

1_L - 2sM _

1 2 3 4 5 6Figure 1. Influence of hypo- and hyper-tonic sucrose solutions on the intensity of light emitted by

luminescent bacteria (5). The original intensity - 1 (ordinate). Abscissa = time in minutes.

the cell is high (120 mm per kg of wet cells, of These have been identified as phosphoryl groups,which 40 mm is acid extractable and 16 mm is perhaps of ribonucleic acid. The binding reac-inorganic) (89). It leaks potassium at a rate of tion is rapid and reversible. Other cations areonly 1 per cent per hr to potassium-free media also bound by the same groups and they can(99). The membranes of ChloreUa and Rhodo- therefore compete with the basic dyes, protecting8pirillum are somewhat less retentive for phos- the cell from deleterious effects. Secondly, thephate, whereas those of some bacteria are bound dye damages the membrane in an oxida-relatively leaky (47). Some bacteria are also relative reaction probably by conversion of sulfhy-tively leaky with respect to potassium (53). r

The retentiveness of the cells varies widely dryl groups to diulfide. In the case of mercury,between species and a few observations sugges only the second reaction is involved. The damagethat it may vary with the physiological state to the cells is exactly the same as with the basicwithin a given species. Nevertheless, every cell dyes.seems to have to some degree a capacity to re- The leakiness of the membrane produced bytain cellular electrolytes, a capacity which de- mercury or by basic redox dyes follows an all-pends at least to some extent on the relative or-none pattern. At any given concentration ofimpermeability of its outer membrane (not the the agent a proportion of the cells lose all of theircell wall, as shown by studies with protoplasts). potassium, while the rest lose little. The cellsThe role of the cell membrane in retention is also that become leaky to potassium also becomeindicated by a number of studies with agents permeabile to dye molecules and can be stained,which destroy its integrity, and thereby allow whereas nonleaky cells do not stain. For thisthe escape of cellular solutes. Among the many reason, the fractional loss of K+ is equal to theagents in this category are certain antibiotics fraction of stained cells (figure 2). With in-(42, 76, 80), detergents (76, 80), bacterial viruses(82), ultraviolet and X-irradiation (2, 37, 96)p croig concentrations of the agents, more and(8) ultavioetand Xrio more cells exceed the threshold and becomeand heavy metals(97).1The release of potassium from yeast by basic eaky. Thus the response curve (concentration

redox dyes and mercury has been studied in versus effect) represents the distribution ofsome detail (78, 79). In the case of dyes, two thresholds in the population of cells. On a plotsteps are involved. Firstly, the dye must com- of the logarithm of dose against effect, a normalbine with fixed negative groups of the membrane. distribution curve is obtained.


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100 inferred that the cell membrane is impermeableto sucrose, to NaCl, and to cellular solutes, but

co 80 not to glycerol, urea, and lipid-soluble sub--J stances. If, on the other hand, the cell is treatedw with lipid solvents or with other substances

60 which can destroy the integrity of the mem-cj brane, the cells will not plasmolyze in NaCl or

z sucrose.; 4 0 Although the cell wall restricts the swelling ofInt microorganisms in hypotonic solutions, some

IL 20 changes in volume can be observed (52, 69).o In E. coli, the volume changes have been meas-

ured quantitatively by observing the changes inae 0 A. . . , . , . . . the turbidity of the suspension (69). In the

0 20 40 60 80 100 presence of isotonic solutions of certain solutesK* LOSS IN % OF TOTAL such as glycerol, a rapid change in turbidity is

Figure 2. The relationship between maximal K+ observed, but with all salts tested includingloss and the staining of yeast cells (79). NaCl, KCI, NH4Cl, MgCl2, NaBr, NaCNS,

KCNS, NH4CNS, Na2SO4, and NaH2PO4, nodemonstrable changes occurred within 1 hr. The

C. Osmotic Behavior: The Penetration or cells behave as though they were permeable toExclusion of Extracellular Electrolytes glycerol, but relatively impermeable to salts.

If the cell membrane is permeable to water Another interesting method of measuringbut relatively impermeable to solutes, then any osmotic effects using luminescent marine bac-alterations of the tonicity of the medium . teria has already been mentioned. With hy-result in changes in the volume of the cells potonic solutions, the cells swell and burst, andpredictable from the van't Hoff equation. T light emission ceases (figure 1). The time re-

cells will swell or shrink until the osmotic pres- quired for extinction has been used as a measure

sures inside and outside are equal. The limiting of the rate of penetration of solutes (40). Withvolume for swelling is determined by the pres- hypertonic solutions, the cells plasmolyze. Theence of restraining walls, or by the bursting of light emission decreases in proportion to thecells stretched to their limit. If, on the other degree of plasmolysis, in a reversible manner.

hand, the solute can penetrate (but more slowly Taking advantage of this phenomenon, Col-than water) then a cell will first respond to the lander (5) found that the cells behaved as though

they were impermeable to sucrose as well as tomovement of water and secondly, as the solute ypenetrates and increases the cytoplasmic osmotic all of the salts tested, including NaCl, NaNO3,pressure, the cell will swell. The cell volume K 'I, LiCl, MgSO4,Na'r, KI, NaHCO3,changes due to penetration of water and of KH2PO4, Na2S04, MgC12, CaCl2, Ca(NO3)2,solutes have been used extensively as a measure K 'S04, and SrCl2.of permeability in animal and plant cells. In Gram-positive orgasms cannot be readilythe latter, the cell wall resists swelling, so the plasmolyzed. Recently, however, techniques for... . . '. ......... removing cell walls have allowed the pre ara-cells are first placed m hypertomc solution to g p p

shrink them (plasmolysis) and the time of re- tion of naked protoplasts (122). With non-

covery to normal volume represents the penetra- penetrating solutes, the protoplasts behave as

tion of the solute osmometers (31, 112, 120), responding to tonicitySome studies using osmotic techniques have changes in the medium with a change in volume

been made with microorgansms. Swelling is according to equationlargely restricted by the cell wall, but some of 7r(V - b) = constant (1)the gram-negative organisms can be plasmolyzedeither by hypertonic solutions of sucrose or of Where r is the osmotic pressure of the medium,salt, but not by solutions of glycerol, urea, or V is the volume of the cell, and b is the volumelipid-soluble substances (51, 69). Thus it can be of the cell which is osmotically inactive (granules,


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0-3-

Volume12+ 4-mWX 10 X '-+

E 012

2 uJ~~~~~~~~~~~~~~~~~~~~~~~.

00ALL Glycerol A

S- 1.0 1*S2-Molality of sucrose or glycerol and equivalent osmolality of NaCI

Figure 4. Stability of the "protoplasts" ofMicrococcus lysodeikticus (NCTC 2665) to sus-pension in NaCl, sucrose, and glycerol solutionsover a range of solute concentration (68). Extinc-tion measured in 1.2 M NaCl containing 0.01 Mphosphate, pH 6.8.

KCl, NH4C1, MgCl2, NaBr, K2SO4, and KH2PO4but are permeable to nonelectrolytes such asglycerol, erythritol, glycine, and urea (68).

C Because the protoplasts of gram-positive or-ganisms behave as though they possess a semi-

2 6 8 permeable membrane, the failure of these cellsFigure 8. Plasmolysis of protoplasts in sucrose to plasmolyze in hypertonic solutions cannot be

as a function of the osmotic pressure of the me- attributed to the leakage of solutes into or outdium (120). The volume of the plasmolyzed proto- of the cells. Rather, a strong adhesion must existplasts is plotted against the reciprocal value of between the cell membrane and the cell wall (69).the corresponding sucrose concentration (M/1).The straight line is drawn by the method of least D. Volume of Distributionsquares.

If a known amount of a solute is added to aetc.). Figure 3 is from a study on protoplasts of thick suspension of cells, it will distribute in allB. megaterium (120). The value of b taken from of the water space accessible to it. By analyzingthe intercept is 36 per cent, which is in good the concentration of the solute in the mediumagreement with that obtained for red blood after centrifuging the cells, it is possible to cal-cells and for marine eggs. culate whether the solute is distributed only inAnother technique using protoplasts is similar extracellular water, or whether it penetrates into

to fragility tests for red blood cells. The proto- the cells. In such experiments, it is necessaryplasts are placed in a series of solutions of a non- to know the volume of the cells. Because packedpenetrating solute (figure 4). As the tonicity is cells trap a certain amount of extracellular fluiddecreased, more and more of the cells reach the between them, direct estimations of cellularbursting point and the decreased turbidity of the water cannot be accurately made. It is usual,suspension can be determined with a colorimeter. therefore, to resort to reference substances, usu-The percentage of lysed cells remains relatively ally macromolecules such as inulin and protein,constant for long periods of time. In contrast, which are assumed not to penetrate into thewith penetrating solutes, lysis proceeds con- cells.tinuously at a rate dependent on the permea- The first detailed study was that of Conwaybility to the solute. By such techniques, the and Downey (11) on the yeast cell. The referenceprotoplasts of M. lysodeikticus and of S. lutea substances, inulin, peptone, and gelatin gave aare found to be relatively impermeable to NaCl, distribution volume of 22 to 24 per cent of yeast


