REDUCTION OF SELENITE BY INTACT YEAST CELLS ANDCELL-FREE PREPARATIONS

GIUSEPPE FALCONE' AND WALTER J. NICKERSONInstitute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication 1 November 1962

ABSTRACTFALCONE, GIUSEPPE (Rutgers, The State Uni-

versity, New Brunswick, N.J.), AND WALTER J.NICKERSON. Reduction of selenite by intact yeastcells and cell-free preparations. J. Bacteriol.85:754-762. 1963.-Nonproliferating cell suspen-sions of Candida albicans rapidly reduced seleniteto red, metallic selenium in the absence of addedsubstrate. Cell suspensions reduced selenite opti-mallyat pH 4.2. Noadded metabolite was found tobe stimulatory; reduction was inhibited bymethionine and formate as well as by fluoride,dinitrophenol, and certain sulfhydryl poisons.Cell-free preparations capable of reducing selenitewere obtained from C. albicans and from baker'syeast disintegrated in a Hughes press. Theenzymatic system had optimal activity at pH 7with 10-2 M selenite. Activity of the system waslost on dialysis but was restored upon the addi-tion of dialyzable substances or of boiled, un-dialyzed extract.

A selenite-resistant strain of Candida albicans(RM806) grew readily in media containing asmuch as 10-2 M sodium selenite. During growthof this yeast, uptake of selenite was both rapidand extensive. The major part of the seleniteaccumulated was reduced and retained withinthe cell as red, metallic selenium. Reduction ofselenite was also accomplished by intact, non-multiplying cells. Enzymatic reduction of seleniteto selenium has been studied with cell-freepreparations obtained from this strain as well asfrom baker's yeast. A preliminary report on thiswork has appeared (Falcone and Nickerson,1960a).Examination of conditions promoting selenite

reduction by intact cells and cell-free prepara-tions showed that the process operates at theexpense of endogenous cellular metabolism.

' Present address: Istituto di Patologia Gene-rale, Universita di Napoli, Naples, Italy.

Through the use of radioactive isotopes, phos-phate ions (H2PO4-) were shown to competewith selenite ions (HSeO3-) for uptake by intactcells; dinitrophenol inhibited uptake of phos-phate and selenite ions alike.

Selenite-reducing activity in cell-free prepara-tions was found to reside in a soluble, non-particulate fraction and to be complex in thatactivity was lost upon dialysis but was restoredby the addition of heat-stable, dialyzablecomponents.

MATERIALS AND METHODS

Microbiological procedures. C. albicans RM806(previously described by Nickerson, Taber, andFalcone, 1956) and Saccharomyces cerevisiae(baker's yeast) were used in this study. Thestrain of C. albicans was maintained by frequentsubcultures at 28 C on agar slants containing10-3 M sodium selenite in a nutrient medium(GGY medium) comprised of: glycine, 10 g;glucose, 20 g; yeast extract, 1 g; agar, 20 g; anddistilled water, 1,000 ml. Cultures were grown inGGY liquid medium at 28 C with continuousagitation on a reciprocal shaker. Baker's yeastwas obtained as pressed cakes, free from starchor other additives, from Anheuser-Busch, Inc.,Old Bridge, N.J. The cakes, wrapped in parafilmsheets and refrigerated at 2 C, were used withina few days of receipt.

Cell-free preparations. Several methods wereemployed to obtain cell-free extracts: plasmolysisof cells with toluene; grinding with alumina orwith glass powder; freezing and thawing; anddisintegration in a Hughes press. The most activeenzyme preparations were obtained by the lattermethod.

For plasmolysis with toluene, 1 lb of pressedbaker's yeast was broken into fragments andmixed in a beaker with 120 ml of warm toluene.The beaker was placed in a water bath at about60 C, and its contents were occasionally mixedwith a rigid spatula. When the yeast suspension

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reached a temperature of 38 C, it liquefied and gaswas evolved. Water (200 ml) was added, the toplayer of toluene was eliminated, and the yeast sus-pension was centrifuged at 18,000 X g for 10 minat 2 C.

