Permeability of the Cell Envelope and Osmotic Behavior inSaccharomyces cerevisiae
WILFRED N. ARNOLD* AND JOHN S. LACYDepartment of Biochemistry, University ofKansas Medical Center, Kansas City, Kansas 66103
Received for publication 4 May 1977
Bakers' yeast (Saccharomyces cerevisiae) was equilibrated with distilled wa-ter and then packed into standardized pellets by centrifugation. The fractionalspace (S value) that was accessible to passive permeation was probed with avariety ofmono- and divalent salts, mono- and disaccharides, polyols, substratesand products of ,-fructofuranosidase (EC 3.2.1.26) and acid phosphatase (EC3.1.3.2), and a cross-linked polymer of sucrose (Ficoll 400). A simple but veryreproducible method was developed to measure pellet volume. At the limit ofzero osmolality for bathing medium, the interstitial space was 0.223 ml/ml ofpellet, and the aqueous volume of cell envelopes was 0.117 ml/ml of pellet. Thusthe cell envelope for this yeast, under these conditions, was approximately 15%of the total cell volume. At a finite osmolality, the space in a yeast pellet thatwas accessible to salt was accounted for by the sum of initial interstitial space,the volume of the cell envelopes, and the volume of water abstracted from thecells by osmosis. Plots of S value versus osmolality were linear for unchargedprobes and curvilinear for all salts. When Ficoll and potassium thiocyanate werepresented to the yeast in admixture, the S values for the salt increased continu-ously over the range of osmolality studied. However, the S values for Ficoll 400(which did not penetrate the cell wall) were lower by an amount equilivalent tothe cell envelopes; they increased in parallel with the S curve for salt up to 1.15osmol/kg and then plateaued. The results support the concept of incipientplasmolysis at 1.15 osmol/kg, and the separation ofprotoplasm from the cell wallis indicated with more concentrated solutions. Such cells were still viable ifslowly diluted in distilled water, but they were injured by the shock of rapiddilution. However, shocking the cells did not release f8-fructofuranosidase intothe medium. The complete accessibility of salts toward killed cells was demon-strated with yeast that had been pretreated with heat, organic solvents, orglutaraldehyde.
The yeast cell envelope is composed of theprotoplasmic membrane, the cell wall proper,and the intervening region, which is called per-iplasmic space. The envelope is metabolicallyactive (6) and is known to contain several en-zymes, of which ,3-fructofuranosidase (EC3.2.1.26) and acid phosphatase (EC 3.1.3.2) arewell documented. For Saccharomyces species,the available evidence supports the hypothesis(2, 4) that these enzymes are not covalentlybound but are mechanically restrained withinthe periplasmic space. This space, which in-cludes invaginations in the protoplasmic mem-brane and irregularities in the inner aspect ofthe cell wall, may be subject to volume changesdepending upon the cell's environment.The relatively thick cell wall is undoubtedly
responsible for the yeast cell's resistance to me-chanical stress, whereas the site of regulationfor solute exchange with the medium resides
primarily in the protoplasmic membrane.Thus, yeast is capable of withstanding concen-trated salt or sugar solutions and, under verydifferent conditions, of accumulating some ofthese same compounds against a concentrationdifference (19). Conversely, the cell wall pre-sents no real barrier to the free diffusion ofsmall molecules and ions (19).Conway and Downey (8) developed a quanti-
tative technique for ascertaining the fractionalvolume in packed yeast that is available to atest solute. About 11% ofthe total cell volume ofresting cells was found to be accessible to arabi-nose, for example, and this region of the cellwas equated with the cell envelope (8). Ger-hardt and co-workers (12, 18) introduced poly-disperse dextrans as probes and were able toestimate threshold pore sizes.
Evidence for plasmolysis in yeast (i.e., sepa-ration of the protoplasm from the cell wall in
hypertonic media) has not been establishedand, indeed, the architecture of even the turgidcell envelope is far from satisfactorily described(7). For these reasons we felt that a physicalapproach that was independent of microscopymight yield interesting and complementary re-
sults. The present work includes elaboration ofthe Conway technique to space measurementsinvolving a salt and a polymer in admixture.This paper also summarizes our backgroundstudies on the behavior of the yeast cell enve-
lope toward a variety of solutes and, in some
cases, over larger concentration ranges thanhitherto reported.