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packed by centrifugation. These values are veryclose to the theoretical value of 26 per cent for .interspace in a system of close-packed spheres. -Thus these substances do not seem to penetrateinto the cellular water. They can, therefore, be £ /used for measuring extracellular space. 2Some solutes such as acetic, propionic, and 0

butyric acids readily distribute into the total a 2'0water of the yeast cell. Other nonelectrolytes E

a

(the sugars) and the electrolytes tested, NaCl S- threshdEand KC1, distribute freely into a space which is

I0

about 12 per cent of the cell volume, but are 1 2 4

restricted from distributing into the rest of the if/osmosivofNaacell. Conway and Downey (11) suggest that the12 per cent volume represents the cell-wall space Fiurme5.Thed ofth e posphAte-of the cell, that the outer surface of the cell wall iprabl volume of E cerationi(A e' ~~~~~~~~~canstrain B) on the NaCl concentration of theis impermeable to macromolecules such as suspension medium (69).inulin and peptone, and that the cell membraneunderlying the cell wall is relatively impermeable NaCl. At salt concentrations above 2 per cent,to salts.Volumeoditbu. i s w bacteria the cells plasmolyze, and as the protoplastsVolume of distribution studies with bacteria rik th phsht-memebevlm.e

have led to some controversial results. In a series .t pof studies, using isotope techniques, one group of creases in accordance with the van't Hoff equa-investigators (86) came to the conclusion that in tion (figure 5), the intercept representing theE..oiandT"heprtolam nonsolvent volume. The cells behave as thoughE.olad Truopisutiis "he roopas the cell wall is permeable to NaCl and to phos-

may be likened to a sponge, the cell membranesto a surrounding hair net unable to exclude the phate, but the protoplast membrane is not. Inentrance or emergence of small molecules." They still another recent study (59) using the same

tested a number of inorganic ions including strain of E. coli, the sulfate space was found to

sulfate,phosphat,1K 1Rb C1 ad Na In be 35 per cent of the cellular volume using inulinsulfate,~~phshae '+.+C .+ an.a.I as the reference substance.each case, the volume of distribution in the cell as thereferencesubtance.'. . . . . ~~~~~In the most recent edition of their book (1957)

even in very short periods of time was equal to Roberts et al. (86) have added an addendum inthe total cellular water content. Mitchell and which they state that "In January, 1955, aMoyle (69) have taken issue with these results. change was noticed in the 'water space' of E. coliUsing the same organism (E. coli, American ,,strain B) and same culture conditions, they con- mesured0by Set the sate ow dhscluded that E. coli "possesses an osmotic barrier tributesm 20 per cent of the water space of thethat is relatively impermeable to sodium phos- cell, cor wit e cent previously. The

phates and to sodium chloride. ... We cannot values for phosphate and sodium, previouslyreconcileorresults ith those o Robe~, ~ ,, 100 per cent, are now 40 and 55 per cent. Noreconceorrreason could be found for the differences before

Mitchell and Moyle carried out several types of and after 1955.studies with E. coli. The swelling experiments, Several other orhave been investi-which measured turbidity changes, have already gated by volume of distribution techniques Inbeen mentioned. By volume of distribution S. aureud (69 71), the extracellular volume of amethods, the extracellular water in the packed centrifuged pad is 25 per cent, using dextran as acells was found to be 22 per cent based on reference substance. Phosphate is excluded fromdextran as a reference substance. The phosphate- about 90 per cent of the remaining "space,"permeable space was only about 10 to 12 per presumably the interior of the cell, for at leastcent of the cellular volume (after correction for 3 hr (figure 6). On the other hand, the chlorideextracellular space). In a further experiment, the space increases slowly in this time, indicating a

phosphate-impermeable volume was measured slow penetration into cellular water, and thio-as a function of the salinity of the medium, using cyanate distributes rapidly into the cellular


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variation is undoubtedly due to variations in(H2PO*+HPo-) * the speed and time of centrifugation. For ex-

w2 -0 ample, with a press, the interspace in yeast canbe reduced from 22 to 4 per cent (11). The

E -'_ atsecond space is one accessible to all small mole-* cules and ions, but not to macromolecules. ValuesE.ws ranging from 12 to 40 per cent or more have>\ been reported. This space is outside of the

permeability barrier of the cell, and probablyE If_CASTS constitutes the cell-wall space. Distribution

E10- --S * -*studies with isolated protoplasts, as compared* with the walled organism, are in accord with0 _ this suggestion. The third space is within the

Time (min.) protoplast. It can be demonstrated only withsolutes that penetrate the cell membrane duringFigure 6. Time course of phosphate-, chloride-, the time of observation.

and thiocyanate-impermeable volumes of Staphy-lococcus aureus (strain Duncan) at 2 C (69). Why is so much variation observed in different

studies? A number of factors can be listed. (a)Differences in age of culture and nutritionalwater. If the cell is treated with butanol, then variation may influence cell size, wall thickness,

the phosphate space increases markedly, whereas and permeability. (b) The technique itself is in-the dextran space does not, again demonstrating herently imprecise. For example, small differencesthat the cell wall is a barrier to large molecules, in concentrations of the solute in the super-whereas the protoplast membrane is a barrier natant become magnified when cellular spacesto phosphate. In B. megaterium, the behavior are calculated. Analysis of the pellet rather thantoward phosphate is similar to that of S. aureus the supernate removes some problems but in-(119). The distribution studies indicate that the troduces others. (c) The cellular compartmentcell wall is permeable to phosphate, whereas the may increase or decrease in size if the outsidecell membrane is not. By the use of protoplasts solution is hypo- or hyper-tonic due to shifts ofwith no cell walls it has been confirmed that the water. (d) If isotopes of ions are used, exchangesphosphate-impermeable barrier is at the outer may occur across the membrane which do notboundary of the cytoplasm (119). In Desulfoibrio represent net solute movements (systems whichde8Wsfuricans (58) the distribution volume in- we have already described in detail). (e) Thedicates no penetration of NaCl or of Na2SO4 assumption is made that a given concentrationinto intracellular water, unless the cell mem- of solute represents the same volume in the bulkbrane is first rendered permeable by treatment solution of the medium as in the cell wall orwith a surface-active agent. If the cells are cytoplasm. This ignores Donnan effects, iontreated with hypertonic solutions, they lose binding, hydration water, and nonaqueouscellular water and the distribution volume of the phases. (f) Volume-surface relationships changesalts increases. This effect is reversible and is drastically with the size of the cell. Very smallapparently due to swelling and shrinking of the changes in the thickness of the cell wall relativecells, although no microscopically visible plas to the diameter of the cell constitute very largemolysis was found. differences in the relative volumes. For example,The volume of distribution studies, except for in a cell 2MA in diameter, with a wall 0.2Mi thick,

the original studies of Roberts et al. (86), all the cell wall occupies 49 per cent of the cellindicate that in a packed pellet of microorgan- volume. If the thickness of the wall is reducedisms, at least three kinds of "spaces" can be to 0.15, 0.10, or 0.05 ,u, the cell wall volumes be-differentiated. The first space, determined by come 39, 27, and 14 per cent, respectively.distribution of macromolecules such as dextran,inulin, and protein, is the interspace between E. Conclusions Based on Osmotic andpacked cells. The theoretical interspace for close Distribution Studiespacked spheres is 26 per cent. The experimental From the studies reported in this section, onvalues range from 6 to 25 per cent. Some of the the retention of cellular solutes, on the exclusion


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of extracellular ions, and on the various osmotic permeable to some degree to monovalent cations.responses, it is inescapable that bacterial cells Because of the anion impermeability, cationpossess membranes which act as diffusion bar- movements always involve an ion exchange.riers, restricting the rates of movements of ions Most of the studies with yeast have beenand other solutes to varying degrees. The concerned with potassium exchanges. The in-observations on plasmolysis and on isolated ternal concentration of K+ is very high, overprotoplasts indicate that the diffusion barrier for 0.2 M, yet cells suspended in water leak potassiumsmall molecules and ions is not the cell wall, but only slowly, in exchange for H+ (99, 100, 109).the outer surface of the protoplast. The wall During active metabolism, potassium is rapidlyitself does exclude macromolecules. It must be absorbed also in exchange for H+ (see next sec-kept in mind, however, that osmotic behavior is tion for details). However, the uptake measureda response to differences in osmotic pressure be- by isotope technique (K42) is larger than thattween the medium and the cytoplasm, not to the measured by chemical analysis, indicating anchemical identity of the solutes. In the case of exchange of K42 for cellular K+, in addition toelectrolytes, only one species of ion (either the net uptake. In other words, even with acation or anion) need be restricted by the mem- large net inward movement of potassium (activebrane. For example, if the membrane is im- transport), some outward movement (efflux)permeable to anions, but freely permeable to with the diffusion gradient could be measuredcations, the osmotic gradient will still be main- (38, 98). This component represents the permea-tained because cations entering the cell must be bility of the membrane to potassium.electrically balanced by equivalent numbers of A new technique for measuring K+-effluxcations leaving the cell. Such exchanges can be without using isotopes has allowed a detailedmeasured by chemical techniques if unlike ions examination of this system (99). The cells areare involved, for instance an exchange of K+ for packed on a column on a sintered glass disc.H+. In many cases, however, the ions are in a Potassium-free solutions are passed throughsteady state situation with respect to each other, under pressure, at a rapid rate, so that the leak-so that no net chemical changes occur, even age of K+ raises its level minimally. The effluentthough particular ions are exchanging rapidly. solutions are collected serially and evaporatedSuch exchanges, K+ for K+, for example, can be sufficiently so that the K+ levels are within themeasured only by the use of isotopes. An evalua- range of the flame photometer. When distilledtion of the permeability of a given membrane to water, or acidified water, is passed through theindividual ions requires chemical measurementsto determine net movements of cations andanions, and also isotope measurements, toevaluate the ion exchanges.

