Biochem. J. (1966) 99, 682

Protoplast Formation and Yeast Cell-Wall StructureTHE ACTION OF THE ENZYMES OF THE SNAIL, HELIX POMATIA

By F. B. ANDERSON AND J. W. MILLBANK*The Brewing Industry Research Foundation, Nutfield, Surrey

(Received 22 November 1965)

1. The digestive juice of the snail Helix pomatia was used in the study of thedegradation of isolated cell-wall preparations from a strain of Saccharomycescarlsbergensis. 2. The crude enzyme system was fractionated by gel filtration andthe activities of the more specific fractions thus obtained were examined. 3.Results are discussed with respect to (a) the nature of various factors that are

essential for protoplast formation and cell-wall dissolution and (b) structuresenvisaged in yeast cell walls that are responsible for the observed variations insusceptibility to attack by snail juice.

Enzyme preparations from several microbialsources have been used in the elucidation of yeastcell-wall composition (e.g. Mendoza & Villanueva,1962; Bacon, Milne, Taylor & Webley, 1965). Oneof the richest sources of lytic enzymes for yeastcell wall is the digestive juice from the snail, Helixpomatia. Variations in susceptibility to attack bysnail juice have been observed between one yeaststrain and another of the same species and alsobetween logarithmic-phase and stationary-phasecells of the same strain (Eddy & Williamson, 1957;Millbank & Macrae, 1964); it seems likely that thesephenomena result from differences in cell-wallcomposition.The present work, where snail juice was used

mainly in the study of 'age' variation, is part of aprogramme of investigation into yeast cell-wallstructure and the mechanism of protoplastformation.

Snail digestive juice is known to be a complexmixture of enzymes (Holden & Tracey, 1950) andsome fractionation is desirable for the elucidationof the essential factors involved in the action onyeast cell wall. Preliminary work showed that afractionation into more specific components couldbe achieved by gel filtration (Millbank & Macrae,1964). The present paper describes the results ofa more effective fractionation than has beenachieved hitherto.

MATERIALS AND METHODSOrganisms. Strains of Saccharomyces cartsbergensis

(N.C.Y.C. 74S) and Saccharomyces cerevisiae (N.C.Y.C. 239)were supplied by the British National Collection of Yeast

* Present address: Department of Botany, ImperialCollege, London, S.W. 7.

Cultures. They were maintained in liquid culture in thegrowth medium and subcultured every 4 weeks. Afterinoculation the stock cultures were incubated at 250 for72 hr. and kept subsequently at room temperature (200).

Growth medium. The medium comprised malt extract(0.3%), yeast extract (0.3%), glucose (1.0%) and peptone(0-5%) in distilled water (Wickerham, 1951).

Growth conditions. Roux flasks containing medium(400ml.) were inoculated with 0-5ml. of a mature cultureand incubated at 25° on a reciprocating shaker (3cm.amplitude, 80cyc./min.). LP cellst were obtained fromcultures after 16hr., when the cell density was approx.0-4mg. dry wt.fml., by centrifugation at lOOOg for 3min.SP cells were obtained by harvesting cultures after42-50hr.,when the cell density was approx. 5 mg. dry wt./ml. Thecells were washed with buffer and resuspended as appro-priate. The citrate-phosphate buffer used throughout wasat pH5.8 and refers to that of Mcllvaine (1921) diluted 1:5.

Preparation of cell-wall material. Washed yeast cells(30-50mg. dry wt.) suspended in 10% (w/v) sucrose soln.(6ml.) were shaken with Chance Ballotini no. 12 glass beads(approx. 6.8g.) for 10-12min. in a Mickle disintegrator.This treatment gave 95-99% disruption estimated byphase-contrast microscopic observation. The resultantturbid suspension was removed from the beads, whichwere then washed with further additions of sucrosesoln. until the supernatant liquid was clear. The cell sus-pension, including washings, was then centrifuged atlOOOg for 5min. to separate the walls from the cell contents.The sediment was washed repeatedly (eight to ten times)with sucrose soln. until microscopic observation showedthe cell walls to be free from protoplasmic debris and otherunwanted material. Cell-wall material was finally washedwith and suspended in distilled water and the dry wt. ofthe suspension determined by heating samples at 1050.Suspensions were stored at 20.