MATERIALS AND METHODSYeast. Bakers' yeast, which was donated by Red
Star Yeast and Products Co. at weekly intervals,was stored under refrigeration. About 1 cm was
removed from each surface of a 454-g cake, and theremainder was crumbled into distilled water. Afterbeing stirred for 15 min and allowed to settle for 5min, the suspension was decanted into tubes andcentrifuged. The supernatant liquid was discarded.The yeast was similarly washed once more in dis-tilled water and then suspended to 30% (wet wt/vol)in distilled water. All operations were at 23 to 25°C,and a fresh suspension was made each working day.
Chemicals. All salts and sugars were analyticalgrade. Pentaerythritol (2,2-bishydroxymethyl-1,3-propanediol) was from Eastman Kodak Co., Roches-ter, N.Y. Ficoll is a synthetic copolymer of sucrose
and epichlorohydrin from Pharmacia Fine Chemi-cals, AB, Uppsala, Sweden. Ficoll 400 has a weight-average molecular weight of 400,000 compared with70,000 for Ficoll 70.
Conway's dilution method. Pellets were obtainedby centrifugation of yeast suspensions at 11,100 x gfor 10 min (standard conditions). Ten-milliliter por-tions of 30% (wet wt/vol) suspension were added totared 15-ml tubes, and a swinging-bucket rotor wasused. The supernatant water was decanted to waste,and tubes were inverted for 1 min. Droplets ofwaterwere removed with absorbent paper. Gross weightswere determined as quickly as possible, and tubeswere then capped with aluminum foil. Each yeastpellet (accurately weighed, but invariably close to 3g) was suspended with 3 ml of a particular testsolution by vortex stirring. After 15 min (standard),the tube was vortexed again and then centrifugedunder standard conditions. Either 1 or 2 ml of super-natant fraction was taken for analysis. An identicalvolume of undiluted test solution was also analyzed.In a few instances involving very dilute test solu-tions, the above amounts were scaled upwardswithin the same proportion.The aqueous space (S) in the original pellet that
is accessible to test solution and consequently lowersthe solute concentration upon mixing is related tothe measured parameters as follows:
s V(Ci - Cf)W,,.Cf
where V, is the volume of solution added, W, is thewet weight of yeast pellet, Ci is the initial concen-tration of test solute, and C, is the final concentra-tion of test solute (i.e., in the supernatant fraction).The dimensions of S are milliliters per gram ofwet, packed yeast. When the density (d) ofthe pelletis known, a secondary value S, (in milliliters permilliliter of wet, packed yeast) follows: S, = S * d.
Analyses. For the majority of solutes tested, thegravimetric method (8) was found to be reliable andaccurate. Small aluminum pans (6 cm in diameterand 1.5 g) were tared, charged with samples, driedovernight in a convection oven set at 95°C, cooled ina desiccator, and weighed on a balance with a sensi-tivity of 0.1 mg. L-Arabinose, .-rhamnose, glucose,fructose, and sucrose (after acid hydrolysis) concen-trations were determined with Sumner's 3,5-di-nitrosalicylic acid reagent (1). Ficoll concentrationswere usually monitored gravimetrically, but whenin admixture with a salt the analyses followed ananthrone procedure (5). Chloride ion was deter-mined by titration with mercuric nitrate (17). p-Nitrophenol and p-nitrophenolphosphate (after acidhydrolysis) concentrations were calculated from theabsorbance of p-nitrophenolate ion at 425 nm (2).Thiocyanate ion was assayed as the ferric complexby the colorimetric method of Cosby and Sumner (9).Small corrections were applied where appropriate.In the gravimetric procedure, distilled-water con-trols were found to contain, on the average, about0.3 mg of extracted yeast solids per ml. In the reduc-ing-sugar procedure, a blank was constructed bymeasuring reducing substances extracted fromyeast by mannitol (nonreducing) at an osmolalityequivalent to that of the test compound.