s0III. PERMEABIMITY TO INDIVIDUAL IONS a uu\wv

The permeability factor in electrolyte metab-olism has been assessed in many animal andplant cells, but in very few microorganisms. Some Zcells, it is true, have been shown to be "leaky"

30

when washed, losing cellular phosphate and \potassium, while other cells are relatively non- X '°/leaky. However, such studies do not constitute 5a very exact description of permeability. The 1 '° 0yeast cell has been most extensively investigated = 0(91). The pattern in this case is rather simple. oThe membrane is relatively impermeable to all 0 20 30 4.0 5. 6.0anions tested, including phosphate, chloride-, pHsulfate, pyruvate7, citrate-, and succinateW. It Figure 7. The effect of pH on K+ efflux withis also impermeable to bivalent cations such as and without glucose and buffer (99). Yeast, 500 toMg+, Ca++, MnH, Sr++, and UO2, but is 800 mg; glucose, 0.1 m; TST buffer, 0.02 M.


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yeast column, the efflux of K+ is balanced by an 109

equivalent influx of H+. For this reason, a higher 8H+ concentration leads to an increased rate of 7K+ efflux (figure 7). Sodium cannot compete 6 /effectively with H+ for exchange, but if the pH Sis raised to 5.0 or higher, so that H+ is very t-& 4 -

low in concentration, then Na+ will exchange for 3 -

K+ (15). Many other factors influence thepermeability of the membrane to cations, in 2

-

sometimes complicated and unpredictable ways.For example, metabolism of sugars increases thepermeability, whereas metabolism of lactate oralcohol does not. Several metabolic inhibitors 0 10 2 3 ; 70-10 0 l 0 4 o 6(azide, cyanide, and dinitrophenol) increase the Time in minutesrate of efflux, but the exclusion of 02 has the Figure 8. The plot of log (1 -Ro)/(l - Ra)opposite effect. The effect of temperature is against t the PH moving into the cells,-@- ;most complicated, with a maximal rate of efflux the P32 moving out of the cells,-O-O-; atat about 28 C. In summary, the permeability of pH 7 and 25 C (64).the yeast membrane to cations is not a fixedproperty, but is influenced by the physiologicalstate of the cell and by many environmental active transport). The exchange system forfactors. phosphate is highly specific for the monovalentMeasurements have been made of the dis- ion, H2POf-, for which arsenate is the only ion

tribution of Nat' and K'2 in a centrifuged pellet that can substitute. The rate of exchange isof cells of E. coli, by isotope techniques alone. influenced by many factors. In particular, theThe distribution of the Nat and K+ was rapid effect of phosphate concentration provides someinto total cellular volume (19). Later, however, insight into the mechanism of the exchange. Asthe same group found a rapid distribution for the concentration is raised, the rate of exchangeNaO of only 55 per cent (86). By isotope tech- increases asymptotically. Implicit in this rela-nique alone it is not possible to differentiate tionship is the concept of "saturation" of abetween net uptake and ion exchange. In the receptor substance or carrier in the membranesame organism (E. coli) in the resting state, with which phosphate must combine during itsdirect measurements of K+ retentivity indicate entry into or exit from the cell. Figure 8 repre-a very low rate of loss in exchange for Na+ and sents data based on such an assumption for Pna somewhat higher rate in exchange for NH,+ influx and efflux. The equation predicts a straight(37). The E. coli was nearly as retentive for line of (1 - Ro) /(1 - R+) against time wherepotassium as the yeast cell. Ro and R+ are the relative specific activities ofSome interesting studies have been carried phosphate initially and at any given time. The

out on the permeability of cell membranes to kinetics of inhibition of the exchange by phenyl-phosphate, using S. aureus (Micrococcus pyogmene) mercuric ion also supports the concept of aand E. coli (64, 69). It has been pointed out membrane receptor in phosphate exchanges.previously that these organisms in the resting In summary, some organisms are relativelystate behave as though impermeable to phosphate impermeable to phosphate. Others are moreby osmotic criteria, or by volume of distribution permeable to phosphate, but because of thetechniques. Nevertheless, P'2-labeled phosphate obligatory exchange imposed by the mechanismwhen added appears in the cell within a short of penetration (exchange diffusion), the celltime. Because the isotope equilibrates into the behaves as though it were relatively impermeablecell with no net movement of phosphate, an in the sense that net movements of the ion doobligatory 1 to 1 exchange of extracellular and not occur. Consequently, any cation movementscellular phosphate must be involved. It should must also be balanced in a cation exchange sys-be noted that in the presence of substrate, the tem. It should be pointed out that no cell mem-rate of efflux is markedly reduced so that a net branes are completely impermeable, nor are theuptake of phosphate occurs (see next section on exchange-diffusion mechanisms 100 per cent


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effective. Thus on washing, some of the cellular prerequisite to entry, then some of the organicelectrolytes diffuse out. phosphate compounds of the cell should have a

higher initial specific activity than the inorganicIV. ACTIVE TRANSPORT OF ELECTROLYTES phosphate of the cell. Evidence of this kind has

.. ~~~~~~~~beenpresented for red blood cells (34, 81) butIf permeability factors alone were to operate, te dataefor or tes (106) and for micro-'the data for other tissues (106) and for micro-then eventually even a cell with a relatively

impermeable membrane would reach an equl- orgis (4)au e s w equivoalrlibrium state with the environment with respect mnybcueo ehia rbes aeetiffumsibe wio. the restirngell (noresub phosphate, after it enters the cell, exchanges intostrates)uthis tend.en is rev nting te.lossbo all of the metabolic pools associated with phos-

phorylation cycles. In addition, the size of thepotassium from yeast and the losses of total pools is altered during active metabolism. Be-electrolytes from other organiss on cause of the complexity of the situation and be-However, when subetrates are supplied, theHowever,whe su'tratesare suppliedth cause the separate phosphate pools are mixedactive transport systems accumulate ions against together after the cell is destroyed by extractants,the concentration gradient.Althcoughntration graccum tiont hasbeedemo interpretation of the P32 data is most difficult.

sltrated ionavarietycofumulaticroorgan detailed Other studies have been carried out by meas-

invstigatdion other of theame,dbranei uring the rate of uptake of phosphate from theinvetigaionsofhe rle o th memblran n under a variety of conditions. In S.the process have been carried out in only a few meum ner a vari of citios. Intspecies. The yeast cell has been investigated aureus net uptake occurs ony with glucose,butextensively, but because detailed reviews of no ihohrsbtae uha lcrlextensively,but because detailed revies o lactate, alcohol, or pyruvate. In some experi-some of these studies have appeared elsewhere ments all of the measurable phosphate wasthey will be oniy summarized here (7, 8, 91). absorbed. Thus the movement is against theNot all investigators have agreed that the concentration gradient, for the cells containmembrane plays an important role in accumula- inorganic phosphate. Because gramicidin in-tion. The specific binding of accumulated ions h t

thcyol. a en rpsd sahibits the uptake of phosphate and becausewithin the cytoplasm has been proposed, as an ...

alternative mechanism. gramncidin is thought to act on the cell surface,it has been suggested that the transport mech-anism for phosphate is in the cell membrane

A. Transport of Phosphate (42). Later studies with S. aureus implicate theThe earlier studies on accumulation of phos- exchange diffusion system of the resting cell as

phate have been reviewed by Kamen and part of the active transport process (67). InSpiegelman (47). They discuss two possible the resting cell, the rate of flow of phosphate intoschemes of entry of phosphate into the cell and the cell is exactly equal to the rate of flow out ofinto the metabolic pools, first, the diffusion of the cell. When glucose is added, the flow ofinorganic phosphate through the membrane, phosphate in the inward direction is unchanged,followed by esterification within the cell and but the outflow approaches zero, so the netsecond, the entry of phosphate by an esterifica- effect is an inward movement against the con-tion process in the membrane, followed by mix- centration gradient. These and other studiesing into metabolic pools. Evidence in favor of indicate that the exchange-diffusion system of thethe latter mechanism includes the following: (a) resting cell is utilized for transport when sub-the temperature coefficient is too large to be strate is present. Thus the properties of theexplained on the basis of simple diffusion, (b) exchange system are also the properties of thethe uptake of phosphate is severely restricted active transport system insofar as kinetics andby such agents as azide, arsenate, and iodoace- specificity are concerned.tate, which block phosphate esterification, and Although the resting yeast cell is impermeable(c) the rate of exchange of phosphate is increased to phosphate (33), whereas S. aureus has anby substrates. exchange-diffusion system, the active transportAttempts have been made to determine the systems are similar in both organisms: (a) a

nature of the entry process for the phosphate by specific substrate, glucose, is required; (b) thedetermining the specific activities of the cellu- phosphate moves against an apparent concen-lar fractions, using P32. If esterification is a tration gradient; (c) the phosphate moves into


