Vol. 50, No. 2INFECTION AND IMMUNITY, Nov. 1985, p. 534-5400019-9567/85/110534-07$02.00/0Copyright C 1985, American Society for Microbiology

Production and Partial Characterization of an Elastolytic Protease ofVibrio vulnificus

MAHENDRA H. KOTHARY AND ARNOLD S. KREGER*Department of Microbiology and Immunology, The Bowman Gray School of Medicine of Wake Forest University,

Winston-Salem, North Carolina 27103

Received 7 June 1985/Accepted 12 August 1985

Conditions are described for the production of large amounts of an extracellular elastolytic protease byVibrio vulnificus. The yield of enzyme was maximal during the late exponential growth phase and was stableduring the stationary growth phase in a medium composed of 2% Proteose Peptone and 1.5% NaCl. Theprotease has a molecular weight of ca. 50,500 (estimated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis), an isoelectric point of ca. 5.8, and a pH optimum range against azocasein and elastin of pH7 to 8. The caseinolytic and elastase activities in protease preparations partially purified by ammonium sulfateprecipitation were inseparable by gel filtration, hydrophobic interaction chromatography, and isoelectricfocusing. Both activities were deleteriously affected by heat, low pH, heavy-metal ions, chelating agents,reducing agents, sodium cyanide, N-bromosuccinimide, ox-2-macroglobulin, and phosphoramidon, but wereunaffected by various trypsin inhibitors, chymostatin, aprotinin, leupeptin, pepstatin A, phenylmethylsulfonylfluoride, and N-ethylmaleimide.

Vibrio vulnificus is a halophilic bacterium that is beingincreasingly recognized as an etiological agent of fulminat-ing, life-threatening wound infections and septicemia inhumans (19, 26, 28). Putative virulence factors produced byV. vulnificus include extracellular cytolysin (8, 11), phospho-lipases (25), and siderophores (2, 23), and a surface anti-gen(s) that confers resistance to phagocytosis and the bac-tericidal activity of normal serum (1, 10, 30) and possessesprotective antigen activity (14). In addition, V. vulnificusproduces extracellular proteases that may be important inhelping the bacterium invade tissues containing elastin andcollagen and may be responsible, at least in part, for theextensive local tissue necrosis often observed during woundinfections caused by V. vulnificus (24; M. M. Carruthers andW. J. Kabat, Abstr. Annu. Meet. Am. Soc. Microbiol. 1981,B59, p. 24; M. D. Poole, J. H. Bowdre, and D. Klapper,Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B155, p. 43).Growth conditions required for collagenase production by V.vulnificus have recently been reported (24). In this paper, wedescribe conditions for the production of an extracellularelastolytic protease by V. vulnificus, and we report some ofthe physicochemical properties of the enzyme.

MATERIALS AND METHODSBacterium and seed culture preparation. V. vulnificus

ATCC 29307 was obtained from the American Type CultureCollection, Rockville, Md. The strain is designated A8694 bythe Centers for Disease Control, Atlanta, Ga., and is thestrain used by Carruthers and Kabat (Abstr. Annu. Meet.Am. Soc. Microbiol. 1981, B59, p. 24). Seed cultures wereprepared as previously described for V. vulnificus E4125 andA1402 (11), except that sheep blood was omitted from theColumbia agar plates.

Assays. Protease activity against azocasein was deter-mined as previously described for the Pseudomonas aerugi-nosa elastolytic protease (13). Elastase activity usually wasestimated by the plate diffusion procedure of Schumacher

* Corresponding author.

and Schill (22), except 0.033 M Tris hydrochloride buffer (pH7.5) was used instead of 0.2 M Tris hydrochloride buffer (pH8.8). Briefly, portions (10 ,ul) of the preparations to beassayed were placed into wells (4-mm diameter) cut in 14 mlof 1% agarose (Bio-Rad Laboratories, Richmond, Calif.)containing 0.08% (wt/vol) elastin (Sigma Chemical Co., St.Louis, Mo.) on glass plates (8.4 by 9.4 cm), the plates wereincubated at 37°C for 3 h, and the clear zones that developedaround the wells were measured. A standard curve con-structed by plotting the square of the clear-zone diameterversus units of porcine pancreatic elastase (Sigma) was usedto estimate the units of V. vulnificus elastase activity permilliliter. Elastase activity in the determination of pH opti-mum and in enzyme inactivation studies was assayed againstelastin-Congo red (Sigma) by a modification of the method ofSachar et al. (20). Reaction mixtures consisting of elastin-Congo red suspension (20 mg in 1 ml of water), 1 ml of 0.1 MTris hydrochloride buffer (pH 7.5), and 1 ml of enzymepreparation in 50-ml flasks were incubated for 1 h at 37°Cwith gyratory agitation (100 cycles per min). Phosphatebuffer (0.7 M, pH 6; 2 ml) was added to each flask, and theA495 of the supernatant fluids obtained by centrifugation ofthe reaction mixtures was determined.The protease preparation obtained by ammonium sulfate