In the freezing and thawing procedure, portionsof yeast cake, broken by hand into small frag-ments, or washed packed cells of C. albicans, wereplaced in a Lusteroid centrifuge tube and alter-nately frozen and thawed three or four times inliquid nitrogen, followed by warming underrunning water. The cell suspension, diluted withwater, was centrifuged at 18,000 X g for 10 minat 2 C.A press (Hughes, 1951) with a cylindrical

inner chamber (38 X 70 mm) and a capacity ofca. 30 ml of cell paste was employed; the pistonwas driven by a guillotine device (Gest andNordstrom, 1956) in which a weight of 38.5 kgwas dropped a distance of 185 cm onto the piston.Portions of pound cakes (about 25 g) werereduced by hand to small fragments, frozen inliquid nitrogen, packed into a cold press (main-tained at -20 C), and crushed without additionof abrasive. Liquid nitrogen was poured liberallyover the press after it was assembled and afterthe plunger had been compressed in the guillotine.The mass extruded from the press was extractedthree times with a total of 100 ml of 8.5% sucrosesolution and centrifuged in the cold at 3,000 Xg. The supernatant liquid portions were pooledand centrifuged at 18,000 X g for 20 min at 2 C;the resulting supernatant liquid was filteredthrough a Millipore filter (pore size 0.45 ,u) toeliminate any residual particulate matter. Theextracts were stored at -20 C until required.

Washed-cell preparations. C. albicans RM806,grown with continuous agitation for 48 hr at 28C in GGY broth, was harvested by centrifuga-tion, washed three times with water, and resus-pended in water. Suspension densities weredetermined, after appropriate dilution, at 420mA in a Klett-Summerson colorimeter. The cellswere incubated at 28 or 37 C for 150 min in0.02 M phthalate-NaOH buffer (pH 4.2) withsodium selenite (0.01 M).

Cell-free extracts. After cellular disruption andcentrifugation, preparations were dialyzed at 2C in 50-ml portions against 6 liters of 0.9%,NaCl solution or tris(hydroxymethyl)amino-methane (tris)-maleate buffer (0.01 M, pH 7.2),with continuous agitation in an Oxford multiple

dialyzer for 24 hr; external solutions were renewedafter 12 hr of dialysis. The extracts were trans-parent after dialysis against tris buL'er. Extractswere slightly turbid after dialysis against NaClsolution, and some protein precipitated duringstorage at -20 C; however, activity of theextract was only very slightly impaired. Extractswere incubated in tris-maleate buffer (0.01 M,pH 7.0) with sodium selenite (0.01 M) for 60 minat 37 C.

Estimation of selenite reduction. Extent ofreduction of selenite to red, metallic selenium bynonproliferating cell suspensions, or by cell-freepreparations, was estimated by the method ofFalcone and Nickerson (1960b). This methodinvolves determination of increase in opticaldensity (OD) at 420 m, (due chiefly to formationof red selenium) and correction for nonspecificincrease in turbidity as measured at 660 min.The corrected OD readings at 420 mA were con-verted to ,g of selenite reduced (Table 1) orto ,ug of selenium by reference to a calibrationcurve (Fig. 1) based on selenite reduced withhydrazine sulfate according to the method of

TABLE 1. Spectrophotometric determination ofelemental amorphous selenium

Optical density at Selenium SeleniteChange in OD produced reduced420 mjs 660 nmju

pg/Itl pg/mi0.054 0.012 0.042 2.4 5.30.124 0.020 0.104 6 13.20.258 0.032 0.225 12 26.50.452 0.068 0.374 20 43.90.544 0.082 0.462 24 52.7

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0.100 0.200 0.300 OAOO 0.500

A OPTICAL DENSITY (420 m,u )

FIG. 1. Calibration curve for estimation of selenitereduced to metallic selenium from change in ODat 420 m,u, calculated as described in the text.