Density of pellets. The open ends of several glasscentrifuge tubes (15 ml) were ground flat on carbo-rundum paper. Tubes were identified with symbolsinscribed by a diamond pencil, and several microcover glasses (no. 1, 22-mm square) were similarlymarked. Tubes and cover glasses were washed,dried, and tared. Each tube was filled with distilledwater and a cover glass was placed across theground-glass mouth, care being taken to avoid airbubbles. Excess water was wiped off, and the filledtube was weighed. The total volume of the tube wasobtained from the weight of water it contained. Apellet of cells was prepared under standard condi-tions and weighed as usual. The tube was thencarefully charged with water and topped with acover glass as before. The volume of added waterwas again determined by weight difference and,when substracted from the total volume of the tube,yielded the volume of the wet yeast pellet. On onetypical batch of yeast, the mean of four separatedeterminations of density was 1.0668 g/ml (standarddeviation = 0.0004), which indicated adequate sensi-tivity and reliability for the method.
Water abstracted by osmosis. When yeast ismixed with a solution of higher osmolality, there isa movement of water from the cells to the mediumand a decrease in cell volume. This is reflected in therelative height of yest columns obtained upon cen-trifugation. We used Wintrobe tubes with a uniformbore of 3 mm and a graduated length of 105 mm
(Arthur H. Thomas Co., Philadelphia). Centrifuga-tion was at 1,440 x g for 10 min, and readings weremade after letting the tubes stand for 1 h. Centrifu-gation conditions obviously affect the resultant "he-matocrit." Also, the "rebound" in the column of par-tially compressed cells after centrifugation is timedependent and apparently is influenced by the di-ameter-to-volume ratio of the column. The aboveconditions were arrived at empirically and are dis-cussed later.
IfV1 is the initial volume of the yeast pellet, V2 isthe volume of solution added, and h is the relativeheight of packed cells (expressed as a fraction of thetotal height). Then
V1 (1 - AV)VI + V2
where AV is the decrease in pellet volume per unitinitial volume. Values of AV are an approximationto the fractional volume of water abstracted by os-mosis.
Osmolality. Values were derived from concentra-tions by reference to standard tables (21) and byinterpolation. Unlisted compounds or mixtures weremeasured on an osmometer (Advanced Instruments,Inc., Needham Heights, Mass.) by freezing-pointlowering.
RESULTSPotassium and sodium chlorides. The rela-
tionship between S value and osmolality ex-hibited by these salts is curvilinear and ap-proaches an asymptote of 0.680 ml/g (Fig. 1).The water content of standard pellets was 0.799(±0.007) ml/g. There is no significant differ-ence between KCl and NaCl, and analysesbased on the titration of chloride ion showgood agreement with those based on the gravi-metric method (Fig. 1).
Ficoll. The relationship between S valuesand concentration (C) for Ficoll 400 is shown in
u
>%c
'n
OSmdoality (OsmolS/Kg)
FIG. 1. Effects ofosmolality on the S values ofKCl(0) and NaCI (A). Data points are based on analysesby the gravimetric method (open symbols) or by titra-tion of chloride ion (O). Insert contains additionaldata points for osmolalities greater than 3.0.