Snail digestive juice. This (suc digestif d'Helix pomatia)was obtained from Industrie Biologique Fran9aise (Genne-

t Abbreviations: LP cells, early logarithmic-phase cells;SP cells, stationary-phase cells.

682

YEAST PROTOPLASTS AND CELL WALLSvilliers, Seine, France) and was found to contain approx.200mg. dry wt. of material/ml.

Fractionation of snail enzymes. A solution of snail-juicematerial (100mg.) in 0.85% NaCl soln. (saline) was appliedto a column (45cm. x 3.1cm.) containing the gel preparedby swelling Sephadex G-100 (bead form, fine grade;Pharmacia Ltd., London, W. 13) in saline. The columnhad been previously equilibrated at 4°. Elution waseffected with saline at 40 at a flow rate of 35ml./hr.; 3ml.samples of the eluate were collected.

Chromatography. Descending paper chromatograms wereprepared at room temperature with Whatman no. 1 paperand either (A) ethyl methyl ketone-acetic acid-water(9:1:1, by vol.; satd. with boric acid) (Rees & Reynolds,1958) as solvent or (B) ethyl acetate-pyridine-water(10:4:3, by vol.) (Whistler & Hickson, 1955).

Aniline phthalate (Partridge, 1949) (1) or AgNO3-NaOH(Trevelyan, Procter & Harrison, 1950) (2) was used asspray reagent.Acid hydrolyis. Acid hydrolysis was carried out by

heating a 0.5% solution of cell-wall material in 2N-H2SO4at 1050 for 3hr. Hydrolysates were neutralized with solidBaCOS and concentrated by rotary evaporation.

Carbohydrate estimations. (a) Total carbohydrate wasestimated by the anthrone method of Hall (1956), themethod being calibrated with both glucose and mannoseas standards; (b) glucose was estimated by the method ofFleming & Pegler (1963); (c) mannose was estimated bydifference.

Protein estimation. Protein was estimated by the methodof Lowry, Rosebrough, Farr & Randall (1951); bovineplasma albumin was used as standard.Enzyme assay. Hydrolysis of cellobiose, gentiobiose and

laminaribiose was estimated by measuring the glucosecontent of assay mixtures by the method of Fleming &Pegler (1963). fi-Acetylaminodeoxyglucosidase (P-2-acetyl.amino-2-deoxy-D-glucoside acetylaminodeoxyglucohydro-lase, EC 3.2.1.30) activity was estimated by measuring theliberation of p-nitrophenol from p-nitrophenyl N-acetyl.B-glucosaminide (Borooah, Leaback & Walker, 1961;Woollen, Heyworth & Walker, 1961). Phosphomono-esterase and phosphodiesterase were estimated by measur-ing E440 of samples of the assay mixtures after treatmentwith an equal volume of O1N-NaOH. Proteinase wasassayed by the method of Anson (1938).

RESULTSAnalysis of cell-wall preparations

(a) Carbohydrate. Total carbohydrate, estimatedin acid hydrolysates of a number of preparationsof either LP or SP cell-wall material of S. carlsber-gens8s, was found to be approx. 85% of the initialweight of the wall. In LP material the glucose/mannose ratio was consistently about 1:1-2; theratio in SP material was 1 :1-7.

(b) Protein. Both LP and SP materials gavevalues of approx. 10-12% for total protein.

Experiments with crude enzymeCell-wall material (2mg./ml.) or whole cells

(5mg./ml.) together with crude enzyme (5mg./ml.)

in citrate-phosphate buffer were incubated at 300for 90min. 0 6M-Mannitol was used as osmoticstabilizer for whole cells.