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FIG. 2. Effect of pH of suspending medium on

selenite reduction by nonmultiplying cells of Can-dida albicans RM806. Incubation at 37 C for 160min with 10-2 M Na2SeO3 in 0.01 M buffers of thefollowing compositions: pH 2, HCl-KCl; pH 3 to6, phthalate-HCl or phthalate-NaOH; pH 6 to 8,tris-maleate; pH 9, tris-HCl; pH 10, glycine-NaOH.Note optimum at pH 4.2. Cells (dry weight)/ml =

11 mg.

700

-600-

'.5000

U4000

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Z200-J

100 .

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,80t

0 30 60 90 120

TIME OF INCUBATION (MINUTES)

FIG. 3. Increase in amount of selenite reducedby nonmultiplying cells of Candida albicans RM806with time of incubation. Phthalate-NaOH buffer(pH 4.2, 0.01 M) containing 10-2m Na2SeO3; incuba-tion at 37 C for times noted. Cells (dry weight)/ml = 15 mg.

Tuller (1954), and estimated spectrophoto-metrically at 420 and 660 m,.

RESULTS

Selenite reduction by nonproliferating cells of C.albicans. Intact cells of C. albicans RM806suspended in an aqueous solution of sodiumselenite reduced selenite rapidly. Reduction ofselenite was markedly influenced by environ-mental conditions, such as selenite concentration,pH, temperature, and time of incubation. Theeffect of pH on selenite reduction by restingcells is shown in Fig. 2; an optimum lies in therange from pH 4.2 to 4.7. As will be presented

TABLE 2. Effect of selenite concentration on selenitereduction by nonproliferating cells of

Candida albicans

Selenite concn Selenite reduced*

M Pg/ml10-1 445410-2 420-48010-3 88-108

* Determined after 150 min of incubation at37 C, with a cell suspension of approximately 13mg (dry wt)/ml.

subsequently in more detail, the existence of anoptimal pH for reduction of selenite by intactcells reflects the effect of pH on selenite per-meation into intact cells. The dissociation con-stant for the first H of selenious acid is 3 x 10-3(pKa = 2.52) and for the second H is 5 X 10-(pKa = 7.30). A maximal concentration of thebiselenite ion (HSeO3-) is attained at pH 4.9;this form may be assumed to be the species ofselenite permeating intact cells.On exposure of washed cells to a solution of

selenite, uptake and reduction of selenite com-menced promptly, without lag or inductionperiod; the amount reduced increased on con-tinued incubation (Fig. 3), reaching a plateauafter about 60 min. With nonmultiplying cells,selenite reduction was extensive at a seleniteconcentration of 10-2 M (Table 2), a concentra-tion ten times higher than that optimal forselenite reduction by multiplying cells.

In Table 3, values are reported for selenitereduction in the presence of a variety of sub-strates incorporated into the incubation mixture.It is obvious that none of the substances testedexerted any significant stimulatory influence onselenite reduction. Thus, metabolism of endoge-nous reserve materials suffices to supply reduc-tants for the conversion of selenite to selenium.It is noteworthy that compounds which have beenimplicated in methylation of selenium (Chal-lenger, 1945) were without stimulatory effect(Table 3). On the contrary, of this group,methionine consistently caused a slight inhibitionof selenite reduction; formate, either alone or inassociation with methionine, homocystine, orbetaine, strongly inhibited selenite reduction;formaldehyde had an even more pronouncedinhibitory erect. No evidence for the formationof volatile selenium compounds was obtained in

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TABLE 3. Effect of methylating agents and substrateson selenite reduction by nonproliferating

cells of Candida albicans

Additionsa Selenite reductionb

Methylating agentscCholine .............................. 95

Inositol... 105

Betaine.......................... 98

DL-Homocystined.82DL-Methionine.74Na-formate.41Na-formate + DL-methionine 45

Na-formate + DL-homocystined 38

Na-formate + betaine.39

Choline + inositol.98

Formaldehydee.22Carbon substratese

Glucose 105

Succinate 95

Ethanol..... 92Pyruvate 90

Citrate 92

Acetate..... 89Lactate..... 92

-The compounds listed were added to cell

suspensions at zero time and incubation continuedfor 150 min at 37 C.

b Activity expressed as percentage of endogen-ous control.

c Added at a concentration of 0.02 M.

d Added at 0.004 M.

