JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 806-814Copyright 0 1975 American Society for Microbiology

Vol. 123, No. 3Printed in U.S.A.

Evidence for Extrusion of Unfolded Extracellular EnzymePolypeptide Chains Through Membranes of Bacillus

amyloliquefaciensR. L. SANDERS* AND B. K. MAY

Department of Biochemistry, University of Adelaide, South Australia, 5001

Received for publication 21 April 1975

The production of extracellular a-amylase and protease by protoplasts ofBacillus amyloliquefaciens has been achieved. The production of enzymicallyactive protease was totally dependent on a high concentration of either Mg'+,Ca2+, or spermidine, but production of active a-amylase was not. This cationdependence of protease production was seen immediately upon addition oflysozyme to intact cells. The cations could prevent the inactivation of proteaseand alter the cytoplasmic membrane configuration of protoplasts. Production ofactive a-amylase and protease by protoplasts was totally inhibited by proteolyticenzymes such as trypsin, a-chymotrypsin, or the organism's purified extracellu-lar protease. The evidence suggests that these degradative enzymes act specifi-cally on the emerging polypeptide of the extracellular enzyme and that thepolypeptide emerges in a conformation different from that of the native molecule.

We are examining the synthesis and secretionof extracellular enzymes by Bacillus amyloli-quefaciens as a model system for investigatinghow some proteins are selectively secretedthrough cellular membranes. This organism hasthe advantage that washed-cell suspensionsrapidly produce large amounts of extracellularprotease, a-amylase, and ribonuclease.No significant amounts of active protease,

a-amylase, or inactive cross-reacting materialcan be detected inside secreting cells of B.amyloliquefaciens (4, 11). In addition, thereexists in the cytoplasm an inhibitor specific forthe extracellular ribonuclease (17), and sincethe formation of the enzyme-inhibitor complexis essentially irreversible (9), it seems unlikelythat the native enzyme could ever have existedas such within the cell. We have thereforesuggested that extracellular enzymes are syn-thesized on membrane-associated ribosomesand that the nascent polypeptides are extrudedthrough the membrane to assume their activetertiary configuration only outside the permea-bility barrier (12).Compatible with this model are our findings,

which suggest that within the cell there arepools of extracellular enzyme-specific messen-ger ribonucleic acid (2, 7). We have suggestedthat these apparent pools are the result ofexcessive transcription, possibly designed tosaturate the membrane "translational-extru-sion" sites with messenger ribonucleic acid

despite its rapid degradation en route from geneto membrane (6).Attempts to directly test the extrusion hy-

pothesis in our system have been difficult be-cause of the apparent inability of protoplasts ofthis organism to secrete active extracellularenzymes, even though intracellular protein andribonucleic acid synthesis continue almost nor-mally (12). However, it has now been shownthat in the presence of sufficient concentrationsof either Mg2+, Ca2+, or spermidine, protoplastswill secrete enzymatically active protease anda-amylase. During secretion these extracellularenzymes exist in a form that is sensitive toproteolytic attack, whereas the released en-zymes are insensitive. This finding is compati-ble with our proposed model for enzyme secre-tion. A preliminary report of this work hasappeared previously (16).

MATERIALS AND METHODSOrganism used. The parent strain of the organism

used in this work was an unclassified strain of Bacillusamyloliquefaciens (11). This strain produces an extra-cellular peptide-lipid molecule, "surfactin," thatlyses protoplasts (13). A mutant unable to producethis compound was isolated as follows and used in thepresent work. Approximately 106 spores in 1 ml ofglass-distilled water were treated with 0.1 ml of ethylmethyl sulfonate for 20 min at 37 C. The spores werewashed once by centrifugation and suspension in 5 mlof 0.9% NaCl and were finally suspended in 1 ml of0.9% NaCl. This suspension (0.1 ml) was used to

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inoculate 500 ml of culture medium (2), and theculture was shaken at 30 C for 25 h. Cultures wereplated for single colonies on blood agar plates andincubated for 16 h at 37 C. Those colonies which didnot produce a hemolyzed zone were picked off andpurified by subculturing four times. The mutantstrain used was cultured as described previously (2);cultures did not produce any lytic factor detectable byprotoplast lysis (13), whereas the production of extra-cellular a-amylase and protease was normal com-pared with wild type.