Effect on cells and cell walls of S. carlsbergensis.This strain is known to form protoplasts readilyunder the appropriate conditions (Eddy &Williamson, 1957; Millbank, 1963).

(a) Whole cells. Microscopic observation showedthat with LP cells complete lysis had occurred,i.e. 100% conversion into protoplasts. No proto-plast formation was observed with SP cells.

(b) Cell walls. With LP cell walls the bulk ofthe material went into solution. After removingthe small amount of residue by centrifugation, thesupernatant liquid and acid hydrolysates of boththe supernatant and the residue were subjected topaper-chromatographic analysis (solvents A andB, spray reagents 1 and 2 respectively). Whereasfree glucose but no mannose was detected in thesupernatant liquid, after hydrolysis both sugarswere found in approximately equal amounts; onlytraces of glucose were observed in the hydrolysateof the residue. In a parallel experiment withcell-wall material from SP cells, only glucose wasobserved in the supematant liquid. Glucose and asmaller amount of mannose were detected in thehydrolysed supematant and glucose plus a greateramount of mannose in the hydrolysed residue.

Effect on cels and cell walls of S. cerevisiae. Thisstrain does not form protoplasts with snail enzymeunder the given conditions (Millbank & Macrae,1964).

(a) Whole cells. Microscopic observation revealedlittle or no attack by snail enzymes; no protoplastswere formed. Chromatographic analysis of thesupernatant liquid and its hydrolysate showed thatonly glucose was released. In these trials mannitolwas omitted.

(b) Cell walls. Material derived from LP cells ofthis strain gave results similar to those obtainedabove with cell wall prepared from SP cells ofS. carlsbergensis.

Experiments withfractionated enzymes

Snail digestive juice was fractionated on acolumn of Sephadex G-100. Fig. 1 shows the E280values of the samples eluted from the column(Unicam SP.500 spectrophotometer, 0-5 cm.cuvette,saline blank); the first 30ml. was discarded.Identical elution patterns were obtained fromnumerous runs.

Effect on cells and cell walls of S. carlsbergensis.The digests were similar to those above with1-5ml. samples of the individual eluates replacingthe crude enzyme. Incubation time and tempera-ture were as before.

(a) Whole cells. Microscopic observation showed

Vol. 99 683

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1 2

*o

0 8

04

0-2A: B:

t0 40 6CSamp

Fig. 1. Gel filtration of snail juiG-100 column (45cm. x 3.1cm.)were collected and E280 valusamples are recorded.

that protoplasts were onlywith samples 60-80. Sampleas were samples 80-90. Vsolutions thus obtained wai

F. B. ANDERSON AND J. W. MILLBANK 1966

possible participants in the degradation of struc-tures known or thought likely to be present in yeastcell wall. All assay mixtures in citrate-phosphatebuffer in a total volume of 10Oml. were incubatedat 300; activities were estimated as given in theMaterials and Methods section.

Cellobiase, gentiobiase and laminaribiase. Assaymixtures containing 0-2ml. of the individualsamples from the Sephadex column were incubatedfor 15 min. together with 200,ug. of the appropriatedisaccharide. Hydrolysis of all three substratesoccurred only with samples 10-40 with maximumactivity in samples 20-30.

,,I ,,, ,BI.-Acetylaminodeoxyglucosidase. Column80 100 120 samples (0.2ml.) together with lmg. of p-nitro-

le no. phenyl N-acetyl-B-glucosaminide were incubatedfor 1 hr. Hydrolysis occurred with samples 15-35;

ice (100mg.) on a Sephadex there was a sharp peak in activity with sample 21.in saline. Samples (3m1.) Pes of the even-numbered Phosphomonoesterase and phosphodiesterase.