6 Added at 0.01 M.

studies with growing cultures of C. albicans. Incultures held for many days, only traces of alkylselenide formation were detected, beginningmany days after both multiplication and selenitereduction had ceased (Falcone and Nickerson,1960b). In the short-term experiments withnonmultiplying cells and cell-free preparations,no evidence for the formation of volatile seleniumcompounds was obtained. Thus, inhibition ofselenite reduction by methylating agents doesnot result from diversion of selenium into volatileorganic combination.The influence of various well-known metabolic

poisons on selenite reduction by intact cells isshown in Table 4. Some sulfhydryl inhibitors,such as Cu , Hg I, Ag+, and iodoacetate (atfinal concentrations of 10-4 M) strongly inhibitedselenite reduction, whereas other sulfhydrylpoisons, including arsenite, p-chloromercuri-benzoate, and iodoacetamide, were only slightly

TABLE 4. Effect of metabolic inhibitors on reductionof selenite by intact cells of Candida albicans*

Concn of testAdditiont substance

10- ms 10-4 Ms

2,4-Dinitrophenol............ 57 27KCN...................... 50 7NaN3...................... 82 63Cu++...................... 100 56Hg+...................... 100 85AsO3=...................... 25 0Ag+...................... 91 73p-Chloromercuribenzoate 49 18Iodoacetate.................. 75 15Iodoacetamide............... 55 0Fluoroacetate................ 0 0Fluoride .................... 15 0Ethylenediaminetetraacetate. 6 0Atabrine .................... 0 0Aminopterin................. 0Sulfathiazole....... 0

* Results are expressed as per cent inhibitionin comparison with the control.

t Test substances were added to the cell sus-pension at zero time. Incubation was for 150 minat 37 C.

inhibitory at concentrations of 10-3 M. Fluorideat 10-2 M, a concentration sufficient to blockglycogen utilization in yeasts (Nickerson andChung, 1952), almost completely inhibitedselenite reduction. At 10-4 M, inhibition bycyanide was slight, but inhibition by sodiumazide was quite extensive; the mechanism ofaction of azide in this instance has been examinedand will be described later. Dinitrophenol(DNP) inhibition of selenite reduction bymultiplying cells can be overcome by the additionof riboflavine 5'-phosphate (FMN; Falcone andNickerson, 1960b); DNP also strongly inhibitedselenite reduction by nonproliferating cells. Theyellow color of DNP was discharged by acidifica-tion before determination of reduced selenium.However, the deep yellow color of riboflavinephosphate could not easily be eliminated, and theeffect of FMN on DNP inhibition of selenitereduction was not investigated with nonpro-liferating cells.The inhibitory effect of phosphate on selenite

reduction in growing cultures of C. albicans hasbeen reported (Falcone and Nickerson, 1960b).As shown in Fig. 4, this effect of phosphate is

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0

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CONCENTRATION OF KHM2P4 (MOLARITY)

FIG. 4. Effect of phosphate concentration onselenite uptake and selenite reduction by nonmulti-plying cells of Candida albicans RM806. Incubationfor 60 mmn at 37 C in 0.01 m phthalate-NaOH buffer(PH 4.2) containing 10-2m Na2SeO3 and KH2PO4in concentrations noted.

very marked with non-proliferating cells;inhibition is maximal at phosphate concentrationsequimolar with that of selenite. Determinationsof the selenite content of the suspending mediumat the beginning and at the end of an experimentshowed that selenite uptake was inhibited byphosphate. From experiments with MUltiDplingTcells, the hypothesis was advanced that suchinhibition of selenite reduction could be ex-plained by a competition between phosphate andselenite for sites of absorption. Support for thishypothesis is given by the present demonstrationof the paralel inhibition of selenite uptake andreduction in the presence of phosphate. Thecurious shape of the curves in Fig. 4 is to benoted. Maximal inhibition (90%) of seleniteuptake was observed repeatedly at equimolarconcentrations of phosphate and selenite (0.01m), with markedly less inhibition (60%) atphosphate concentrations either one-tenth orten times that of selenite.