precipitation was assayed for protein by the method ofBradford (4), with bovine gamma globulin as the standard.The standard and the assay reagent were obtained fromBio-Rad Laboratories.

Protease production. The ability of V. vulnificus ATCC29307 to produce extracellular protease in various basalmedia was examined by inoculation of 5 ml of medium in50-ml flasks with 8 to 10 ,ul of a seed culture suspension (0.25units of optical density at 650 nm [OD650]; ca. 2.5 x 108CFU) and assay of the culture supernatant fluids after thecultures were incubated for 24 h at 30°C on a gyratory shakeroperating at 220 cycles per min. The effects of Na+ ionconcentration, peptone concentration, initial pH of the me-dium, incubation temperature, and supplementation of themedium with carbohydrates, buffers, and metal ions on

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V. VULNIFICUS ELASTASE 535

TABLE 1. Effect of carbohydrates, buffers, and divalent cationson growth and extracellular protease yield by V. vulnificus

Addition' 0D6'50 O

Final pH c̀ProteaseAdditiona cultureb (U/mlf

None (basal Proteose Peptone 5.3 7.2 37brothd)

Glucose 1.6 4.5 0Fructose 2.3 4.5 0Mannitol 8.5 6.3 0Xylose 5.6 7.3 17Lactose 4.8 7.0 26Sucrose 5.2 7.0 28Glycerol 5.2 5.3 13TESe-KOH (0.1 M, pH 7.4) 4.8 7.3 38Glucose plus 0.1 M TES-KOH (pH 7.2 6.9 16

7.4)K2HPO4-HC1 (0.05 M, pH 7.4) 4.7 7.2 30Glucose plus 0.05 M K2HPO4-HCI 6.1 6.6 1(pH 7.4)

Ca2+ 4.5 7.1 29Mg2+ 4.5 7.0 26Zn2+ 3.2 7.1 0

a The final concentration of the carbohydrates was 0.5% (wt/vol), exceptthe glycerol was 1% (vol/vol). The final concentration of metal ions was 10-3M.

b OD650 was determined after the cultures were incubated with agitation at30°C for 24 h.

c The pH and caseinolytic activity of culture supernatant fluids obtained bycentrifugation were determined after the cultures were incubated with agita-tion at 30°C for 24 h.

d Other media tested included (i) various commercially available formulas,such as marine broth, heart infusion broth, and synthetic broth AOAC(Difco), and Trypticase soy broth and brain heart infusion (BBL MicrobiologySystems, Cockeysville, Md.); (ii) various peptone-based media (1% peptoneand 2% NaCI) containing commercially available peptones, such as Casitone,Proteose Peptone no. 3, tryptone, tryptose, and neopeptone (all from Difco);Lactalysate, Thiotone, Phytone, Biosate, Gelysate, Polypeptone, Trypticase,and Myosate (all from BBL); and Bacteriological Peptone (Oxoid Ltd.,London, England); and (iii) other media such as heart infusion diffusate broth(12), 1% Casamino Acids (Difco) supplemented with 2% NaCl, and 1% nonfatdry milk (Carnation Co., Los Angeles, Calif.) supplemented with 2% NaCI.

e N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.

protease yield were examined with cultures prepared by theinoculation of 150 ml of broth in 2-liter flasks with 7.5 OD650units of seed culture suspension (7.5 x 109 CFU) andincubation for 24 h (220 gyratory cycles per min).Ammonium sulfate precipitation. Unless otherwise noted,

all steps were done at ca. 4°C. Twelve 2-liter flasks contain-ing 150 ml of Proteose Peptone broth (2% Proteose Peptone[Difco Laboratories, Detroit, Mich.] and 1.5% NaCl) wereinoculated as described above, and the culture supernatantfluids were obtained by centrifugation (16,000 x g, 20 min)after incubation for 16 h at 30°C on a gyratory shakeroperating at 220 cycles per rnin. Ammonium sulfate (enzymegrade; Schwarz/Mann, Cambridge, Mass.) was dissolved inthe culture supernatant fluids to a final concentration of 60%saturation (420 g/liter), and the precipitate was collected bycentrifugation (23,000 x g, 20 min) after 16 to 18 h anddissolved in 10 ml of cold 0.1 M ammonium bicarbonate(AB; pH 7.8). A small amount of insoluble residue wasremoved by centrifugation (25,000 x g, 20 min), and thesolution was stored at -60°C.