The digests consisted of 0-5ml. of the columnsamples plus 2.5mg. of either disodium p-nitro-phenyl phosphate or sodium bis-(p-nitrophenyl)phosphate and were incubated for 30min. Enzymic

formed from LP cells activity towards either substrate was confined to)s 50-60 were combined, samples 23-45 withmaximum activity in sample 33.Vhereas neither of the No proteolytic activity was detected in any of,scapable of producing the samples.

protoplasts when acting on its own, when theywere mixed together in equal proportions 1-5ml.samples of the mixture effected protoplast forma-tion under the given conditions.

(b) Cell walls (LP). After incubation any un-dissolved material was removed by centrifugationand the supernatant liquid was desalted by treat-ment with ion-exchange resins. The samples (15-30) comprising the initial peak (fraction A) pro-duced limited attack on the cell-wall material.Chromatographic analysis (solvent A, spray reagent1) showed glucose but no mannose in either thesupernatant liquid or its hydrolysate. Samples30-50 (fraction B) again exhibited limited attack;only glucose was detected with the earlier samples,whereas the later samples of this group yielded freeglucose in the supernatant liquid and glucose plusa smaller amount of mannose in its hydrolysate.Samples 50-90 (fraction C) produced almost com-plete solubilization of the cell-wall material, inparticular samples 60-80. With these fractions twofurther components (RG0C0-28 and 0-14 in solventA; mannose has RB0, 1.40) were detected in thesupernatant liquid. The identities of these sub-stances are considered below. Acid hydrolysatesof the supernatant liquid contained glucose andmannose in approximately equal amounts. Theremainder of the samples (fraction D) exhibitedvery limited activity.Enzymic activitie8 of the 8amples. The activities

examined were selected on the basis of their being

A number of replicate fractionations was carriedout and by reference to the E28o distributionpattern (cf. Fig. 1) the corresponding samples werecombined. The combined samples were thenfurther grouped into four fractions A-D corre-sponding to the fractions described above. Thesematerials were concentrated by freeze-drying,desalted by passage of the concentrated solutionsthrough columns containing ion-retardation resinAG-i lA8 (Bio-Rad Laboratories, Richmond, Calif.,U.S.A.) and the desalted solutions freeze-dried.Examination of the u.v. spectra of aqueous solu-tions of these material showed that, whereasfractions A, B and C exhibited an absorptionmaximum at 280m,t, fraction D showed no specificabsorption at this wavelength.

Activity of the freeze-dried fractione. (a) Cells andcell walls of S. carlabergen8i8 (LP). The action ofthe freeze-dried fractions on cells and cell wallswas found to be similar to that described abovefor the individual samples comprising the corre-sponding groups, i.e. protoplasts were only formedfrom whole cells under the action of fraction Cand, whereas this fraction brought about almostcomplete dissolution of isolated cell walls, fractionsA and B showed only limited attack. Fraction Dwas virtually inactive and was not examinedfurther.

(b) Model substrates. The assay procedures wereas given above with freeze-dried preparations(0-5mg./ml.) in place of the individual samples of

YEAST PROTOPLASTS AND CELL WALLS

the column eluate. The activities were found to beas expected, i.e. hydrolysis of cellobiose, gentio-biose, laminaribiose and p-nitrophenyl N-acetyl-P-glucosaminide occurred mainly with fraction Awith less activity in fraction B and no activity infraction C. The phosphatases were mainly locatedin fraction B with slight activity in fraction A andno activity in fraction C; similar results wereobtained for lipase activity as measured with Tween20 as substrate (Bier, 1955).