Addition of radioactive selenite (Na2Se7503) tocarrier selenite, to give a final concentration of0.01 M, served to confirm the fact that phosphatecaused inhibition of selenite uptake. In contrastto results obtained by chemical analyses forresidual selenite in the suspending medium (seeFig. 4), little difference was noted in the extentto which uptake of labeled selenite was inhibited

Time (minutes)FIG. 5. Effect of phosphate concentration on up-

take of radioactive selenite by nonmultiplying cellsof Candida albicans RM806. Incubation for timesnoted in 0.01 M phthalate-NaOH buffer, pH 4.3, insuspending medium containing Na2Se7503 andcarrier Na2SeO3 to give 0.01 M final concentrationand concentrations of KH2PO4 as follows: *, zero;*, 10-3 m; A, 10-2 M; 0, 10-1 M.

by phosphate over a concentration range from10-3 to 10-1 M (Fig. 5). In view of competitionbetween phosphate and selenite permeation intothe cell, one might anticipate that a selectiveinhibitor of phosphate permeation would alsointerfere with uptake of selenite. Such, indeed,was found to be the case. Inhibition of phosphateuptake by DNP is well known (Hotchkiss, 1944).Marked inhibition of selenite uptake by DNPwas also found in studies with Se75 (Fig. 6);5 X 10-5 M DNP inhibited selenite uptake by50%.

Effect of selenite on glucose assimilation.Sodium selenite strongly inhibited glucoseoxidation by C. albicans 806; at the same time,carbon dioxide was produced in excess of theoxygen consumed. With a selenite concen-tration of 10-3 M, oxygen uptake was inhibited84%, whereas CO2 production was inhibitedonly 63%; the RQ, meanwhile, was elevated from1.0 to 2.3. In the selenite-resistant strain RM806,on the other hand, respiration was considerably

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0c0

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Time (minutes)FIG. 6. Effect of dinitrophenol on selenite uptake

by nonmultiplying cells of Candida albicans RM806.Incubation at 37 C for times noted in 0.01 Mphthalate-NaOH buffer (pH 4.3) containing 10-2 MNa2SeOa, and concentrations of 2,4-dinitrophenolas follows: O, zero; O, 5 X 10-5; ,5 X 104 M.

less sensitive to inhibition by selenite, and in no

instance was an excess in CO2 production ob-served (Falcone and Nickerson, 1960b).Endogenous and exogenous (glucose) fer-

mentation of C. albicans RM806 was insensitiveto selenite. In contrast, endogenous fermentationof selenite-sensitive strain 806 was stimulatedmarkedly by selenite, and fermentation ofglucose went to completion in the presence ofselenite; in other words, anaerobic assimilationof glucose in this strain was blocked by selenite(Fig. 7). The action of selenite on respiration andfermentation of strain 806 was remarkablysimilar to that observed after the addition ofDNP.

Reduction of selenite by cell-free extracts.Undialyzed cell-free extracts of C. albicansRM806 and of baker's yeast were found to becapable of reducing sodium selenite to metallicselenium. Extracts prepared by autolysis withsodium acetate or by freezing and thawing showedvery little activity, whereas extracts prepared bygrinding or crushing cells and by autolysis withtoluene were very active. Extracts of baker's

THEORETICAL_VALUE

120 180TIME (MINUTES)

240 300

FIG. 7. Suppression of anaerobic assimilation ofglucose in presence of selenite. Nonmultiplying cells[7.8 mg (dry weight)/vessell of Candida albicans 806studied manometrically for time noted under N2atmosphere at 37 C with 1 mg of glucose and the fol-lowing concentrations of Na2SeO3: 0, zero; @, 10-3;and O, 10-2 M. Endogenous fermentation in theabsence of added glucose or selenite shoum by i.Theoretical value for complete fermentation of 1 mgof glucose shown by dotted line. Note resemblance totypical "dinitrophenol effect" on assimilation.

yeast obtained with a Hughes press were usedextensively, since fresh material was available inquantity, and special precautions against in-fection were not necessary.