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1700

p21600 -

20

1Z500 -

z ~ ~~~ ~~~~ CHEMCAL P 19 EIL~

0. \ \ P32J 1400 - \-I 1400

emRELATIVE SPEGFIC ACTMTVI

4~~~~~~~~~~~~,_1300

5200

O00~~~~~~~~~~~~~~~~~~~~~~~01100 -

0 30 60 90 520TIE IN MINUTES

Figure 9. Disappearance of p32 and of chemical phosphate from the medium during the fermentationof glucose by yeast (33). The yeast concentration was 50 mg per ml of suspension; pH, 7.0; glucose, 0.2 m;and initial phosphate, 0.022 M.

the cell with almost none moving out, and total electrolyte and acid-base balance in thetherefore the specific activity of the extracellular cell. As H2P0j- moves into the cell, the equi-phosphate remains constant, undiluted by un- librium,labeled phosphate from the cell (figure 9); (d) H+ + HPO4- H2P04-only the monovalent ion H2P0- is absorbed; in the is shifted to the right and the(e) the kinetics of uptake are of the "saturation"type and can be fitted by the Michaelis-Menten medthe recon whte cllis st to theequation, giving Michaelis constants of 4 X tlme the reaction wth the cell sshifted to the10-moreasan 8X 1-4 fo S.aurw;left with an increase in acidity (the normal

.. ~M fo es n O o. aues average cytoplasmi pH of fermenting yeast is(f) inhibitors of phosphorylation reactions such avrg yolmipH ffeenngests

.inhiitrof phophryan areeati as 5.8 to 6.2) (12). A potassium-rich cell, however,asocdinitropenol, has a more allaline cytoplasmic pH and a largebokssing cagn (47).dly stimulate phosphateamount of fixed base. It can, therefore, absorbPotassium can markediy stimulate phosphate muc moepopaeta 'po elmuch more phosphate than a K+-poor cell,uptake by yeast (89, 108), by E. coli (84), and without upsetting the acid-base balance.

by Bacterium lactis aerogenes (26). In yeast (33) If K+ and phosphate are present at the samethe action of K+ is largely dependent on pH time, the potassium is absorbed more rapidiy(figure 10). The uptake of phosphate, with or ..without K+, is restricted at high pH because the iAl but after a period of time both ionstransported anion H2PO is reduced in con-

are absorbed at about the same rate. The two4on uptakes tend to balance each other out, eachcentration. It is restricted at low pH because of .i b a

reduced metabolism. At intermediate values of from tec ( ha nd e posphatepH the stimulating action of K+ is considerable.The action of K .is indirect. Indeed, the potas- being associated with the gain of a H+ by thesium does not even have to be present at the cell. Thus the two ion movements are linked,time of the phosphate uptake (94, 95). If cells even though the mechanisms of transport are

are first exposed to potassium plus glucose, the entirely independent.potassium is rapidly absorbed. The K+-rich cells B. Transport of Monovalent Cationswill subsequently absorb considerably greater Microorganisms in general require potassiumamounts of phosphate. The problem is one of for growth. Although rubidium will substitute,


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2

I- 160

120

042

Xso

I.- NO~~~~~K*

0~~~~

I 2 3 4 5 6 7 S 9 O

pH

Figure 10. Effect of pH on the uptake of phosphate in the presence and absence of K+ (33). The yeastconcentration was 50 mg per ml; the glucose, 0.2 m; and the K+, 0.02 m.

sodium will not (23, 56). The actual absorption measure K+ uptake in B. lacti aerogenes (23, 24,of potassium has been measured in few organisms. 26). In this organism, a predominant factor is Ht.The first observations in yeast (38, 83) have For example, when K+ is absorbed, the mediumbeen followed by others in E. coli (57, 85) B. becomes acidified, suggesting a K+-H+ exchange.lactis aerogenes (23, 24, 26), Alcaligenes faecalie If the initial pH of the medium is low, the K+(53), and marine bacteria (60). In each case, the uptake is reduced. The uptake of P'2-labeledabsorption occurs only after the addition of phosphate is somewhat parallel to that of potas-substrate, but no source of nitrogen is necessary. sium, but the amount absorbed is less. Again itAny utilizable substrate will do (77). Rubidium is not possible from the isotope measurementscan be absorbed in place of potassium, but Nat to determine the contributions of the net uptakeis poorly absorbed. All of the potassium that is of potassium and of the K+ exchanges.absorbed may not be retained after the sub- Studies of cation absorption by yeast arestrate is exhausted, but all that is retained is rather numerous. Fortunately, much of the ma-readily exchangeable for KC3 added to the terial has already been reviewed (7, 91), so thatmedium. only the latest material need be dealt with in

In E. coli (85) the uptake of potassium is any detail. The impetus to studies of this or-dependent on (a) glucose concentration, (b) K+ ganism was the simultaneous but independentconcentration, (c) temperature, (d) pH, (e) the observation by two groups that K+ was ab-nature of the buffer, (f) aeration, and (g) meta- sorbed in a 1 to 1 exchange for H+ (16, 100). Inbolic poisons. Many of these factors also in- fact, with high K+ concentrations, the amountfluence the outward movement. In this study, of H+ released is sufficient to reduce the pH ofpotassium distributions were measured by the medium to 1.7. Although the H+ is derivedisotope techniques alone, so that it is not possible from metabolic reactions, no accumulation ofto separate the net movements of potassium H+ occurs within the cytoplasm (12). In fact,from the exchanges. somewhat more H+ is secreted than is produced,

Isotope techniques alone were also used to so that the pH inside the cell increases. Another


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indication that the driving force for exchange is 4

not the accumulation of intracellular H+ is thefact that K+ and H+ exchange proceeds rapidlywhen the extracellular pH is below 4.0 and the 4

potassium level is below 1 X 10-3 M. The intra- Ccellular pH is over 6.0 and the K+ level over 3 UV0.15 M. Thus each of the ions involved in the pHexchange moves against an apparent concentra-tion gradient of over 100 to 1 (98). Conway et al. 3

have suggested that the H+ must be producedin an outer zone of the cell near the membraneacross which the exchanges occur (17). This sug-gestion is in accord with other studies whichindicate that the glycolytic reactions in the yeastcell are located in a structure underlying the TIME IN MINUTESmembrane (105). 4 50 60

Various properties of the K+ transportingsystems have been investigated. The relative Figure 11. Changes in the pH of the mediumeffectiveness of ions other than K+ to be taken during fermentation of glucose by yeast, as in-up in exchange for 1+ can be demonstrated by asimple experiment in which the degree of acidifi-cation of the medium is used as a measure of 1.5ion exchange (figure 11). The pH, in unbufferedsolutions, drops rapidly with K+, less rapidly 1-4with Rb+, and slowly with Na+ and Cs+ (91).Conway and Duggan have investigated the ion 1 3specificity in much greater detail (13). They find i.2_by competition studies that the affinities of ions 1Iyfor the transporting system is in the order of 1.1K+ > Rb+ > Cs+ > Nat > Lit with relativevalues of 100:42:7:4:0.5. As the pH of the 1-0medium is raised, Nat uptake is increased be-cause of reduced competition by Ht, which has 09a high affinity for the transport system. In the I I I Iabsence of K+, large amounts of Nat can be 0 0.1 02 0*3 04taken up, largely replacing cellular K+ (15). The 1/[RbfJsodium-yeast thus prepared can be used forstudying Na+-K+ exchanges. Conway et al. Figure 12. Double reciprocal graph 1/V againstsuggest the cell has two transporting systems, 1/(Rb+), where V is the rate of uptake of Rb+one in the inward direction, highly specific for ions (mEo/kg/nin) by fermenting yeast, andK+, and one in the outward direction, highly (Rb+) the external rubidium concentration (13).specific for Nat (18). The two systems respond The yeast was suspended (1 g per 20 ml) in fluidspecifirentlfor inah..Tentwote pond which contained 5 per cent of glucose (w/v) anddifferently to inhibiting agents, the potassium was buffered to approximately pH 6.system being sensitive to cyanide, azide, ordinitrophenol. Foulkes, on the other hand, doubtsthat a separate Na+ system is necessary to ex- where V is the rate of the reaction, S the sub-plain available data (29). strate concentration, VM the maximal rate, andThe kinetics of K+ uptake follows a typical Km the Michaelis constant. Figure 12 shows

"saturation" curve that can be fitted by the data for Rb4 uptake. The intercept representsMichaelis-Menten equation for enzyme kinetics, l/Vm and the slope, KM/Vm. At pH 4.0, theImKm for K+ is 5 X 103 M (98) but at pH 6.0,

1 = K 1 +1 the value is about 3 X 1ow- M(13). The value atV VM S ViM pH 4.0 is higher because of the competition of