Washed-cell and protoplast suspension experi-ments. PMC medium contained tris(hydroxy-methyl)aminomethane (Tris) (25 mM), (NH4)JHPO,(3.8 mM), KCl (5 mM), sodium citrate (4.25 mM),CaCl, (0.125 mM), ZnSO4 (0.0125 mM), 0.025%(wt/vol) Casamino Acids (Difco), 0.25 ml of tracemetal solution (2) per liter, 1% maltose, and 22%(wt/vol) sucrose adjusted to pH 7.3 with HCI. Cellsafter 25 h of growth were harvested, washed once withPMC medium lacking sucrose, and finally suspendedin the same volume of PMC medium. Protoplastswere prepared by shaking washed-cell suspensions at30 C for 45 min with 133 jig of lysozyme per ml of cells.In some experiments, samples (0.8 ml) were removedat various times during protoplast formation andcentrifuged, and the supernatants were assayed fora-amylase and protease activity. In other experi-ments, protoplasts were resuspended in fresh PMCmedia and further incubated at 30 C with shaking.Samples were withdrawn at appropriate times andcentrifuged, and the supernatants were assayed forenzyme activity. Ribonuclease could not be measuredin these experiments because of inhibitor released bylysis of a small proportion of protoplasts (17).Assay of enzymes. Protease activity was assayed

by using a Remazol brilliant blue-hide powder assay(15). One unit of activity is defined as the amount ofenzyme giving an increase in absorbance at 595 nm of5.7 in 40 min at 37 C and corresponds to the unitdefined earlier (11). a-Amylase was assayed as previ-ously described (2).Measurement of total protein synthesis. The

incorporation of L- ["C ]phenylalanine (460 mCi/mmol) into total trichloroacetic acid-precipitable ma-terial was measured as described earlier (2). Totalextracellular protein represents only about 5% of thetotal cellular trichloroacetic acid-precipitable mate-rial.

Preparation of antibodies. B. amyloliquefaciensextracellular protease was purified to homogeneity asdescribed earlier (2), and its antibody was preparedby inoculating New Zealand rabbits with weekly,multisite, subcutaneous injections of protease in com-plete Freund adjuvant. Rabbit antisera were fraction-ated by precipitation with 50% (wt/vol) ammoniumsulfate, followed by dialysis of the precipitate toremove salt and fractionation on diethylaminoethyl-cellulose columns. The gamma globulin fractions werepooled and concentrated by using aquacide. Goatanti-rabbit gamma globulin was obtained by injectinggoats with normal rabbit gamma globulin.

Inactivation of trypsin and a-chymotrypsin.Trypsin (or a-chymotrypsin) in cell or protoplast

supernatants was inactivated before the determina-tion of B. amyloliquefaciens protease activity. Tryp-sin was inactivated by incubation for 4 h at 30 C with1-chloro-3-tosylamide-7-amino-2-heptanone hydro-chloride (TLCK) at a final concentration of 30 jig/ml.For a-chymotrypsin, L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) at 10 jig/ml was used(added as a methanolic solution of 1 mg/ml) andincubated at 30 C for 5 h. These procedures com-pletely inactivated both enzymes but did not affect B.amyloliquefaciens protease.

Polyacrylamide gel electrophoresis. Electro-phoresis of protease was carried out as describedearlier (2). To detect ptotease and 3H radioactivity,gels were frozen in solid CO2 and sliced longitudinally,and corresponding halves were cut into 1-mm slices.The enzyme was eluted overnight from the slices with1.5 ml of 50 mM Tris-hydrochloride (pH 7.8), and theeluate was assayed for protease. Radioactivity wasdetermined by dissolving gel slices for 16 h at 20 C in2.5 ml of a solution of 0.3% 2,5-diphenyloxazole and0.03% 1,4-bis-[2]-(4-methyl phenyloxazolyl)-benzene,12% (vol/vol) NCS tissue solubilizer (Amersham/Searle Corp.), and 0.08 M NH4OH, followed by liquidscintillation counting in a Packard Tri-Carb spec-trometer.

Covalent coupling of a-chymotrypsin to Sepha-rose 4B beads. a-Chymotrypsin was coupled to cya-nogen bromide-activated Sepharose 4B in 0.1 MNaHCO,, and after coupling was completed theproduct was extensively washed with six cycles eachof 0.1 M sodium acetate (pH 4.0) and 0.2 M NaHCO,(pH 8.5), each containing 1 M NaCl. The product wasstored in distilled water with 0.1% (wt/vol) sodiumazide and thoroughly washed before use. Using acasein assay (11), it was established that each 1.0 mlof the settled beads possessed proteolytic activityequivalent to 2.5 mg of free a-chymotrypsin.