Gel ifitration. A sample (3 ml) of the protease preparationobtained by ammonium sulfate precipitation was applied to awater-cooled (5°C) column (2.6 by 95 cm) of Sephadex G-100(Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) equili-brated with 0.1 M AB and was eluted at a flow rate of 20 ml/h(ca. 3.8 ml/cm2 per h) with the equilibrating buffer. The

fractions (ca. 5 ml) were assayed for A280 and for caseinolyticand elastase activities. The protease peak fractions (ca. 12fractions) were pooled and lyophilized.

Hydrophobic interaction chromatography. A sample (3 ml)of the protease preparation obtained by ammonium sulfateprecipitation was applied to a water-cooled (5°C) column (1.6by 15 cm) of phenyl Sepharose CL-4B (Pharmacia) equiii-brated with 0.1 M AB. The column was washed (ca. 50 ml/h;5-ml fractions were collected) with ca. 7 bed volumes (210ml) of 10% ethylene glycol in 0.1 M AB, and the proteasewas eluted by washing the column (30 to 40 ml/h; 3- to 4-mlfractions were collected) with ca. 5 bed volumes of 30%ethylene glycol in 0.1 M AB. After elution of the enzyme, thecolumn was washed (ca. 20 ml/h; 5-ml fractions were col-lected) with 50% ethylene glycol in 2 mM glycine-NaOHbuffer (pH 9.8). The fractions were assayed for A280 and forcaseinolytic and elastase activities. Five protease peak frac-tions (ca. 18 ml) were pooled.

Isoelectric focusing. Protease preparations obtained bysequential ammonium sulfate precipitation and gel filtrationwere fractionated by preparative high-speed electrofocusing(29) in pH 3.5 to 10 and pH 5 to 7 sucrose density gradientsformed at 15 W for 18 h with an LKB 8100-1 column (LKBInstruments, Inc., Gaithersburg, Md.). The pH of eachfraction (4 ml) was determined at 4°C, and the fractions wereassayed for caseinolytic and elastase activities.

Triplicate samples of the pool obtained by hydrophobicinteraction chromatography were examined by analyticalthin-layer isoelectric focusing in a polyacrylamide gel withthe LKB 2117 Multiphor electrophoresis apparatus andcommercial PAG plates (pH 3.5 to 9.5) as recommended bythe manufacturer. Previously described isoelectric pointstandards (8) were obtained from Pharmacia. After electro-phoresis, the gel was divided into three parts. One part of thegel was stained with Coomassie brilliant blue R-250, and theother two parts were soaked in 0.4 M Tris hydrochloridebuffer (pH 7.5) for 5 min and examined by a proteasezymogram technique with an overlay of 1% soluble casein inagarose (16) and an overlay of 0.08% elastin in agarose (thesame as that used for the plate diffusion assay for elastase).Caseinolytic and elastase activities were detected after thegels were incubated at 37°C for ca. 1 and 3 h, respectively.

Molecular weight estimation by SDS-PAGE. The Coomas-sie blue-staining band corresponding to caseinolytic andelastase activities in PAG plates was excised, and acetic acidand ethanol were removed and the gel was incubated indisruption buffer as described by Gardi and Lungarella (7).The gel was then examined by slab sodium dodecyl sulfate(SDS)-polyacrylamide gradient gel electrophoresis (PAGE)by a modification (8) of the Laemmli method (15). Themolecular weight of the denatured and reduced protease wasestimated by the relative mobility method of Weber et al.(27).Determination of pH optimum. Samples (0.2 U of

caseinolytic activity in 100 ,ul and 4 U of elastase activity in1 ml) of the protease preparation obtained by ammoniumsulfate precipitation were added to assay mixtures contain-ing either azocasein substrate solution (0.5 ml) or elastin-Congo red suspension (1 ml) and 1 ml of the different testbuffers (0.1 M acetate, pH 4; 0.1 M phosphate, pH 6; 0.1 MTris hydrochloride, pHs 7, 7.5, 8, and 9; and 0.1 M glycine-sodium hydroxide, pH 10). Caseinolytic and elastase activ-ities were assayed as described above.