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H+ with K+. The kinetic data, the competition hand, much less is known about the transportbetween pairs of ions, and the inhibition by of bivalent cations such as Mge, Ca++, andmetabolic inhibitors indicate that K+ combines Mn++ except for a few studies with yeast,with a membrane receptor or carrier during its which are summarized below, and some studiestransport. One estimate of the carrier concen- of plant roots (27).tration is 0.09 mm per kg (wet weight) based on Magnesium ions can be transported into thethe displacement of the potassium by Rb+ (13). yeast cell during active metabolism, providedA second, in good agreement, is 0.1 mm per kg that the pH is above 6.0 to 7.0 and that nobased on binding of azide in sufficient quantity potassium is present (9). The mechanism is theto inhibit K+ transport (29). The chemical nature same as that which transports potassium, andof the carrier is not known. Because Mg++ com- which has been described in the previous section.petes ineffectively against K+, Conway and The transport of Mg& is inhibited by H+ andDuggan believe that a polyphosphate (for which K+, because these ions have a much greaterMge could compete effectively) is not involved. affinity for the carrier. In fact, the relativeLargely because of the inhibitory effects of affinity of K+ to Mg++ is 200 to 1. The transportaside and cyanide, they do believe that an iron of Mg++ by this mechanism probably has littlecompound (for which these inhibitors would physiological significance because conditions forhave an affinity) is involved (13). its operation are so restricted (high pH, low K+,The net movements of potassium are condi- and high Mg)).

tioned by many factors. Already mentioned are: The yeast cell also possesses a specific trans-the presence of other competing ions, especially port mechanism for bivalent cations whichH+; the substrate; the presence of certain in- operates at any pH and which is not inhibitedhibitors (at concentrations that do not inhibit by potassium (44, 95, 104). Because no con-metabolism); the uptake of phosphate; and the venient isotope of Mg++ is available, most of therate of K+ efflux. Other influential factors in- work was carried out with Mn++, using MnI asdude the K+ content of the cell (98), and the a tracer. As in other transport systems, a specificredox potential of the medium (14). In assessing carrier seems to be present in the membrane withvarious factors it must be kept in mind that which the transported ions can combine. Foralthough the initial rate of K+ uptake is prob- example, the kinetics (rate of uptake versusably a function of the transport system, the concentration of cation) is a typical saturationamount of K+ taken up is conditioned as well by curve with a Michaelis constant of approxi-the total balance of ions in the cell. Under mately 1 X 103 m. Furthermore, pairs of similaroptimal conditions a cell may rapidly double its ions compete for the carrier. Not all cations areK+ content. The extra potassium must be equally effective. Mg is preferred over Mn-balanced either by an uptake of phosphate, or by by a factor of 3, over Ca++ by a factor of 10,metabolic anions such as bicarbonate and suc- and over Sr++ and UO2++ by a factor of 50 tocinate (10). In the latter case, an equivalent 100 (figure 13).amount of H+ must be excreted. If a source of The transport of Mn++ requires the substrate,nitrogen is present, the cell will grow and use glucose, and it is also dependent in a peculiarthe extra electrolytes, but in the absence of a way on phosphate. Resting, starved cells have nonitrogen source the cell will simply increase in transport capacity even if supplied with glucose.osmotic pressure and in turgor against the cell However, if phosphate is added, then Mn'+ iswall. When the substrate is gone and the trans- rapidly absorbed. The higher the phosphateport system shuts down, some of the K+ dif- concentration, the more rapid the absorption.fuses back out of the cell but at a low rate com- The two ions are not, however, absorbed at thepared with the uptake (99, 100, 109). same rate, phosphate uptake being more rapid

than that of Mn- | (figure 14). Furthermore,C. Transport of Bivalent Cation Mn++ absorption continues even after no phos-

phate is left in the solution. In fact, the cells canInformation concerning the active transport be pre-exposed to phosphate and glucose and

of Nat, K+, and phosphate has been derived then washed. They will then have acquired thefrom many kinds of cells from animal and capacity to absorb Mn+. Thus the phosphateplants as well as microorganisms. On the other and Mn'+ do not have to be present at the same


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This fact, plus (a) the linkage to glycolysis reac-MIOC ~ tions (glucose specifically supplies the energy for

both phosphate and Mn. transport) and (b)80 the sensitivity of the Mn+ transport system to

arsenate (which specifically competes with phos-0 / phate in esterification reactions) suggest thatz6C ~ / ^Pn++^g the MnH carrier is a phosphorylated componentW / / of the membrane. The specificity pattern, on< 40_ the other hand, is similar to that of phosphorylat-

ing enzymes, suggesting the presence of a pro-Z tein component. One other feature of the system020

+42 C ~_/requires comment. Bivalent cations can be bound

co W on the surface of the yeast cell. The bound ionsC0 20 40 60 80 100 120 are completely exchangeable (101). The ionsX16 20 40 60 -80 100 120 that are transported into the cell are, on the other

TIME IN MIUTES hand, no longer exchangeable. In figure 15, theFigure 18. Specificity pattern in the absorption Mn54 taken up in the absence of phosphate repre-

of bivalent cations by respiring yeast (104). The sents surface binding. Addition of large amountsyeast concentration was 50 mg per ml; all ions, of unlabeled Mn+ results in a complete de-5 X 10-'4 M; glucose, 0.1 M. Corrections were made sorption of Mn". However, the large amount offor surface binding, by controls with no glucose. MnM4 taken up in the presence of phosphate and

potassium cannot be exchanged. The transportmechanism is essentially irreversible, allowingno back diffusion. In this regard, it resemblesthe transport systems for bivalent cations inplant cells (27).The relationship of potassium to the trans-

z port of Mn+ follows an interesting pattern.LO Potassium does not compete with Mn++ for

transport. In fact, potassium stimulates the0\k transport to a marked degree (figure 15) with

an optimal effect when the ratio of K+ to Mn++0 10 20 30 40 is approximately 2 to 1. Yet, the K+ is almost

TIME IN MINUTEcompletely absorbed before the uptake of Mn++

Figure 14. The disappearance of K+, Mn", starts. Thus, as in the case of phosphate, it isand phosphate from the medium of respiring yeast the absorption, not the presence of the K+ thatcells (104). The yeast concentration was 100 mg is imporptant In tepexse dto pthaper ml and glucose, 0.1 M. The glucose, K+, Mn++, Is important. In fact, cells exposed to potah-and phosphate were added at zero time. sium plus glucose, which are washed free of this

cation, will later show a markedly enhancedMnul uptake. The stimulatory action of potas-

time. The Mn++ transporting system is activated, sium is an indirect effect. Potassium increaseswhenever phosphate is absorbed. If the phos- the phosphate uptake. In turn, phosphate uptakephate-treated cells are allowed to stand, the is necessary for the formation of the Mn++ability to absorb Mn++ decreases at a rate de- carrier. If the ratio of K+ to MnH is muchtermined by temperature (at low temperatures, greater than 2 to 1, then the stimulatory effectthe absorptive capacity is retained longer) and is markedly reduced. Apparently a cell with anby the metabolic activity (with glucose, the excessive amount of K+ has a reduced capacityabsorptive capacity is rapidly lost). to absorb other cations.The cell has large supplies of internal phos-

phate, but these will not serve to activate the D. Mechanisms of TransportMn++ transport system. Only when extracelular The various transport systems in micro-phosphate is transported through the membrane organisms and in other cells as well, have certaindoes synthesis of the Mn+ carrier take place. common features:


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192 ASER ROTHSTEIN VOL. 23

100 i s-80 s .03M Me'

z°|$ iL j ~~~~ADDED

GLUCOSE ONLY

C 20 L-OR P ONLY

0 10 20 30 40 50 60 7O 80 90 TIME IN MINUTES

Figure 15. Binding and absorption of Mn+ by yeast cells (95). The yeast concentration was 100 mgper ml; Mn^, 7.5 X 10-' M; PO4, 5 X 10- m; K+, 1 X 10-' M; and glucose, 0.3 m. At 60 mintheMn"concentration was raised to 3 X 102. The uptake of MnH was measured by y emission from Mn'added in constant amount to each flask at zero time.