Materials. Radiochemicals were obtained fromSchwarz/Mann. TLCK and TPCK were both pur-chased from Cyclo Chemical Corp., Los Angeles,Calif. TPCK-treated trypsin and TLCK-treated a-chymotrypsin were purchased from Worthington Bio-chemical Corp., Freehold, N.J. Chloramphenicol wasa product of Parke-Davis and Co., Sydney. Egg whitelysozyme, three times recrystallized, was a product ofSigma Chemical Co., St. Louis, Mo. The Remazolbrilliant blue was a generous gift from FarbwerkeHoechst, AG, Frankfurt.

RESULTSEffect of lysozyme on extracellular enzyme

secretion by washed-cell suspensions.Washed-cell suspensions were incubated in a22% (wt/vol) sucrose medium (PMC medium)containing 1 mM Mg2+ (which is optimal forextracellular enzyme production by intactcells), and the effect of lysozyme on enzymesecretion was examined. Production of activeprotease was inhibited almost instantly (Fig. 1).The first protoplasts were not visible until 20min, and electron microscopic examination

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showed that complete cell wall removal re-quired 45 min of incubation. The inhibitoryeffect of lysozyme was reduced by about 50%when the medium contained 10 mM Mg2+ (Fig.1). Although Mg2+ did have some effect ongeneral protein synthesis (Fig. 2), incorporationof ["'Ciphenylalanine into total protein in thepresence of lysozyme was not dependent on 10mM Mg2+, as was protease synthesis. Theselectivity of the Mg2+ effect for protease syn-thesis in the presence of lysozyme was furtherreinforced by the observation that extracellular

Incubation time (mm)

FIG. 1. Effect of Mg2+ concentration on productionof active protease by lysozyme-treated cell suspen-sions. Cells were incubated with lysozyme (added atzero time) in the presence of I or 10 mM Mg2+ (O, A)or without lysozyme in the presence of either 1 or 10mM Mg,+ (O, 0).

3.

0).2.

I,

Incubation time (min)FIG. 2. Effect of Mg2+ concentration on the incor-

poration of L-["4CJphenylalanine into trichloroaceticacid-precipitable material by lysozyme-treated cellsuspensions. Cells were incubated with lysozyme(added at zero time) in the presence of either I or 10mM Mg2+ (A, 0) or without lysozyme in the presenceof either 1 or 10 mM Mg2+ (0, A).

a-amylase synthesis after lysozyme additionwas the same in 1 or in 10 mM Mg2+.The inhibitory effect of lysozyme on protease

production by cells in 1 mM Mg2+ was immedi-ately alleviated by adding Mg2+ to 10 mM atany time up to at least 45 min. Similar resultswere obtained with Ca2+ or spermidine.To test the possibility that cells treated with

lysozyme in 1 mM Mg2+ secreted an inactiveform of the protease molecule, a cell suspension(8 ml) was incubated in PMC medium contain-ing 1 mM Mg2+ and lysozyme for 8 min, atwhich time 5 ltCi of L- [C "4]phenylalanine (460mCi/mmol) and 15 gCi of L-[14CJleucine (312mCi/mmol) were added. A control suspensiondid not contain lysozyme. Samples were takenat 8 min and again after 40 min of incubationand centrifuged, and the supernatants wereassayed for material that cross-reacted withprotease gamma globulin as described (8). Theresults suggest that lysozyme-treated cells in 1mM Mg2+ produce a form of the proteasemolecule capable of reacting with proteasegamma globulin (Table 1).

Effect ofcations on protease and a-amylaseproduction by protoplasts. Protoplasts wereincubated with shaking in PMC medium con-taining 10 mM Mg2+, and the production ofprotease was compared with that by intact cellsunder conditi6ns identical except for the omis-sion of lysozyme. The rate of synthesis ofprotease by protoplasts was about 30% of that ofintact cells, and production of enzyme waschloramphenicol sensitive (Fig. 3). (Separateexperiments showed that the enzyme produced

TABLE 1. Precipitation by protease gamma globulinof radioactively labeled material secreted by cellsincubated with and without lysozyme in the presence

of 1 mM Mg2+

Radioactivity precipitateda

Cells Time of incubationNet

8 min 40 min

Lysozyme treatedb 180 1,314 1,134(244) (1,624) (1,380)

Control .......... 220 5,500 5,280(411) (4,328) (3,917)

aRadioactivity precipitated by protease gammaglobulin followed by goat anti-rabbit gamma globulin,expressed as counts per minute per milliliter ofsupernatant. All values have been corrected for non-specificity by addition of normal rabbit gamma globu-lin instead of protease gamma globulin. Results arefrom two separate experiments.

b Lysozyme was added at zero time and "4C-labeledamino acids were added after 8 min of incubation.