(c) Degradation of carbohydrate components ofcell wall. Cell-wall materials (2mg./ml.) derivedfrom either LP or SP cells of S. carl8bergen8i8 wereincubated with each ofthe freeze-dried preparationsA, B and C (lmg./ml.) in citrate-phosphate bufferat 300 for 90min.; the supernatant liquids andresidues were analysed chromatographically forcarbohydrate constituents. Fraction A liberatedonly glucose from either cell-wall preparation,leaving the bulk of the glucan and the mannanundissolved. The action of fraction B was similarto that of fraction A except that a small amountofmannan was liberated, i.e. mannose was detectedin acid hydrolysates of the supernatant liquid.With fraction C, however, there was virtuallycomplete dissolution ofLP cell-wall material. Here,in addition to glucose and mannan, other reducingsugars were found in the supernatant liquidincluding those with RGICO28 and 0-14 in solventA and RG1C0-74 and 0-42 in solvent B. Acid hydro-lysates of these components showed only glucose.Authentic samples of gentiobiose and laminaribiosehad RGIC0 14 and 0-28 respectively in solvent Aand R0l,0-42 and 0 74 respectively in solvent B.On more prolonged incubation (3-4hr.) of the cell-wall material with fraction C, further componentsof the supernatant liquid with RG,0c1-30 and 0-52in solvent B were detected; the latter was notcharacterized. The component with RGIC 1@30,which was slow to react with spray reagent 2, gavea purple-violet colour with the Elson-Morganreagents (Partridge, 1948) and its chromatographicmobility was comparable with that of N-acetyl-glucosamine. Only glucose was detected in acidhydrolysates of the small amount of residue afterattack on LP cell-wall material by fraction C. Theproducts of attack by fraction C on SP cell-wallmaterial were similar to those from LP material.In this case there was considerable residue foundto be rich in mannan.

(d) Cell-wall protein. The residues after incuba-tion of the cell-wall preparations with the variousfreeze-dried fractions were washed thoroughly toremove any contaminating material and the proteincontents examined. Whereas with fractions A andB little or no protein was solubilized from eithercell-wall preparation, i.e. the residues containedthe bulk of the original protein present in the

untreated walls, with fraction C virtually all theprotein went into solution from LP cell-wallmaterial. However, about 80% of the proteinoriginally present in SP cell wall remained un-dissolved by fraction C.

(e) Action of the freeze-dried fractions on solublelaminarin. Assay mixtures containing laminarin(0.5mg./ml.) and each of the snail-juice fractionsindividually (0.5mg./ml.) in citrate-phosphatebuffer were incubated at 300. Samples were with-drawn at intervals, the reaction was stopped byheating and the glucose content of samples wasdetermined by the method of Fleming & Pegler(1963). The glucose content of the substrate wasmeasured in acid hydrolysates. Whereas fractionsA and B gave almost complete conversion intoglucose in 3 and 2 hr. respectively, fraction Cliberated only about 80% of the available glucosein 3 hr. and this value did not increase on prolongedincubation. The products of the action of snail-juice fractions on laminarin were studied. Mixturesof substrate (8mg./ml.) and enzyme (1mg./ml.) incitrate-phosphate buffer were incubated at 30°.Samples were withdrawn at intervals, the reactionwas stopped by heat treatment and samples sub-jected to paper-chromatographic analysis withsolvent B and spray reagent 2. It was found thatthroughout the course of the reactions glucose wasthe only detectable product under the action ofeither fraction A or B. Fraction C, on the otherhand, gave rise to glucose, laminaribiose, laminari-triose (the chromatographic mobilities of whichwere comparable with authentic samples) andhigher oligosaccharides in the early stages of thereaction (up to 3 hr. incubation) and yielded glucoseand laminaribiose as the main products on pro-longed incubation (18hr.).

(f) Action of the freeze-dried fractions on thepolysaccharide luteose. A series of experimentswas carried out with luteose (kindly supplied byProfessor D. J. Manners) as substrate; the assaymixtures and conditions were similar to thoseused above with soluble laminarin. Fractions A,B and C acting individually released glucose moreslowly than from laminarin; thus the amount wasabout 6, 6 and 15% of the theoretical maximumrespectively after 1 hr. and approx. 75, 75 and 40%respectively on prolonged incubation (18hr.).However, a combination of all three fractions (each0-5mg./ml.) eventually produced almost completeconversion into glucose (53% after 1 hr. incubation).Again with fractions A and B glucose was the onlydetectable end product throughout the course ofthe reactions. With fraction C, in addition toglucose, gentiobiose (the chromatographic mobilityof which was comparable with that of an authenticsample) and other reducing components (RGl0C0.23and 010 in solvent B) were detected in the early

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F. B. ANDERSON AND J. W. MILLBANK

stages ofthe reaction and were present on prolongedincubation (18 hr.).