Fig. 2. The straight line of best fit is describedby S = 0.209 + 0.00424 C, where C is given aspercent (wt/vol). Ficoll 70 at a final concentra-tion of3.82% yielded an S value of 0.231, whichwas not significantly different from the re-sponse to the larger polymer.Sugars and polyols. In general, the S values
for uncharged test compounds showed a linearresponse to final osmolality. The curve for L-
arabinose is depicted in Fig. 2 and is represent-ative of this class of compounds. Equations de-scribing the individual lines of best fit are as-sembled in Table 1, together with a line de-scribing the combined data for the sugars andpolyols.KSCN. The convenience of the colorimetric
assay for thiocyanate, and the similarity of theS curve for potassium thiocyanate (KSCN)(Fig. 2) with that for KCl (Fig. 1), prompted ouradoption of KSCN as a reference compound.The smooth curve of Fig. 2 is drawn to the
O.81
I0.6
0.4
00.2
0.O5 LO .5Osmolality (Osmol.s/Kg)
0 3 10~~~~~~~~~~~~~~~~~~
2.0 2.5 30
0 5 10Concentration (% wVo)
FIG. 2. Effects of osmolality on the S values forKSCN (0), L-arabinose (a), and Ficoll-400 (A).TABLE 1. S values as a function of osmolality for
unmetabolized sugars and polyolsLinear
Compound" (mol wt) S value" (ml/g of correla-wet packed yeast) tion coef-ficient
combined data of 10 separate trials on differentbatches ofyeast over a 6-month period. Consist-ent behavior by the yeast and reliability in themethod are indicated. S values for KSCN areslightly higher than those for KCl at equivalentosmolality. The difference, although consist-ent, was never greater than 5%.
Divalent cations and anions. The S valuesfor a variety of salts are plotted in Fig. 3. Thedashed curve in Fig. 3 is the KCl line (cf. Fig.1). In general, the S values for salts of divalentcations and anions are slightly lower thanthose for monovalent salts at equivalent osmo-lality. The activity coefficients for several ofthedivalent ions are significantly removed fromunity and different from each other (21). Conse-quently, plots (not shown here) of S valuesversus molarity for the above salts were quitedisparate. On the other hand, the reasonableagreement of S values on the osmolality scale(Fig. 3) argues in favor of a predominant influ-ence of osmosis on the magnitude of S.Water abstracted by osmosis. A series of
standard yeast pellets was mixed with a rangeofKSCN concentrations, and the S values weredetermined. These were converted to Sv valuesafter ascertaining the density of standard pel-lets. An additional portion of each suspensionwas removed and subjected to packed-cell vol-ume measurement. Calculated values for AVare listed in Table 2, together with correspond-ing values for S,. The magnitude ofS,-AV was0.354 on the average and was fairly uniforn(standard deviation = 0.014). This parameter isnot significantly different from the S, value(0.340 + 0.006) for sugars and polyols at zeroosmolality. (The latter value is the product ofthe intercept of the S curve [Table 1] and pelletdensity.) These results indicate that the in-
I
Is
0.5 1.0
Osmololity (Osmolts/Kg )
2.0 2.5
FIG. 3. Effects of osmolality on the S valuesMgCl2 (0), Na2SO4 (0), MgSO4 (A), K2SO4 (V), ar
NaK tartrate (0). The dashed line represents tequivalent response for KC1.
crease in S value with solute concentration isprimarily due to osmosis.Concurrent measurements of KSCN-acces-
sible and Ficoll-accessible spaces. A stock so-lution was made to contain 2.5 M KSCN and 5%(wt/vol) Ficoll 400. Serial dilutions were madeto a lower limit of one-tenth. These solutionswere individually mixed with standard yeastpellets. The highest final concentration of Fi-coll in these experiments was 3.45%. Comparedwith the companion salt, Ficoll concentrationscan be neglected in calculating the osmolalityof the medium. A modification was involved inone such trial in that the Ficoll concentration oftest solutions was kept constant at 5% while theKSCN concentration was varied. There isgood agreement among the results of all threetrials (Fig. 4). The smooth line which is drawnto the KSCN data is curvilinear, and S val-ues show a continuous increase over therange examined. The Ficoll line is parallel tothe KSCN line up to an osmolality of 1.15, butthereafter there is a plateau.Condition of cells. It was important to know
whether the yeast had suffered any deleteriouseffects. In particular, we asked whether themost concentrated salt solutions might causemeasurable damage to the cell envelope. Wefound that the KCl-accessible space approacheda maximum value of 0.680 ml/g at high osmo-lalities, whereas the water content of standardpellets was 0.799 (+0.007) ml/g. On the otherhand, killed cells (see below) exhibited com-
TABLE 2. Relationship between S, values and thedegree of water abstracted from yeast by osmosis
KSCN (open symbols) and Ficoll (admixture. The different symbols rrate experiments.