Only the soluble part of the extract was activein reducing selenite. With extracts prepared bythe different procedures described, and exercisingthe utmost care in preserving the integrity ofmitochondrial particulates, removal of cellularparticulates did not cause any impairment of theactivity of the extract; the isolated mitochondrialparticulates were inactive (Table 5). The cell-freeextract was stable; its activity remained almostunchanged after 15 days of storage at -20 C, or

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TABLE 5. Selenite-reducing activity of variousfractions of cell-free preparations*

SeleniteFraction reduced

Cell-free extract..................... 100Supernatant (18,000 X g) ............ 106Mitochondrial particulate............ 1Dialyzed extract..................... 2Dialyzed extract + boiled yeast

extract............................. 92

* Enzymatic assays were carried out in tris-maleate buffer (0.01 M, pH 7) with 0.01 M Na2SeO3.Activity was measured after 60 min of incubationat 37 C, and is expressed as per cent of value forcell-free extract.

TABLE 6. Effect of phosphate and metals on selenitereduction by cell-free extract of Candida

albicans RM806*

Additions Concn Activity

M

Control................... 100Phosphate...... 5 X 10-2 49Cu ............10 10

10-4 18p-Chloromercuribenzoate. 10-4 73Mn++................... 10-4 73Zn++................... 10-4 18

* Results are expressed as percentage of activ-ity in comparison with the control. Incubation for60 min at 37 C with 0.01 M NaSeO3 in 0.01 M tris-maleate buffer (pH 7.0).

after 3 days at 2 C, but activity was lost after 5min at 60 C. Enzymatic activity was stronglyinhibited by copper or zinc ions and partiallyinhibited by p-chloromercuribenzoate, phosphate,and manganous ions (Table 6).The effect of pH on selenite reduction by

cell-free extracts is shown in Fig. 8; an optimumat about pH 7 is apparent, suggesting that thepH optimum observed with nonproliferatingcells is not due to a direct effect of hydrogen ionconcentration on the enzymatic activity of thecell, but probably reflects the concentration ofthe biselenite ion, HSeO -. With tris-maleatebuffer at pH 7, it was observed that high concen-trations of buffer inhibited selenite reduction.With a final concentration of 5 X 102 M, 50%Oinhibition was observed; in all other experiments,

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FIG. 8. Effect of pH on selenite reduction by cell-free enzyme preparation from baker's yeast. Incuba-tion for 60 min at 37 C with 0.01 M Na2SeO3 in 0.01 Mbuffer of composition as given in legend of Fig. 2.Note optimum at pH 7.

30

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30 60 90 120 150TIME OF INCUBATION (MINUTES)

FIG. 9. Increase in amount of selenite reduced bycell-free enzyme preparation from baker's yeast withtime of incubation at 37 C in tris-maleate buffer(0.01 M, pH 7.0) containing 0.01 M Na2SeO3.

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70

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FIG. 10. Effect of selenite concentration on selenitereduction by cell-free enzyme preparation frombaker's yeast. Incubation for 60 min at 37 C in 0.01 mtris-maleate buffer (pH 7.0) with concentrations ofNa2SeO3 noted.

80,-

the buffer concentration was kept constant at10-2 M. The amount of selenite reduced increasedwith time; after an initial sharp rise, the ratedeclined to a plateau (Fig. 9). Enzymatic activitywas maximal with 10-2 M buffer (Fig. 10) andvaried directly with enzyme concentration(Fig. 11).Dialyzed extract did not reduce selenite,

whether dialysis was carried out against distilledor running water, NaCl solution, or tris-maleatebuffer (pH 7.2). Activity of the dialyzed extractcould be restored by the addition of boiled yeastextract (Table 5). Boiled extract, by itself,reduced selenite to a very limited extent, and ablank for evaluation of reduction from thissource was always included in enzymatic assays.Various known cofactors added to the dialyzedextract neither restored activity nor enhancedthe activity of a dialyzed preparation to whichboiled extract had been added. The substancestested, singly and in combinations, included:FMN, flavin adenine dinucleotide, di- and tri-phosphopyridine nucleotides, folic acid, biotin,coenzyme A, lipoic acid, and thiamine pyrophos-phate.