Inactivation studies. The protease preparation obtained byammonium sulfate precipitation was diluted with variousbuffers (pHs 4, 6, 7, 8, and 10) to a final concentration of 10

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536 KOTHARY AND KREGER

6

5

4

3

iEc0LOcoCu

a)0

0

ca)0

cJ

0

40

430

a)0.C,,20

a)cua)

10 o

00 4 8 12 16 20 24

Incubation Time ( Hours )

FIG. 1. Growth and protease production by V. vulnificus ATCC 29307. Two-liter Erlenmeyer flasks containing 150 ml of broth (2%Proteose Peptone and 1.5% sodium chloride) were inoculated with a seed culture suspension (7.5 OD650 units; ca. 7.5 x 10' CFU) andincubated at 30 and 37°C on a gyratory shaker operating at 220 cycles per min. Growth was followed turbidimetrically (1-cm light path) witha model DB-GT spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.), and the culture supernatant fluids were obtained bycentrifugation and assayed for protease activity against azocasein.

U of caseinolytic activity per ml, and the diluted prepara-tions were assayed for residual caseinolytic and elastaseactivities after storage at 4°C for 24 h.The effects of heat (4, 25, 37, 56, and 100°C for 30 min),

metal ions, and enzyme inhibitors on the caseinolytic activ-ity in preparations obtained by ammonium sulfate precipita-tion were examined as previously described for a Serratiamarcescens metalloprotease (16). The effects of heat, metalions, and inhibitors on elastase activity were examined byincubating ca. 4 U of elastase in 2 ml of 0.05 M Trishydrochloride buffer (pH 7.5) at the above-mentioned tem-peratures for 30 min and with the reagents at 25°C for 30 min,and assaying for residual activity against elastin-Congo red.

RESULTS AND DISCUSSIONProtease production. Of the 23 basal media tested (Table

1), Proteose Peptone broth, heart infusion broth, and heartinfusion diffusate broth were optimal for extracellular prote-ase yield by V. vulnificus ATCC 29307 (data not shown).Proteose Peptone broth was selected for further examinationof the variables affecting protease yield because it containsless high-molecular-weight contaminants than heart infusionbroth and is less expensive and simpler to prepare than heartinfusion diffusate broth. Maximal amounts of extracellularcaseinolytic activity (35 to 40 U/ml) were obtained fromProteose Peptone broth cultures (2% Proteose Peptone and1.5% NaCl, pH 6.8; 150 ml) incubated with vigorous agita-tion at 30°C for 10 to 24 h (late exponential to stationarygrowth phase; Fig. 1). The amount of extracellular proteaseactivity obtained from V. vulnificus grown under theseconditions is two- to ninefold more than that previouslyobtained with strongly proteolytic isolates of P. aeruginosaand S. marcescens (13, 16). Adjusting the medium to an

initial pH of 5, 6, 8, or 9 did not affect protease yield (datanot shown), and supplementing the medium with variouscarbohydrates, buffers, and metal ions either reduced or didnot affect protease yield (Table 1).

Elastase activity was not detected in the culture superna-tant fluids. However, elastase activity was detected whenthe culture supernatant fluids were concentrated by ultrafil-tration with a Centricon-10 microconcentrator (AmiconCorp., Danvers, Mass.) and diluted with 0.1 M AB prior tobeing assayed. The elastase activity in the preparationscould be inhibited by the addition of heated (100°C, 30 min)or unheated ultrafiltrate of Proteose Peptone broth or by theincorporation of Proteose Peptone (ca. 0.4% [wt/vol], finalconcentration) in the assay mixture. Our observations sug-gest the presence of a thermostable, low-molecular-weight(<10,000) elastase inhibitor in the Proteose Peptone used forprotease production. The presence of this inhibitor or otherinhibitors in some growth media may be responsible for thereported absence of elastase production by V. vulnificus (5).The enzyme preparation obtained by ammonium sulfateprecipitation had ca. 40 U of elastase activity and 200 U ofcaseinolytic activity per mg of protein. About 98% of thecaseinolytic activity present in the culture supernatant fluidswas recovered in the preparation obtained by ammoniumsulfate precipitation.