Effect of 2-mercaptoethanol. Samples of a suspen-sion of SP cell-wall material from S. carl8bergens8i(5mg./ml.) in tris-hydrochloric acid buffer, pH 7-5(002M), with orwithout2-mercaptoethanol (0-06M),were incubated at 300 for 1 hr. The wall materialwas then recovered by centrifugation, washed twiceon the centrifuge with citrate-phosphate bufferand samples (2 mg./ml.) were treated with snail-juice fraction C (1 mg./ml.) in citrate-phosphatebuffer for 90min. at 30°. Whereas the suspensionsof cell wall that had not been pretreated withmercaptoethanol remained turbid, those of thetreated material became greatly clarified under theaction of fraction C. Microscopic observationrevealed that the former contained material withthe general shape of the original cell wall; no suchmaterial was observed in the treated samples. Ina parallel experiment intact SP cells (5mg./ml.)were similarly treated with 2-mercaptoethanol intris-hydrochloric acid buffer. On subsequentincubation with snail-juice fraction C in citrate-phosphate buffer plus mannitol the treated cellsreadily formed protoplasts, whereas SP cellspreincubated with tris-hydrochloric acid buffer inthe absence ofmercaptoethanol did not form proto-plasts on subsequent treatment with fraction C.

DISCUSSION

The action of snail juice on cell-wall materialderived from yeast cells in a state where protoplastscan be readily formed results in virtually completedegradation of the cell-wall glucan with consequentdissolution of the mannan. The mannan does notappear to be extensively degraded. Millbank &Macrae (1964) reported that under similar condi-tions mannose occurred among the reactionproducts. However, it has since been shown(F. B. Anderson & J. W. Millbank, unpublishedwork) that the free hexose arises from degradationof mannan as a result of the deproteinization pro-cedure employed in the earlier work. That mannanis not extensively degraded is in agreement withthe findings of Eddy (1958a). With cell-wallmaterial from yeast in a state where protoplastsare not formed under the action of snail juice (SPcells of S. carlsbergen8si; LP cells of S. cerevsiae)the glucan is again degraded. In this case, however,a residue rich in mannan remains undissolved.These results indicate differences in the nature ofthe mannan between the two types of cell wall;analysis of the wall materials also shows a highermannan content for the resistant cell wall.Whole snail juice subjected to gel filtration yields

three major fractions characterized by their actionon yeast cells, cell-wall materials and model

substrates. Only one of these (fraction C) has theability to form protoplasts from LP cells of S.carlsbergen8is and to bring about extensive degrada-tion of cell-wall material derived from such cells.As this fraction is devoid of the lipase and phos-phatases, present in the original snail juice andlocated in fractions A and B, and of proteinase, itseems that such enzymes do not play an essentialrole in cell-wall degradation.The products of the action of fraction C on cell-