plete accessibility to salts basecontent of such pellets.The methylene blue test, w
indicator of viability when pprescribed conditions (3), reveigree of integrity even in cellsmixed with concentrated salt s
experiment, triplicate pelletsmixed with approximately equaM KSCN. Analysis on one tubeconcentration of 1.534 M KSCNS value of 0.656, which is typicThe contents of the second tulabout 1,000-fold by the dropwdistilled water to the stirredappropriate volume was thebuffered methylene blue solutioined in a hemocytometer. Eig(about 40 cells each) were obspercentage of dead (blue-stainecorded. The average was 7 (-with 1 (+3)% for cells from thiyeast that had only receivedwashes. It is worth pointing oudilution of cells (from a concendium) is mandatory for the mairbility. When a third pellet of celtreated with KSCN as ab"shocked" by rapid dilution in 1
water, the fraction of dead cel(+6%). However, this treatmerthe status of yeast fl-fructofursthat were exposed to KSCN (2.'then washed retained 94% ofuntreated controls no matter h(were diluted.With one batch of yeast, S va
sured at four concentrations of0.4 to 2.7 osmol/kg) and witl
presentation times. Compared with the stan-dard time of 15 min, the S values were: (i) notsignificantly different at 7.5 min; (ii) about 2%
AsOf higher at 30 min; and (iii) about 5% higherwhen the incubation was extended to 60 min.Controls. Pellets prepared from cells that
FIQOLL had been killed by pretreatments with organicsolvents (ethyl acetate or chloroform), heat, orglutaraldehyde exhibited complete accessibilityto KSCN (judged by their water content). All ofthese pretreatments were lethal, as indicatedby 100% of the cells taking the stain in the
2.0 2.5 &, methylene blue test. Although the conclusionswere clear-cut, experiments on killed materialyielded S values that displayed somewhat morethe S values for variability.
'filled symbols) in The S values for killed cells were independ-refer to three sepa- ent of external concentration. For example,
with heat-killed cells (90°C/3 min), KSCN Svalues of 0.701, 0.693, and 0.699 ml/g (wet,
,d on the water packed yeast) were obtained at osmolalities of0.713, 1.412, and 2.889 osmol/kg, respectively.
vhich is a good The water content of pellets of heat-killed yeasterformed under was 0.640 ml/g (wet, packed yeast). Likewise,aled a high de- for cells pretreated overnight with 3% glutaral-ithat had been dehyde in 0.1 M collidine-HCl (pH 7.0), the Solutions. In one values over a series of KSCN concentrationsof yeast were had an average of 0.707 (+0.076) ml/g (wet,
Ll volumes of 2.5 packed yeast), which was close to the waterrevealed a final content of those pellets: 0.678 (+0.001) ml/gI and yielded an (wet, packed yeast).cal for this salt. Substrates of 3-fructofuranosidase and acidbe were diluted phosphatase. Acid phosphatase exhibits maxi-rise addition of mal activity at pH 3.8 and negligible activity atsuspension. An pH 7.5, which was used here to study the ac-*n mixed with cessibility of conventional substrates and theirn (3) and exam- corresponding products of hydrolysis (Table 3).ght large fields Commercial bakers' yeast has a high concen-3erved, and the tration of f8-fructofuranosidase, and the enzyme3d) cells was re- catalyzes the hydrolysis of sucrose over a broad:5%), compared range of pH values. It is well known that thee same batch of products, glucose and fructose, are readily ab-distilled-water sorbed by the protoplasm. To overcome both
it that the slow problems in the assessment of passively pene-itrated salt me- trable space, we took advantage of the inhibi-ntenance of via- tory effect of uranyl acetate on ,B-fructofurano-[Is was similarly sidase as well as the glucose transport systemove but then (10). The yeast was washed twice in 10 mM ura-liter of distilled nyl acetate-acetic acid (pH 5.0), and test solu-Ils then was 41 tions were also fortified with the inhibitor. Pre-it did not affect liminary trials showed that the inhibitor wasinosidase. Cells effective at this concentration. The results are7 osmol/kg) and summarized in Table 3, along with referencethe activity of values. They demonstrate that substrates andow quickly they products ofboth enzymes penetrate a space that
is comparable to that found for salts and non-dlues were mea- metabolized sugars. The one exception, p-nitro-r KSCN (range, phenol, exhibits large S values, which indi-i four different cate accumulation by the yeast.