1. The ions may move against the electro- anions produced by metabolism enter into thechemical gradient. In studies with microor- total electrolyte balance, the acid-base regula-ganisms the gradient is not known precisely tion of the cell is also involved.because the membrane potential has not been A number of specific and general mechanismsdetermined. However, in the K+-H+ exchange have been proposed, none of which will be dis-system, both ions may move against 100 to 1 cussed here in detail. Two problems must beconcentration gradients. Because they move in met, the first being the mechanism of ion move-opposite directions, the electrical gradient for ment through the membrane and the second thethe pair cancels out. The concentration of free coupling of the energy source necessary forK+ in the cell is not exactly known, but binding active transport. Proposals to meet the firstcan account for only a fraction of the total problem include: fixed carriers, diffusible car-potassium. riers, carriers dissolved in the lipid phase of the

2. In some cases (Mn+ and phosphate in membrane, contractile proteins, and reorientingyeast) the ions move in the inward direction proteins, among others. Metabolic coupling hasonly. An essentially irreversible step is involved. been suggested via phosphorylation reactions

3. A source of metabolic energy is required and by redox reactions. In the case of phosphate,but growth conditions with a source of nitrogen direct incorporation into metabolic compoundsare not. In some cases a specific substrate is is an obvious solution. With bivalent cations,required (phosphate or Mn++ require sugars) combinations with protein-adenosine triphos-and in others any substrate will do (K+). phate complexes might do. With K+ and Nat,

4. That the absorptive process involves a however, the possibilities are not so obvious.combination with specific receptors or carriers is Although some biological substances such asindicated by (a) saturation kinetics, (b) compe- adenosine triphosphate or nucleic acids will formtition between pairs of ions, (c) a high degree of complexes with either Nat or K+, the discrinmina-specificity (25 to 1 between K+ and Nat), and tion between the ions is negligible (93), whereas(d) specific inhibition by traces of inhibitors such the transport may discriminate by a factor ofas azide for K+, or arsenate for phosphate. 25 to 1. It is true that certain enzymes are ac-

5. Although the transport of anions and tivated by Ki and not by Nat, particularlycations proceeds via different mechanisms, the phosphorylation reactions involving adenosine-systems are linked in the sense that the number triphosphate. However, activation per se is notof positive and negative groups within the cell by any means a measure of combination, formust be balanced. Because H+ and organic Na+ may combine without effect on the en-


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zymatic capacity. It is possible, however, that of binding. It is not important here to reviewthe transport system involves a reorientation of these studies in detail because very few correla-the protein molecule in a reaction analogous to tions have been made between the fixed chargesan enzyme reaction, which would allow the and ion transport. Furthermore, it is- not cleartransport of K+ but not of Nat even though both which of these binding groups are located in thewere able to combine. Another possibility for cell wall material and which in the cell mem-discrimination between Na+ and K+ is by a brane. In the yeast cell, the cation-bindingcarrier dissolved in the lipid phase of the mem- groups include phosphoryl groups and carboxylbrane, which increases the partition coefficient groups (101), which can bind Na+ and K+. Bothof one of the ions far more than the other. For are weakly bound and no discrimination betweenexample, phospholipids will specifically increase them can be demonstrated. Moreover, thethe partition coefficient of K+ into a lipid phase cation-binding groups are not involved directly(111). in transport of K+, because they can be blockedActive transport requires metabolic energy. off with Mge, for which they have a high af-

The exact nature of the enzymatic coupling is finity, without diminishing the rate of K+not known. It has been demonstrated, however, transport (91). The binding sites can also bethat the cell membrane contains in its structure blocked off with UO2+ without interfering witha number of specific enzymes (90). Recent studies Mn+ transport (104).of a variety of cells including yeast, suggest that In the case of phosphate transport, becausethe glycolytic system of the cell is peripherally glycolysis reactions seem to be specificallylocated, perhaps in a zone directly under the linked, the glyceraldehyde-3-dehydrogenase stepmembrane, where coupling to active transport has been suggested as playing a central roleprocesses might occur (92, 105). In the bac- (33). This reaction is the only one in glycolysisterial protoplast, after osmotic lysis, a "ghost" of sugar, in which inorganic phosphate is esteri-can be isolated which is presumably the mem- fied. Data on relative specific activities of phos-brane of the cell and perhaps underlying struc- phate fractions in red blood cells during PnO4tures (66, 121). In S. aureus, B. meaterium, and uptake are certainly consistent with this hypoth-M. lysodeikticus, the "ghost" contains 90 per esis (34, 81).cent of the cytochrome and 90 per cent of the Mitchell (65) has proposed a general theory ofsuccinic dehydrogenase activity of the cell, as active transport based on studies of phosphatewell as lactic dehydrogenase and phosphatase uptake in various bacteria. He suggests that theactivity (32, 67, 117, 118). membrane contains an enzyme which acts as aThe presence in the membrane of fixed charges, phosphate carrier. It can be phosphorylated and

with which ions can combine, has also received dephosphorylated, with the translocation of themuch attention. Numbers of studies have been phosphate being due to reorientation of thecarried out with artificial membranes composed enzyme in the membrane in connection with itsof ion-exchange resins. If, for example, the resin enzymatic activity. He states, ". . . enzymes areis negatively charged, such a membrane will be the conductors of bacterial membrane transportimpermeable to anions, but permeable to cations -that metabolic energy is generally convertedby a cation exchange process (3). Such mem- to osmotic work by the formation and openingbranes will even discriminate to a degree be- of covalent links between translocators in thetween Na+ and K+ (75). membrane and the carried molecules exactly asThe membranes of microorganisms may, at in enzyme-catalyzed group-transfer reactions."

least to some degree, resemble ion-exchange Another schema is that of Conway (6-8) basedmembranes because they often contain a large in large part on his studies of K+-H+ ion ex-number of fixed negative charges. Membranes changes in yeast. It has similar features toare composed of the same kinds of constituents mechanisms proposed for gastric acid secretionas the cytoplasm, i.e., proteins, phospholipids, and for salt uptake by plant cells (1, 73). Thisetc. The charged terminal group of these con- schema involves the presence of oxidation-reduc-stituents will act as ion-binding sites, as demon- tion systems in the membrane, with a flow ofstrated by several techniques, such as electro- electrons through iron-containing enzymes,phoretic measurements, agglutination properties, similar to electron transfer in the cytochromeand virus binding, as well as direct measurements system. When the metal-protein complex is


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400 ,o process is, by definition, connected with the380 -

J membrane. Estimations of inside and outside30- are based largely on such criteria as distribu-

_ 320 - 6 tions of substances and estimations of cellular2300-280 - volumes. Knowledge of the location and thick-260 ness of the membrane is therefore limited by the

0 220-* accuracy of the measurement.220-, In microorganisms several barriers may be160 - w involved. The barrier to large molecules is more140-1 peripheral than that for small molecules. In the120 _ yeast cell it has been shown that the barrier to+..100g 80 - , bivalent cations is more peripheral than that for

o0 _ K+ and H+ (92) and that glycolytic reactions may20 - x be located in an inner zone of the cell surface

100 120 140 160 180 200 220 240 260I 300 (105). Volumes of distribution are not the same90 110 130 iSO 170 190 210 230 250 270 290 for all external substances.

4b (my) Some correlations can be made between theFigure 16. Effect of redox dyes on K+ uptake "physiological membranes" and the "cytological

during fermentation of yeast buffered by 0.04 M membranes." Knaysi (50, 51) has discussed thepotassium hydrogen succinate (14). The dyes used visual and electron microscopic observations onwere: Nile blue, *; safranine T. X; phenol-indo- bacteria. The outer part of the cytoplasm is2,6-dichlorophenol, V; o-cresol-indo-2,6-dichloro- sheathed by a cell wall and this in turn may bephenol, '0; phenol-indo-2,6-dibromophenol, V; covered by a slime layer. The cell wall can beo-chloro-indo-2,6-dichlorophenol, *; o-chloro- distinguished from the outer membrane of thephenol-indophenol, 0; Janus green, +; concen- protoplast by differential staining with certaintration of dyes, 10

*M.

basic dyes (88) and by appropriate electronmicrographic techniques (48). It seems reason-

reduced, an extra negative charge is produced able that the barrier to large molecules is theon which an ion is specifically absorbed. When it cell wall, while the barrier to small molecules isis oxidized, the ion is released. By appropriate an outer membrane of the protoplast, so that thecontrol of oxidation-reduction reactions on the differences in distribution represent "cell walltwo sides of the membrane, the system will ac- space." Consistent with this correlation are thecomplish an active transport of ions. In this increased volumes of distribution of substancescase the carrier itself is a redox system, its in plasmolyzed cells correlated with the visibleability to combine with the ion being dependent shrinking of the cytoplasm from the cell wallon its state of oxidation. The energy for trans- (69). Insofar as transport functions are con-port comes from metabolism which ultimately cerned, no correlations of physiological behaviordonates or accepts electrons. Conway et at. and cytological appearance can yet be made.believe that the redox system of the membrane The outer membranes of a variety of cells, asis an iron-containing enzyme, largely because of visualized by electron microscopy, may be quiteits affinity for azide (17, 29). They support the complex and interpretation most difficult (87).concept with many kinds of experiments. For Techniques have been developed for isolatingexample, the rate ot K+ and Nat transport in walls (126) and even membranes (32, 66, 67, 70,yeast (14) is directly related to the redox po- 117) of bacteria. The membrane fractions aretential of the medium (figure 16). isolated by mechanically disintegrating the cells

or by formation of "ghosts" by osmotic lysis ofV. NATURE OF THE MEMBRANE: CHEMICAL protoplasts. Components that have been identi-

AND CYTOLOGICAL OBSERVATIONS fied include complex lipoproteins, ribonucleicIn all of the physiological studies the cell acid, and a glycerophosphoprotein, as well as a

membrane is defined in operational terms, as a number of enzymes. Studies with intact cellsbarrier separating the inside and outside of the indicate the presence in the membrane of variouscell. If a process is influenced by extracellular enzymes such as phosphatases (102, 103) andrather than by intracellular factors, then that saccharases (22, 74, 125); of carboxyl and phos-