wall materials differ from those of the whole snailjuice in that the glucan is incompletely hydrolysedto glucose. The two other fractions A and Bcontain the activities necessary for the hydrolysisof the ,-linked glucose disaccharides remainingafter attack by fraction C. Fractions A and Bexhibit only limited attack on the cell-wall glucan,glucose being the sole detectable product. Theresults are explicable if fractions A and B attackcell-wall glucan by exo-glucosidase action, suchaction being curtailed by the presence of branchpoints in the molecule. Fraction C, on the otherhand, may act in a random fashion and in thisway by-pass such branch points; it may alsohydrolyse any branch-point linkages. Support forthis hypothesis has been gained from the study ofthe action of the snail-juice fractions on the#-(1--3)- and fl-(1 -6)-linked glucose polysac-charides laminarin and luteose (Anderson, Haworth,Raistrick & Stacey, 1939) respectively. Whereasfractions A and B degrade both polysaccharidesextensively in a stepwise manner with the liberationof glucose alone, fraction C acts in a randomfashion. The approximately complete conversionof luteose, which is known to be a branchedstructure, into glucose by the combined action ofthe three fractions may be indicative of somede-branching activity being present in fraction C.Both linear and branched structures have beensuggested for yeast cell-wall glucan (for a review,see Phaff, 1963); however, the present postulationof a branched structure is supported by the recentfindings of Manners & Patterson (1966). Theendo-8-(l -3)- and endo-fl-(1 -6)-glucanase pre-sent in fraction C may be analogous to thoseisolated by Tanaka & Phaff (1965) from the yeastcell-wall lytic enzymes produced by a strain ofBacillus circulan&. A separation of the snail-juiceactivities is being undertaken to determine whetherboth are essential for cell-wall degradation andprotoplast formation. It was observed that,whereas neither the initial nor the last eluatesamples included in fraction C were capable ofprotoplast formation on their own, they were activein this respect when combined. This indicates thatat least two enzymic activities are essential forprotoplast formation. Such activities may well beendofl.(1 --*3)- and endo.fl.(1 -*6)-glucanase and

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Vol. 99 YEAST PROTOPLASTS AND CELL WALLS 687

perhaps a partial separation has been achieved onthe Sephadex column.The release of N-acetylglucosamine on treatment

of cell wall with snail-juice fraction C is consistentwith the findings of Eddy (1958a). The role of theamino sugars in yeast cell-wall structure is as yetunestablished. Various references to the possiblepresence of chitin in yeast cell wall are to be foundin the literature (for reviews, see Eddy 1958b;Phaff, 1963). It has also been suggested that theamino sugar may provide the link between proteinand carbohydrate in the walls (Eddy, 1958a;Korn & Northcote, 1960).The fact that fraction C dissolves all the protein

in LP cell walls but only 20% in SP cell wallssuggests either that the protein itself differs in thetwo types of cell wall, or that it is differently linked.It has been shown by Davies & Elvin (1964) thatprotoplast formation from Saccharomyces fragii8under the action of snail juice is enhanced by pre-incubation of the cells with 2-mercaptoethanol.The present work shows that pretreatment of SPcells of S. carlebergensi8 and cell-wall materialsderived from such cells with 2-mercaptoethanolresults in protoplast formation and cell-wall dis-solution respectively on subsequent incubationwith snail-juice fraction C. From this it seems thatthe thiol has in some way modified the mannanand perhaps the protein of the SP cell-wall, render-ing it as susceptible to attack by the snail-juiceenzymes as is the LP cell wall. Bacon et al. (1965)from their studies on the effect of2-mercaptoethanolon the lytic enzymes from (ytophaga john8oniipostulate that two structural systems exist, eitherof which when intact preserve the integrity of theyeast cell wall. One of these structures is theglucan, the other composed of mannan-proteincomplexes associated through disulphide linkagesas suggested by Nickerson & Falcone (1956).Extending this hypothesis to the present findings,perhaps the difference between cell wall fromeither LP or SP cells ofS. carlsbergens8s and perhapsbetween all yeasts that are either susceptible ornon-susceptible to protoplast formation under theaction of snail juice lies in the nature of the secondof the two proposed structural systems. It ispossible that in the susceptible yeasts the di-sulphide linkages are incompletely formed anddegradation of the glucan is sufficient to effectsolubilization of the complete cell wall. On theother hand, in the resistant cell wall it is only after

the disulphide linkages have been disrupted bysuch agents as 2-mercaptoethanol that the glucan-ases can bring about complete dissolution.

The authors thank Dr A. H. Cook, F.R.S., for his adviceand encouragement.

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