DIscussION

Uptake of selenite by nonproliferating cells ofa selenite-resistant strain of C. albicans ensues

E 60 0 without lag on exposure to selenite. In contrastto uptake of H2PO4-, for which exogenous glucose

x o metabolism is essential (Mullins, 1942), uptakeO of HSeO3- occurs in the absence of added sub-U strate. In fact, no exogenously supplied sub-O 40 stance has been found to promote HSeO3-CZ / uptake, and, therefore, endogenous metabolismw / of some (CH20)n reserve is implicated as a

source of energy for uptake and reduction ofw / HSeO3-. Fluoride inhibition of such processesLaJ 20 - o may be interpreted in this light. Nevertheless,/uptake of both H2P04- and HSeO3- is inhibited

by DNP. Uptake of the biselenite ion (HS03-)is competitively antagonized by the phosphateion (H2P04-). Conversely, selenite interferes

° 2 3 4 5 6 with phosphate uptake and causes an inhibitionPROTEIN CONCENTRATION (mg/ml) of oxidative assimilation that is comparable tG

the effect of DNP on glucose metabolism.

FIG. 11. Relationship between amount of selenite .

Selenite taken up by intact cells of C. albicansreduced and amount of protein added in cell-free is reduced to red, metallic selenium to such an

enzyme preparation from baker's yeast. Incubation extent that suspensions become bright red.for 60 min at 37 C in 0.01 M tris-maleate buffer (pH Although no evidence for the production of7.0) containing 0.01 M Na2SeO3. volatile selenium compounds was obtained,

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either with cultures of C. albicans or with non-proliferating suspensions, methylating agentsand other substances involved in one-carbonti ansfer reactions were found to inhibit formationof elemental selenium. Whatever the mechanismof this inhibition may be, it does not involvediversion of selenite to form dimethyl selenide,or other volatile selenide.

Cell-free enzyme preparations capable ofreducing selenite to elemental selenium wereobtained from C. albicans and from baker'syeast. The particulate-free fraction obtained byhigh speed centrifugation comprised the activityof cell-free extracts; the mitochondrial particulatefraction was inactive. Formation of elementalselenium ensued promptly upon exposure of theenzyme system to selenite, even with extracts ofbaker's yeast which had not previously beenexposed to selenite. Enzymatic activity was lostupon dialysis, but restored by the addition ofdialyzed suspension or of boiled cell-free extract.No intermediate states of reduction were detectedbetween Se+4 and Seo.

ACKNOWLEDGMENTS

This work was supported in part by a grantfrom the U.S. Public Health Service (E-251)and a grant under the NATO Research GrantsProgram. The authors thank E. Palumbo ofAnheuser-Busch, Inc., Old Bridge, N.J., forsupplies of pressed yeast. The large Hughespress employed in these studies was made tospecification by R. Makin, Faculty of Medicine

Workshops, Sheffield University, England. Theguillotine device for operating the Hughes presswas constructed for us by G. C. Gatewood,Machinist at the Institute of Microbiology.

LITERATURE CITED

CHALLENGER, F. 1945. Biological methylation.Chem. Rev. 36:315-361.

FALCONE, G., AND W. J. NICKERSON. 1960a. Enzy-matic reduction of selenite. Bacteriol. Proc.,p. 152.

FALCONE, G., AND W. J. NICKERSON. 1960b. Metab-olism of selenite and mechanism of inhibitoryaction of selenite on yeasts. Giorn. Microbiol.8:129-150.

GEST, H., AND A. NORDSTROM. 1956. A plunger-hammer for the Hughes microbial press. NewsLetter, Society of American Bacteriologists22(2): 15-16.

HOTCHKISS, R. D. 1944. Gramicidin, tyrocidine,and tyrothricin. Advan. Enzymol. 4:153-200.

HUGHES, D. E. 1951. A press for disrupting bac-teria and other microorganisms. Brit. J. Exptl.Pathol. 32:97-109.

MULLINS, L. J. 1942. Permeability of yeast cells toradiophosphate. Biol. Bull. 83:326-330.

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