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phoryl groups (20, 101) and of sulfhydryl groups a Donnan distribution would obtain for those(78, 79). ions that can penetrate the membrane by diffu-But in toto the present state of our knowledge sion. In muscle, certain ions do distribute

of the chemical structure of the cell membrane according to the Donnan equilibrium (7) butis at best fragmentary, so that the mechanisms in microorganisms this factor has not beenof transport at the molecular level must remain evaluated.speculative and somewhat theoretical. Unlike Perhaps the most important extramembranemetabolic systems, which can be elucidated by factor entering into the equilibrium state is thethe isolation in turn of each of the individual binding of ions by cellular constituents. In fact,biochemical reactions with its responsible en- ion binding has been proposed to explain ionzymes, the transport system can function only in distributions and ion accumulation, as an alter-the integrated system, the intact membrane native to the concept of active transport by theitself. Even if the membrane can be isolated membrane. For example, K+ uptake by E. coliand broken down to constituent substances, the has been attributed to binding by hexose phos-means of directly identifying any of these sub- phates (85). Others have not been so specificstances with the transport machinery are meager. in identifying the "binding substances" (24,

53, 86). Actually, no direct assessment hasVI. EXTRAMEMBRANE FACTORS IN ION been made of the contribution of ion binding

DISTRIBUTION within the cytoplasm to ion distribution (93).A number of intracellular factors are of pri- Information concerning ion binding by cellular

mary importance in electrolyte distribution. constituents including macromolecules such asIt has already been pointed out that the general proteins and nucleic acids is, however, pertinent.metabolism of the cell must ultimately supply The nucleic acids present the simplest situation.the energy for transport, but metabolism is also The phosphoryl groups are located serially alongimportant in other ways. For example, metabolic the molecule in such a position that chelationreactions supply the H+ for exchange with K+, can occur, particularly with polyvalent cations.and the anions such as HCOj- and succinate Bivalent cations such as Ca++ and Mgd arewhich balance the extra K+ absorbed (10). firmly bound (but are exchangeable). UnivalentIn the case of phosphate, phosphorylation reac- cations are weakly bound with no particulartions also play a role. When phosphate is ab- discrimination between Nat and K+. Polyphos-sorbed, it does not increase the inorganic phos- phates, both inorganic and organic (adenosinephate content of the cell. Rather, it is stored in triphosphate for example) behave in a similarvarious phosphorylated intermediates and in the fashion. Simple esters of phosphate do not formyeast cell, largely as inorganic polyphosphates complexes with Nat and K+.(46, 124). Any form of metabolism is bound to It is beyond the scope of the present discussioninfluence ion distributions because of production to review the large literature on the protein-ionor destruction of ions. For example, glycolytic interactions (35, 49). A few general remarks arereactions produce organic acids from nonelec- germane. There are two classes of interactions.trolytes (sugars) and redox reactions produce The first type is relatively nonspecific and isor use up Ht. associated with terminal groups of single amino

Ions are not homogeneously distributed in cells. acid residues, the carboxyl groups of glutamicFor example, mitochondria are able to concen- and aspartic acids, the imidazole group of histi-trate K+ (72). Exchange studies in various dine, the sulfhydryl group of cysteine, and thecells indicate that K+ and phosphate exist in amino group of lysine. These groups are capablethe cells in pools that exchange with extracellular of binding a variety of ions. The alkaline earthions at different rates. At the present time, the metals are bound by carboxyl groups, Zn+quantitative contribution of internal non- largely by imidazole, Hg and Cub by sulf-heterogenity to ion distributions cannot be hydryl, Cl- by amino groups, etc. Such boundeasily assessed. ions are exchangeable. At physiological pH noThe equilibrium distribution of ions in the ab- measurable binding of Nat and K+ can be

sence of transport and metabolism is not a measured by such single amino acid residues.simple equality of concentrations. Because of the The second type of binding involves no correla-presence of nondiffusible ions in the cytoplasm, tion with particular amino acid ligands. Rather a


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unique configuration of the amino acids residues carboxyl groups of cellular proteins and phos-is involved which conveys a high degree of speci- phoryl groups of nucleic acid, which do not formficity. The binding of prosthetic groups of pro- specific K+ complexes. (d) When the membranetein enzymes falls into this category. No specific is injured by agents such as butanol, or by redoxbinding of Nat or K+ by such a special configura- dyes, or by drying or freezing or thawing, vir-tion has been demonstrated. It is true that K+ tually all of the cellular potassium is released.may specifically activate enzymes and often (e) The high internal osmotic pressure of theNa+ antagonizes. However, almost all such cell requires that most of the internal solutes becases involve adenosine triphosphate in phos- free. In nerve cells direct conductivity measure-phorylation reactions, and adenosine triphos- ments indicate that the K+ of the cytoplasm isphate is known to form complexes with Nat unbound. (f) Ions can be actively transportedand K+. Furthermore, where enzyme activation across layers of cells (frog skin or intestine) intoindicates binding, lack of activation does not solutions that have no possible binding sub-necessarily imply absence of binding. The Nat stances. (g) In cells in which measurements canenzymes might be inactive and the K+ enzyme, be made, the electrical potentials on the cellactive. membrane can be quantitatively accounted forWhat, then, is the contribution of ion binding by the ion permeability and active transport

to ion distribution? It can be calculated that in phenomena.cells in general the protein and nucleic acid con- In summary, ion binding within the cytoplasmtent of cells is sufficiently high that most of the might account for a small amount of potassiumbivalent cation content would be bound (93). accumulation, but membrane transport plays aNevertheless, in the only microorganism in predominant role.which uptake of Mg& and MnH has beenstudied, the yeast cell, binding cannot account VII. COMENTfor uptake because the ion cannot diffuse through Knowledge of inorganic electrolyte metabolismthe membrane to reach the binding sites. The and particularly the role of the cell membraneonly way that the ions can enter the cell is by has developed rapidly in the past decade. Thetransport through the membrane by the mecha- central role of the active transport systems isnism that has been described in detail previously well established. Many characteristics of the(44, 104). In other cells more permeable to membrane systems have been described in termsMg++ and Ca++, it is quite possible that ion of kinetics, inhibitors, substrates, specificitybinding is a primary factor in their distribution. patterns, and other criteria. Undoubtedly, manyOn the other hand, attempts to explain the more details will be described in years to come.accumulation of potassium purely on the basis of However, the basic problem of determining, ation binding within the cell, face serious problems the molecular level, the series of events thatwhich have been discussed in the body of the occurs in the membrane during active transportpapers cited and will be only summarized here. or exchange-diffusion or even penetration of(a) The assumption that the cell has no effective ions by diffusion, remains a little beyond us.

permeability barrier to ions is untenable in the The major obstacle in our path is our inadequateface of the many studies reviewed previously. knowledge of membrane structure and of the(b) No specific binding substances for K+ have chemistry of its constituent macromolecules.been demonstrated. (c) The logistics of the situa- As pointed out earlier, the biochemist can studytion are not compatible with the ion-binding the separate pieces of the metabolic machinery,hypothesis. The surplus of K+ over Nat in cells the enzymes; but the physiologist cannot takemay be equivalent to 0.2 M or more, and there- the membrane apart and recognize in any simplefore the binding substances would have to be way any of its functional pieces. He must trypresent in at least that concentration. Yet up to see its insides without being able to dismantleto half of the K+ is balanced off against diffusible it. He must therefore work blindly, using foranions such as HCOS , acetate, succinate, inor- "eyes" the various substances which influenceganic phosphate, or simple phosphate esters, membrane function. For example, lipid-solublenone of which form specific K+ complexes. Fur- substances "see" only the lipid phase of thethermore, most of the nondiffusible anions are membrane. Ions "see" charged groups. Mercury


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"sees" the sulfhydryl group; uranyl, the phos- I. Quantitative relations of succinic andphoryl groups; azide, the K+ carrier; arsenate, carbonic acids to the potassium and hydro-the phosphate carrier, etc. However, piecing gen ion exchange in fermenting yeast.together the total membrane structure from the Biochem. J., 47, 360-369.peeks that we get with our various "eyes" is 11. CONWAY, E. J. AND DOWNEY, M. 1950 Anmost difficult. outer metabolic region of the yeast cell.The situation is reminiscent of the old Per- Biochem. J., 47, 347-355.

fables

of the beindsentan

the eldPephan 12. CONWAY, E. J. AND DOWNEY, M. 1950 pHsian fable of the blind men and the elephant. values of the yeast cell. Biochem. J., 47,The sultan, as a treat, had the blind men taken 355-360.to the zoo, where they visited an elephant. 13. CONWAY, E. J. AND DUGGAN, F. 1958 AOne felt the tail and exclaimed, "It is like a cation carrier in the yeast cell wall. Bio-rope!" Another felt a leg, "like a post." Another chem. J., 69, 265-274.felt the side, "like a wall;" and still another the 14. CONWAY, E. J. AND KERNAN, R. P. 1955ear, "like a basket." Now each man was objec- The effect of redox dyes on the active trans-tive and correct within the context of his past port of hydrogen, potassium and sodium

experiences an knowled. YeT, t n together, ions across the yeast cell membrane. Bio-experiences and knowledge. Yet, taken together, chem. J., 61, 32-36.the rope, post, wall, and basket do not make a 1 C'..'' . . ~~15. CONWAY, E. J. AND MOORE, P. T. 1954 Avery convincing elephant. In a similar way, sodium-yeast and some of its properties.

all of our peeks at the cell membrane through Biochem. J., 57, 523-528.indirect eyes do not yet add up to a very realistic 16. CONWAY, E. J. AND O'MALLY, E. 1946 Thestructure. nature of the cation exchanges during yeast

VIII. REFERENCES fermentation, with formation of 0.02 N-Hion. Biochem. J., 40, 59-67.

1. BROWN, R. AND DANIELIJ, J. F. (Editors) 17. CONWAY, E. J., BRADY, T. G., AND CARTON,1954 Active transport and secretion. Sym- E. 1950 Biological production of acid andposia Soc. Exptl. Biol. No. 8, 1-516. alkali. 2. A redox theory for the process in

2. BRUCE, A. K. 1958 Response of potassium yeast with application to the production ofretentivity and survival of yeast to far-ultra- gastric acidity. Biochem. J., 47, 369-374.violet, near-ultraviolet and visible light and 18. CONWAY, E. J., RYAN, H., AND CARTON, E.X-irradiation. J. Gen. Physiol., 41, 693 1954 Active transport of sodium ions from702. the yeast cell. Biochem. J., 58, 158-167.