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PROTEINS THROUGH MEMBRANES 809

by protoplasts was electrophoretically identicalto that from cells.) The production of extracel-lular protease by the protoplasts could not beaccounted for by the presence of intact cellssince viable counts showed that there were lessthan 5 cells per ml of protoplast suspension andelectron microscopy revealed negligible levels ofcells. The protoplasts were devoid of both cellwall material and mesosomal structures asobserved by electron microscopy of sectionedspecimens.The lowered rate of extracellular protease

secretion observed was not due to lysis ofprotoplasts or reduced general metabolism sinceincorporation of L- [IC ]phenylalanine into totaltrichloroacetic acid-precipitable material byprotoplasts was essentially the same as that byintact cells. The production of active extracellu-lar protease by protoplasts was dependent onthe presence of 10 mM Mg2+ as found earlierwith lysozyme-treated cells. When protoplastsprepared in PMC medium containing 10 mMMg2+ were resuspended in the presence of 1 mMMg2+, no active protease was produced (Fig. 4).A separate experiment established that thiseffect was selective since the incorporation byprotoplasts of L- ["C]phenylalanine into totalprotein was almost identical in the presence of 1or 10 mM Mg2+. The Mg2+ required for activeenzyme production by protoplasts could bereplaced by Ca2+ at 10 mM or spermidine at 7.5mM. The rate of production of a-amylase byprotoplasts in PMC medium containing 10 mMMg2+ was comparable with that of protease,

Incubation t.m(mh)

FIG. 3. Effect of chloramphenicol on the produc-tion of protease by intact cells and protoplasts. Cellsin the presence and absence (A, 0) of chlorampheni-col. Protoplasts in the presence and absence (U, 0) ofchloramphenicol.

Incubation tim.(n )

FIG. 4. Effect of Mg2+ concentration and chloram-phenicol on the production of a-amylase and proteaseby protoplasts. Protoplasts were prepared in PMCmedium containing 10 mM Mg2' and then resus-pended in fresh PMC medium containing either I or10 mM Mg2+. a-Amylase production in 1 mM Mg2+(0), 10 mM Mg2+ (0), 1 mM Mg2+, and 20 ug ofchloramphenicol per ml (A). (An identical result withchloramphenicol was observed in the presence of 10mM Mg2+.) Protease production in 1 mM Mg2+ (0),10 mM Mg2+ (U), 10 mM Mg2+, and 20 Mg ofchloramphenicol per ml (A). (An identical result withchloramphenicol was observed in the presence of 1mM Mg2+.)

representing approximately 25% that of intactcells (Fig. 4). This production was sensitive tochloramphenicol, and separate experimentsshowed that the enzyme was electrophoreticallyidentical to that from cells. However, in markedcontrast to protease, a-amylase production pro-ceeded unimpaired in the presence of only 1mM Mg2+.

Effect of cations on protease inactivation.Performed protease molecules remained com-pletely active in PMC medium containing 1mM Mg2+, and it seemed possible that theaddition of high concentrations of cations toprotoplasts (or lysozyme-treated cells) was re-quired to prevent inactivation of protease mole-cules during secretion. To see whether thesecations prevent inactivation of protease, puri-,fied B. amyloliquefaciens protease (2) was incu-bated in 25 mM Tris-hydrochloride (pH 7.3) at30 C in the absence of cations. A 50% loss ofactivity occurred in 12 h, but this inactivationwas totally prevented by either Mg2+, Ca2+, orspermidine at concentrations as low as 0.1 mM.electrophoresis on sodium dodecyl sulfate gelsshowed that no detectable autodigestion ofprotease occurred during this inactivation.