a Nonmetabolized polyols or KCl at an osmolalityequivalent to that of the test compound.
b Yeast ,3-fructofuranosidase and sugar transportsystems were inhibited by 10 mM uranyl acetate-acetic acid (pH 5.0).
c In the presence of 10 mM phosphate buffer (pH7.5).
d In the presence of 10 mM acetate buffer (pH 3.8).
Effects of glutaraldehyde. In addition to us-ing pellets prepared from glutaraldehyde-fixedcells as controls, some determinations of den-sity, volume, and water content were per-formed on them. A batch of fresh, washed yeastwas stirred overnight in 3% glutaraldehyde at23 to 25°C, washed in copious amounts of dis-tilled water, and then made up to 30% (wet wt/vol). The cell concentration was determined ona suitably diluted sample in a hemacytometer.Another batch of yeast was similarly treated,except that 0.1 M collidine-HCl (pH 7.4) wasincluded during fixation. The results are sum-marized in Table 4; measurements of volumeand water content are compared among equalnumbers of cells for each treatment. The resultsdemonstrate that considerable shrinkage ofcells results from fixation in the glutaralde-hyde-collidine mixture. Smaller changes werenoted when unbuffered glutaraldehyde was em-ployed.
DISCUSSIONBy definition, all S values refer to initial
pellets, which were equilibrated with distilledwater. The magnitude of the S value dependsupon the final concentration of test solute.However, extrapolation to zero concentrationyields a limiting value for S that is independentof, for example, an osmotic effect on the cell.The data for Ficoll 400 (Fig. 2) and for nonme-
tabolized polyols (Table 1) yield linear relation-ships, and the limiting values of S, as concen-tration or osmolality approaches zero, are read-ily computed. A summary that is based on
these values and a conversion to unit volumeis given in Table 5. Only the interstitial waterin a yeast pellet is accessible to the polymerFicoll (line 1, Table 5). This value is 0.223 ml/ml of pellet, and the space that cells occupy is0.777 ml/ml of pellet (by difference). Relativelysmall molecules permeate the cell envelope andare additionally diluted by that volume (line 4,Table 5). From the data ofTable 5, we calculatethat the cell envelope is, on the average, 15% ofthe total cell volume. Conway and Downey (8)obtained a comparable estimate of 11% for theiryeast.The total space that is accessible to small
molecules may be viewed as the sum of theinitial interstitial water, the cell envelopes,and the water abstracted from the cells by os-
mosis. Our results substantiate this simpleworking hypothesis in that a great variety ofsalts, mono- and disaccharides, and polyols fol-low (reasonably well) the same general re-
sponse curve for S value versus osmolality incontrast to molarity.The increase in S value with osmolality can
be accounted for by an independent estimate ofthe water abstracted by osmosis (Table 2).There was no indication that increased accessi-bility at higher salt concentrations was due toirreversible damage of the protoplasmic mem-
brane. Instead, exposed cells were shown to bein good condition, as judged by the methyleneblue test, and S values were not greatly af-fected by a fourfold increase in the presentationtime.The S curves for salts were curvilinear, but
showed agreement with polyols at zero osmolal-ity and approached similar values at higherosmolalities (see Fig. 2). The intervening re-gion in which S values, at equivalent osmolal-ity, are higher for salts than for sugars is mostlikely related to the ionic environment of thecell wall, to which covalently bonded carboxyl(13) and phosphate (11) groups have been at-tributed. A related finding may be the demon-stration of salt-induced contractions in isolatedbacterial cell walls (15). It has been shown (16)that negatively charged particles (e.g., kaolin-ite) exhibit larger hydrogen ion and cation con-centrations at their surfaces than in the sur-rounding medium, and absorbed enzymes areinfluenced by this "double layer" surroundingthe particle (14). In this context, we were inter-ested in the S values exhibited by KCl at lowconcentrations. No evidence of exclusion or ad-sorption was discernible, and good agreementbetween gravimetric analyses of the salt andtitrimetric analyses of chloride ion was ob-tained (Fig. 1).Yeast cells are osmotically responsive, and