3. CLAM, H. T. AND NACHMANSOHN, D. (Edi- 19. COWIE, D. B., ROBERTS, B., AND ROBERTS, Z.tors) 1954 Ion transport across nem- 1949 Potassium metabolism in Escherichiabranes. Academic Press, Inc., New York. coli. I. Permeability to sodium and potas-

4. CLARK, J. A. AND MAcLEOD, R. A. 1954 Ion sium ions. J. Cellular Comp. Physiol. 34antagonism in glycolysis by a cell-free bac- 243-258.terial extract. J. Biol. Chem., 211, . DAVIES J. T. AND HAYDON, D. A

5., COLLANDER, R. 1956 Permeability studies 20 'AIS .T N ADN .15

5ConluAnDER Rac.195 . Prmeably ,tui Surface behavior of Escherichia coli. Proc.On luminous bactera. Protoplasma, A, Roy. Soc. (London) Ser. B, 145, 375-383.

6. CONWAY, E. J. 1951 The biological per- 21. DAVSON, H. AND DANIELLI, J. F. 1943 Theformance of osmotic work. A redox pump. permeability of natural membranes. Cam-Science, 113, 270-273. bridge Univ. Press, Cambridge, England.

7. CONWAY, E. J. 1954 Some aspects of ion 22. DEMUs, J., ROTHSTEIN, A., AND MEIER, R.transport through membranes. Symposia 1954 The location and function of inver-Soc. Exptl. Biol. No. 8, 297-324. tase in the yeast cell. Arch. Biochem.

8. CONWAY, E. J. 1955 Evidence for a redox Biophys., 48, 55-62.pump in active transport of cations. In- 23. EDDY, A. A. AND HINSHELWOOD, C. N. 1950tern. Rev. Cytol., 4, 377-396. The utilization of potassium by Bacterium

9. CONWAY, E. J. AND BEARY, M. E. 1958 lactis aerogenes. Proc. Roy. Soc. (London)Active transport of magnesium across the Ser. B, 138, 544-558.yeast cell membrane. Biochem. J., 69, 24. EDDY, A. A. AND HINSHELWOOD, C. 1951275-280. Alkali-metal ions in the metabolism of

10. CONWAY, E. J. AND BRADY, T. G. 1950 Bacterium lactis aerogenes. III. GeneralBiological production of acid and alkali. discussion of their role and mode of action.


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Proc. Roy. Soc. (London) Ser. B, 138, effect of Roentgen rays and ultraviolet237-240, radiation on the permeability of yeast.

25. EDDY, A. A. AND WILLIAMSON, D. H. 1957 Acta Radiol., 27, 316-327.A method of isolating protoplasts in yeast. 40. HILL, S. E. 1932 The effects of ammonia,Nature, 179, 1252-1253. of the fatty acids, and of their salts on the

26. EDDY, A. A., CARROLL, T. C. N., DANBY, C. luminescence of Bacillus fischeri. J. Cel-J., AND HINSHELWOOD, C. N. 1951 Alkali- lular Comp. Physiol., 1, 145-158.metal ions in the metabolism of Bacterium 41. HODGKIN, A. 1958 Ionic movements andlactis aerogenes. I. Experiments on the up- electrical activity in giant nerve fibers.take of radioactive potassium, rubidium Proc. Roy. Soc. (London) Ser. B, 148, 1-37.and phosphorus. Proc. Roy. Soc. (London) 42. HOTCISS, R. D. 1943 Gramicidin, tyroci-Ser. B. 138, 219-228. dine and tyrothrycin. Advances in Enzy-

27. EPSTEIN, E. 1956 Mineral nutrition of mol., 4, 153-199.plants: mechanisms of uptake and trans- 43. JACOB, F. AND FUERST, C. R. 1958 Theport. Ann. Rev. Plant Physiol., 7, 1-24. mechanism of lysis by phage studied with

28. FEW, A. V., FRASER, M. J., AND GILBY, A. R. defective lysogenic bacteria. J. Gen.1957 The intracellular catalase of Micro- Microbiol., 18, 518-525.coccus lysodeikticus. Biochim. et Biophys. 44. JENNINGS, D. H., HOOPER, D. C., AND ROTH-Acta, 24, 306-314. STEIN, A. 1958 The participation of phos-

29. FOULKES, E. C. 1956 Cation transport in phate in the formation of a 'carrier' for theyeast. J. Gen. Physiol., 39, 687-704. transport of Mg++ and Mn a ions into the

30. GILBY, A. R. AND FEW, A. V. 1958 Per- yeast cell. J. Gen. Physiol., 41, 1019-1026.meability of Micrococcus lysodeikticus to 45. JOHNSON, F. H. AND HARVEY, E. N. 1937hydrochloric acid. Biochim. et Biophys. The osmotic and surface properties of mar-Acta, 30, 421-422. ine luminous bacteria. J. Cellular Comp.

31. GILBY, A. R. AND FEW, A. V. 1959 Osmotic Physiol., 9, 363-379.properties of protoplasts of Micrococcus 46. JUNI, E., KAMEN, M. D., REINER, J. M., ANDly8odeikticus. J. Gen. Microbiol., 20, 321- SPIEGELMAN, S. 1948 Turnover and dis-326. tribution of phosphate compounds in yeast

32. GILBY, A. R., FEW, A. V., AND MCQUILLAN, metabolism. Arch. Biochem., 18, 387-408.K. 1958 Chemical composition of the 47. KAMEN, M. D. AND SPIEGELMAN, S. 1948protoplast membrane of Micrococcus lyso- Studies on the phosphate metabolism ofdeikticus. Biochim. et Biophys. Acta, 29, some unicellular organisms. Cold Springs21-29. Harbor Symposia Quant. Biol., 13, 151-163.

33. GOODMAN, J. AND ROTHSTEIN, A. 1957 The 48. KELLENBERGER, E. AND RYTER, A. 1958active transport of phosphate into the yeast Cell wall and cytoplasmic membrane ofcell. J. Gen. Physiol., 40, 915-923. Escherichia coli. J. Biophys. Biochem.

34. GOURLEY, D. R. H. 1952 The role of adeno- Cytol., 4, 323-326.sine triphosphate in the transport of phos- 49. KLOTZ, I. M. 1954 Thermodynamic andphate in the human erythrocyte. Arch. molecular properties of some metal proteinBiochem. Biophys., 40, 1-12. complexes. In A symposium on the mecha-

35. GURD, F. R. N. 1954 The specificity of nism of enzyme action. Johns Hopkinsmetal protein interactions. In Ion transport Univ. Press, Baltimore.across membranes. Edited by H. T. Clark 50. KNAYSI,G. 1946 Ontheexistence,morphol-and D. Nachmansohn. Academic Press, ogy, nature, and functions of the cytoplas-Inc., New York. mic membrane in the bacterial cell. J.

36. HARRIS, E. J. 1956 Transport and accu- Bacteriol., 61, 113-121.mulation in biological systems. Academic 51. KNAYSI, G. 1951 Elements of bacterial cy-Press, Inc., New York. tology. Comstock Publ., Ithaca, New

37. HARRISON, A. P., JR., BRUCE, A. K., AND York.STAPLETON, G. E. 1958 Influence of X-ir- 52. KOTYK, A. AND KLEINZELLER, A. 1959radiation on potassium retentivity by Es- Movement of sodium and cell volumecherichia coli. Proc. Soc. Exptl. Biol. changes in a sodium rich yeast. J. Gen.Med., 98, 740-743. Microbiol., 20, 197-211.

38. HEVESY, G. AND NIELSEN, N. 1941 Potas- 53. KEEBS, H. A., WHITTAM, R., AND HEMS, R.sium interchange in yeast cells. Acta 1957 Potassium uptake by AlcaligenesPhysiol. Scand., 2. 346-354. faecalis. Biochem. J., 66, 53-59.

39. HEVESY, G. AND ZERAHN, K. 1946 The 54. LEDERBERG, J. 1950 The beta-D-galactosi-


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