Effect of proteolytic enzymes on extracellu-lar enzyme production by protoplasts. Tryp-sin at 2 gg per ml of protoplast suspensioncompletely inhibited the production of a-amy-lase (Fig. 5). The appearance of protease (mea-sured after 60 min of incubation with trypsin)

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was also inhibited, 70% by 2 Ag and 98% by 10jg of trypsin per ml. In these experiments,trypsin had no effect on total protein synthesisby protoplasts. A trivial explanation for thiseffect on a-amylase and protease elaborationwas that the native enzymes were trypsin sensi-tive. However, this was not so since the activi-ties of both a-amylase and protease, present ina protoplast supernatant, were not affected byincubation for 24 h at 30 C with 100 ,ug oftrypsin per ml. In contrast to the protoplastfindings, the production of a-amylase and pro-tease by whole cells was unaffected by concen-trations of up to 1 mg of trypsin per ml.However, their appearance was completely andalmost immediately inhibited by trypsin uponthe addition of lysozyme to cells suspended inPMC medium containing 10 mM Mg2+. Theresult for a-amylase is shown in Fig. 6.To distinguish whether trypsin affects the

emerging enzyme or the protoplast itself, proto-plasts were shaken with 2.5 Mg of trypsin per mlfor 15 min at 30 C, centrifuged, and resus-pended in the absence of trypsin. a-Amylaseproduction recommenced immediately upon re-moval of trypsin, although at a slightly reducedrate, presumably because traces of trypsin re-mained (Fig. 7). Protease production similarlyrecovered. a-Chymotrypsin at 10 jg/ml gaveresults similar to those for trypsin; productionof active a-amylase and protease was inhibitedabout 85%, although total protein synthesis wasalmost unaffected and the inhibition could bereversed immediately by centrifugation andresuspension of cells.

It was of interest to examine the accessibilityof the proteolytically sensitive forms of theextracellular enzymes to a-chymotrypsin cou-

04-

~E 0-3-

a

I0-1

Incubation time (min)

FIG. 5. Effect of trypsin added at zero time on

a-amylase production by protoplasts. Protoplastswere incubated without (0) and with trypsin at 0.1(0), 0.5 (0), and 2 ug of protoplasts (A) per ml.

pled to Sepharose 4B beads. The appearance ofactive extracellular a-amylase was inhibited80% by the highest concentration of a-chymo-trypsin-Sepharose 4B complex tested (Fig. 8).(The appearance of active protease was simi-larly inhibited at this concentration.) This ef-fect was not due to the presence of unlinkeda-chymotrypsin in the preparation since theinhibition of a-amylase appearance was imme-diately and completely reversed after the selec-

12

14

Incubation time (rnen)

FIG. 6. Effect of trypsin and lysozyme on a-amy-lase production by washed-cell suspensions. Cellswere incubated in PMC medium containing 10 mMMg2+ together with (0) lysozyme + trypsin; (A)lysozyme alone; (0) trypsin alone at 10 gg/ml; (A) noaddition.

Incubation time (min)FIG. 7. Recovery of a-amylase production by pro-

toplasts after trypsin removal. Protoplasts in thepresence of 10 mM Mg2+ were previously incubatedwith 2.5 jgg of trypsin per ml for 15 min. Suspensionswere centrifuged and resuspended in fresh medium,and a-amylase production was followed in the pres-ence and absence of 2.5 Mg of trypsin per ml (U, A). Acontrol suspension of protoplasts, previously incu-bated for 15 min in the absence of trypsin, wassimilarly resuspended and incubated in the absence oftrypsin (-), and a-amylase production was followed.

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PROTEINS THROUGH MEMBRANES 811

tive removal, by centrifugation, of the coupledenzyme from a protoplast suspension (Fig. 9).The coupled enzyme had no effect on total 0.25protein synthesis by protoplasts nor extracellu-lar enzyme production by intact cells, whereasuncoupled Sepharose 4B had no effect on en-zyme production by protoplasts. E