their survival in distilled water depends on theability of the cell wall to withstand a substan-tial turgor pressure. Numerous reports in theliterature, as well as the present investigation,suggest that the envelope will initially contractas a unit when the yeast cell is placed in solu-tions of increasing osmolality. However, plas-molysis has not been previously reported foryeasts. With reference to Fig. 4, we suggestthat incipient plasmolysis has occurred at anosmolality in the vicinity of 1.15 osmol/kg. Be-low this point the Ficoll-accessible space andthe KSCN-assessible space increase in approxi-mately parallel fashion, which is consistentwith conjoint contraction of the cell wall andthe protoplasm. Above 1.15 osmol/kg, S valuesfor KSCN continue to increase (further contrac-tion of protoplasm), but those for Ficoll remainconstant (no further change reflected at theexternal surface of the cell wall). The simplestexplanation is that plasmolysis has occurred.An osmolality of 1.15 osmol/kg is given by0.637 M KCl, and it should be noted that con-centrations of 0.6 to 0.8 M KCl are recom-mended (20) for the preparation of protoplastsfrom S. cerevisiae. Our results offer a plausiblerationale for this empirical recommendation.
It was anticipated that in higher osmolalitiesthan that required for incipient plasmolysis,
the normalized heights ofpacked yeast columnswould exhibit a plateau in analogous fashion tothe S values for Ficoll (Fig. 4). This was notfound (Table 2). Possibly, the cell walls of plas-molyzed cells are sufficiently flaccid to becomeseverely deformed under centrifugation andthus yield falsely low hematocrits. The condi-tions for packed-cell volume measurement werearrived at by preliminary trials in which dis-tilled-water suspensions ofyeast were subjectedto different centrifugal forces in Wintrobetubes. Relative heights of yeast columns wererecorded immediately after centrifugation andthereafter at 15-min intervals. It was desirableto match these packing characteristics withthose used in the preparation of standard pel-lets (which had different dimensions). Once thedensity of the latter was determined (see Mate-rials and Methods), we were able to predict thehematocrit by calculation. Conditions thatwould match this value were deemed to be agood approximation to equivalent packing.However, for cells equilibrated with test solu-tions, there was a progressive change in col-umn height with post-centrifugation interval,and the rate of change was slower for the cellsfrom the more concentrated salt solutions.These results are consistent with the abovesuggestion that plasmolyzed cells give falselylow hematocrits.Our results with the substrates and products
of f-fructofuranosidase and acid phosphatase(Table 3) indicate that the cell envelope is ac-cessible to all of them. This is consistent with aperiplasmic locale for these enzymes and withthe assessment by Sherrer et al. (18) that thecell wall presents no real barrier to the passivediffusion of molecules up to a size of approxi-mately 620 daltons. Live yeast cells are com-monly assayed for acid phosphatase activitywith nitrophenol phosphate as substrate. Ab-sorption of nitrophenol by cells (as indicated bythe data of Table 3) is not a problem, becausebase is added at the termination of incubationand redistribution of nitrophenol to the me-dium is achieved.Our results indicate shrinkage by cells dur-
ing glutaraldehyde fixation. The common prac-tice of fortifying fixatives with 0.1 to 0.2 Mphosphate, collidine, or cacodylate buffers isnot encouraged by our cell volume measure-ments. Meaningful ultrastructural evidence forplasmolysis in live cells will be predicated onobtaining fixation of specimens with retentionof native proportions.
ACKNOWLEDGMENISThis work was supported by a grant from Research Cor-
poration and by Public Health Service grant Al 13177 fromthe National Institute ofAllergy and Infectious Diseases.
We thank Philip Bestic for helpful discussions and Phil-lip Gerhardt for comments on the manuscript.
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