Effect of extracellular protease from B. *--amyloliquefaciens on extracellular enzyme I2,appearance by protoplasts. The appearance of Xa-amylase was inhibited by treatment of proto-plasts with the organism's own purified extra-cellular protease; 90 U of protease per ml was X ,needed for complete inhibition (Fig. 10). Simi- 0 5 10 15 20

larly, 90 U of protease per ml inhibited the Icubationl time (min)formation of active protease by protoplasts (Fig. FIG. 9. Reversibility ofa-chymotrypsin-Sepharose11). To determine the latter, protoplasts were 4B inhibition of a-amylase production by protoplasts.incubated for 90 min with 50 M.Ci of L-[45- Protoplasts were incubated with a-chymotrypsin-H ]leucine per ml together with 90 U of protease Sepharose 4B at a concentration equivalent to 200H eucine

perml.A controget lerwith 9Uof rontease ug of free a-chymotrypsin (0) per ml of protoplastsper ml. A control sample did not contain and an equivalent concentration of unlinked Seph-protease. Supernatant samples were filtered arose 4B (a). At 5 min (arrow) the a-chymotrypsin-through a membrane filter (0.45 gm, 47 mm; Sepharose 4B was rapidly removed by low-speedMillipore Corp.) and dialyzed for 4 h against 10 centrifugation for 50 s, and the protoplast suspensionmM Tris-hydrochloride (pH 8.5) containing 1 was incubated fora further 15 min.mM CaCl2, and 50,l was subjected to poly-acrylamide gel electrophoresis followed by pro-tease and tritium determinations as described 04in Materials and Methods.

Protease at 90 U/ml did not significantly O 3affect total protein synthesis by protoplasts and E

intact cells, nor did it affect the secretion of |

02~~~~~~~~~~~~~~~~~~~~~0-2S - /l

10 20 30 40 sO 60Incubation time (min)

FIG. 10. Effect of purified extracellular protease_ /< from B. amyloliquefaciens on the production of a-s1 //amylase by protoplasts. Protoplasts were incubated0. without protease (0) and with 4 (0), 10 (A), 20 (A),

30 (0), and 90 U(0) of protease per ml.

active a-amylase and protease by intact cells.Protoplast a-amylase and protease moleculeswere completely stable in the presence of 90 U of

0-1!| protease per ml; protease stability was deter-ncubation time (min) mined by using radioactively labeled proteaseIncubatin time (min) and polyacrylamide gel electrophoresis as de-

FIG. 8. Effect of a-chymotrypsin linked to Sepha- scribed in Fig. 11. Again, the protease inhibitionrose 4B on the production of a-amylase by proto- of a-amylase production by protoplasts wasplasts. The final concentration of a-chymotrypsin- immediately reversed upon suspension of proto-Sepharose 4B added per milliliter of protoplasts

p lats resh rsedium.was equivalent in proteolytic activity to 0.1 (0), plasts in fresh medium.(A), or 250 (O) ug of free a-chymotrypsin. Control It is apparent from these studies that it is(0) did not contain the a-chymotrypsin-Sepharose only possible to detect extracellular enzyme4B complex. production by protoplasts in PMC medium

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'

.2

I

'a

*12

Top Gel Slice NumbeFIG. 11. Effect of purified extracellular protease from B. amyloliquefaciens on the production of protease by

protoplasts. (A, *) 3H radioactivity in the presence and absence of added protease. (0) Activity of protease.(The first peak of radioactivity was due to the incomplete removal of free [3H]leucine.)

containing 10 mM Mg2+ because the amount ofprotease produced by protoplasts (see Fig. 3) isinsufficient to significantly inhibit the appear-ance of either a-amylase (see Fig. 10) or pro-tease itself.

DISCUSSIONThis work shows that protoplasts of B. amylo-

liquefaciens can synthesize and secrete extra-cellular enzymes. The complete absence of cellwall in electron micrographs of sectioned proto-plasts shows that this structure is not obligator-ily involved in enzyme secretion, unlike thesituation in a streptococcus strain (10), wherethe activation of a protease zymogen is depend-ent on cell wall.The effect of cations on a-amylase and pro-

tease secretion is somewhat surprising in thatactive protease production is dependent oneither 10 mM Mg2+, 10 mM Ca2+, or 7.5 mMspermidine, but a-amylase production is not. Inthe presence of 1 mM Mg2+ active a-amylase isproduced but not active protease, althoughprotein capable of reacting with proteasegamma globulin is produced. The simplestexplanation, that the cations are needed toprevent inactivation of the protease moleculesduring secretion but are not needed in such highconcentrations for a-amylase, was supported bythe finding that the cations prevented inactiva-tion of purified protease incubated at 30 C.

Irrespective of whether the cations function toprevent inactivation of protease molecules, allof them appear to alter the properties of theprotoplast membrane. Protoplasts in 10 mMMg2+ are stable and centrifuge easily, whereasin 1 mM Mg2+ they are more susceptible to lysisand centrifuge with difficulty; Ca2+ and spermi-

dine have similar effects. The cations are mostlikely to react with the negative charges on themembrane phospholipid heads, and evidencefor this comes from the spin-labeling studies ofEhrstrom et al. (5), using B. subtilis mem-branes. An apparent thickening of the mem-brane has been observed in sectioned proto-plasts exposed to these cations (unpublisheddata) and may be related to this. Of possiblesignificance is the fact that the cation effectsappear to be somewhat specific for the mem-brane, as far as can be judged, since they do notaffect general "intracellular" metabolism asmeasured by L- [14C phenylalanine incorpora-tion into protein. It is particularly noteworthythat whereas the cation effects are not seen atall in intact cells, they appear immediatelyupon addition of lysozyme. This observation issurprising since 45 min of incubation withlysozyme is required to completely remove thecell wall, and one speculative possibility is thatthe intact cell wall retains a high concentrationof cations that are instantly released on lyso-zyme addition.The most important conclusion to be drawn

from this work is that molecules of extracellularenzymes emerging from the protoplast mem-brane are in a conformation different from thatof molecules immediately after their releasefrom the protoplast. This was shown by the factthat the addition of proteolytic enzymes tosecreting protoplasts completely prevented ap-pearance of active a-amylase and protease. Bycontrast, enzyme in solution that has beenproduced by protoplasts is completely stable tothe same proteases. Similar results have beenreported for penicillinase secretion by proto-plasts of B. licheniformis (1). The immediate

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reversibility of this inhibition in the presentwork is demonstrated convincingly by the ex-periments in which Sepharose-bound a-chymo-trypsin was removed by centrifugation andappearance of a-amylase recommenced at once.This shows that the effect of trypsin and a-chymotrypsin is on the emerging enzyme mole-cules and not on the protoplast itself, a conclu-sion confirmed by the insensitivity of generalcellular protein synthesis to the added proteo-lytic enzymes. The inhibition of extracellularenzyme production by Sepharose-bound a-chymotrypsin eliminates the possibility of theextruding polypeptide chain assuming its finalconformation within a membrane "pocket."There are several possible explanations for

the proteolytic sensitivity of emerging enzyme.The extracellular enzyme nascent polypeptidechain maybe extruded through the membraneand cell wall to assume its active tertiaryconfiguration outside the cell and be sensitive toproteases during the folding process. In view ofthe present work we consider this unlikely sinceit is difficult to see why the emerging polypep-tide is not digested by protease in the extracel-lular medium and yet is digested during proto-plast secretion. Another possibility is that thepolypeptide chain assumes an intermediateconfiguration in the membrane and its finaltertiary structure only outside the membrane.From the present work one would predict thatthe intermediate form would be sensitive toproteases as it emerges into the aqueous envi-ronment from protoplasts. Although we cannoteliminate this idea, we are unable to detect anyenzyme associated with isolated membranes.The final possibility and the one which we favoris that the extended polypeptide chain is ex-truded through the membrane assumes itsnative tertiary structure immediately uponemerging from the membrane. During the fold-ing process the polypeptide chain would besensitive to proteolytic attack.The probability that the polypeptide chain

emerges from cells (as compared with proto-plasts) in a fully native form implies that nativeprotease can diffuse out through the cell wall. Ifit can also diffuse back freely, there is theproblem of why the very high level of proteaseachieved in cultures of the organism do notlimit further extracellular enzyme production.

Braatz and Heath (3) have recently proposedthat a polysaccharide is secreted in associationwith nascent alkaline phosphatase polypeptidechains from intact cells of Micrococcus sodo-nensis and that this renders them insensitive toproteolytic attack. However, these workers didnot examine whether this polysaccharide would

protect the enzyme from trypsin attack as itemerges from protoplasts. Their conclusion wasbased partly on the selective inhibition of alka-line phosphatase secretion by cells in the pres-ence of either glucosamine or bacitracin, but nosuch effect by these compounds was detected inthe present system. An alternative but specula-tive possibility is that passage of proteasethrough the cell wall is unidirectional by someundefined mechanism. An indication of such arestriction is the finding that although trypsinhas no effect- on the synthesis of active extracel-lular enzymes by intact cells, there is an imme-diate inhibition when lysozyme is added. Evenif there is unidirectionality, there is still theproblem that extracellular enzyme polypeptidechains would emerge through the membraneinto an environment containing newly formedactive protease en route to secretion through thecell wall. Whether the effective concentration ofprotease is kept very small by a rapid exitmechanism through the cell wall or some otherunknown protective mechanism exists is notknown.The conclusions from the present studies

support the proposal (12) put forward earlierthat bacterial extracellular enzymes emergefrom the cell by extrusion of the nascent poly-peptide chain and implies synthesis by mem-brane-associated ribosomes. This is analogousto the mechanism for secretion through theendoplasmic reticulum of animal cells proposedby Redman and Sabatini (14).

LITERATURE CITED

1. Bettinger, G. E., and J. 0. Lampen. 1971. Evidence forthe extrusion of an incompletely folded form of penicil-linase during secretion by protoplasts of Bacillus li-cheniformis 749/C. Biochem. Biophys. Res. Commun.43:200-206.

2. Both, G. W., J. L. McInnes, J. E. Hanlon, B. K. May, andW. H. Elliott. 1972. Evidence for an accumulation ofmessenger RNA specific for extracellular protease andits relevance to the mechanism of enzyme secretion inbacteria. J. Mol. Biol. 67:199-217.

3. Braatz, J. A., and E. C. Heath. 1974. The role ofpolysaccharide in the secretion of protein by Micrococ-cus sodonensis. J. Biol. Chem. 249:2536-2547.

4. Coleman, G., and W. H. Elliott. 1962. Studies ona-amylase formation by Bacillus subtilis. Biochem. J.83:256-263.

5. Ehrstrom, M., L. E. G. Eriksson, J. Israelachvili, and A.Ehrenberg. 1973. The effects of some cations andanions on spin labelled cytoplasmic membranes ofBacillus subtilis. Biochem. Biophys. Res. Commun.55:396-402.

6. Glenn, A. R., G. W. Both, J. L. McInnes, B. K. May, andW. H. Elliott. 1973. Dynamic state of the messengerRNA pool specific for extracellular protease in Bacillusamyloliquefaciens: its relevance to the mechanism ofenzyme secretion. J. Mol. Biol. 67:199-217.

7. Gould, A. R., B. K. May, and W. H. Elliott. 1973.Accumulation of messenger RNA for extracellular en-

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zymes as a general phenomenon in Bacillus amyloli-quefaciens. J. Mol. Biol. 73:213-219.

8. Gould, A. R., B. K. May, and W. H. Elliott. 1975. Releaseof extracellular enzymes from Bacillus amyloliquefa-ciens. J. Bacteriol. 122:34-40.

9. Hartley, R. W., and J. R. Smeaton. 1973. On the reactionbetween the extracellular ribonuclease of Bacillus amy-loliquefaciens (Barnase) and its intracellular inhibitor(Barstar). J. Biol. Chem. 248:5624-5626.

10. Liu, T. Y., and S. D. Elliott. 1965. Activation of strep-tococcal proteinase and its zymogen by bacterial cellwall. Nature (London) 206:33-34.

11. May, B. K., and W. H. Elliott. 1968. Characteristics ofextracellular protease formation by Bacillus subtilisand its control by amino acid repression. Biochim.Biophys. Acta 157:607-615.

12. May, B. K., and W. H. Elliott. 1968. Selective inhibitionof extracellular enzyme synthesis by removal of cellwall from Bacillus subtilis. Biochim. Biophys. Acta166:532-537.

13. May, B. K., and W. H. Elliott. 1970. Synthesis andproperties of a protoplast-bursting factor from Bacillusamyloliquefaciens. Biochem. Biophys. Res. Commun.41:199-205.

14. Redman, C. M., and D. D. Sabatini. 1966. Vectorialdischarge of peptides released by puromycin fromattached ribosomes. Proc. Natl. Acad. Sci. U.S.A.56:608-615.

15. Rinderknecht, H., M. C. Geokas, P. Silverman, and B. J.Haverback. 1968. A new ultra-sensitive method for thedetermination of proteolytic activity. Clin. Chim. Acta21:197-203.

16. Sanders, R. L., E. V. Semets, B. K. May, and W. H.Elliott. 1974. Vectorial transport of proteins throughmembranes: evidence for extrusion of nascent polypep-tide chains through membranes. Proc. Aust. Biochem.Soc. 7:56.

17. Smeaton, J. R., and W. H. Elliott. 1967. Isolation andproperties of a specific bacterial ribonuclease inhibitor.Biochim. Biophys. Acta 145:547-560.

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