Stability and physicochemical properties. Caseinolytic andelastase activities were heat labile (90 to 100% of bothactivities was lost after heating for 30 min at 100°C) andunstable at pH 4 (4°C for 24 h), but were stable at pH 6 to 10.Various heavy-metal ions, chelating agents, reducing agents,sodium cyanide, N-bromosuccinimide, a-2-macroglobulin,and phosphoramidon inhibited both caseinolytic activity(Table 2) and elastase activity (data not shown). Phos-

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TABLE 2. Effects of metal ions and enzyme inhibitors on V. iidlnificuis protease

Reagent' ~~~~~~Residual RaetResidualReagenta activity (%) cvReagentaciityduMg2 ............................. 100 Sodium cyanide ............................... 70Ca2+.1 N-Bromosuccinimide ........................... 20Mn2 ............................. 100 Sodium azide ................................ 100Ba2 ............................. 100 PMSF ..................................... 100Ag2+.70 N-Ethylmaleimide .............................100Pb2A ...................0..........0 N 0Co2............................... 50 Bovine trypsin inhibitor ......................... 10Fe2 ............................. 40 Ovomucoid trypsin inhibitor ...................... 100Zn2 ............................. 0 Lima bean trypsin inhibitor ....................... 100Cu2+.............................. 0 Soybean trypsin inhibitor ........................ 100Hg2+.0 Human a-1-antitrypsin .......................... 100Hgnotaett...... ........................... 70 Human..................... C hms ttnt0Nitrilotriacetate.... .................. 70 Chymostatin. .. . . ....Apr o t i n i n 100Et,a'-Dipyridyl ....................... 60 Aprotinin................................... 100EDTA ............... 60 Leupeptin...................100EGTA............................ 30 Pepstatin A.................................. 90OPA .............................. 0 ct-2-Macroglobulin....a-2-Macroglobulin 0Cysteine hydrochloride ..... ............ 30 Phosphoramidon. ............................. 0Dithiothreitol ........................ 30

" Final concentration in assay mixtures (1.6 ml) was 1o0' M, with the exception of mixtures containing the naturally occurring protease inhibitors (whichcontained 0.5 mg of the reagent per assay tube). Pepstatin A, chymostatin, soybean trypsin inhibitor, lima bean trypsin inhibitor, ovomucoid trypsin inhibitor,bovine pancreatic trypsin inhibitor, disodium nitrilotriacetate, a,o'-dipyridyl, 1,10-orthophenanthroline (OPA), PMSF, N-ethylmaleimide, and N-bromosuccini-mide were obtained from Sigma. Aprotinin, cx-2-macroglobulin, and leupeptin were obtained from Boehringer-Mannheim Biochemicals, Indianapolis, Ind. Humana-1-antitrypsin was obtained from Worthington Diagnostics, Freehold, N.J. Phosphoramidon was kindly supplied by Walter Troll, Department of EnvironmentalMedicine, New York University School of Medicine.

b Residual protease activity was determined against azocasein.

phoramidon also is known to inhibit the P. aeruginosaelastase (18). Trypsin inhibitors and serine-protease andthiol-protease inhibitors (phenylmethylsulfonyl fluoride[PMSF] and N-ethylmaleimide, respectively), did not affecteither activity. The pH optimum range for both activitieswas pH 7 to 8.The caseinolytic and elastase activities in the protease

1.0

0

0

CIO

.0

Ec0cocM

0.8

0.6

0.4

0.2

to.120 160 200 240 280 320 360 400 440

preparation obtained by ammonium sulfate precipitationwere inseparable by gel filtration with Sephadex G-100 (Fig.2) and by hydrophobic interaction chromatography withphenyl Sepharose CL-4B (Fig. 3). More than 98% of the twoactivities present in the preparations applied to the columnswas recovered in the peak fractions. The caseinolytic andelastase activities also were inseparable by isoelectric focus-

200

175

1500 4

125 EE0.0.

C CDO

75 a)C. YY~~~~a

25

0600

ml of Effluent

FIG. 2. Sephadex G-100 gel filtration of an elastolytic protease preparation obtained from culture supernatant fluids of V. vulnificus ATCC29307 by ammonium sulfate precipitation. Fractions were assayed for caseinolytic and elastase activities and for A,t;o. The column voidvolume and bed volume were ca. 175 and 505 ml, respectively.

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538 KOTHARY AND KREGER

Elution with 10%EG in 0.1 M AB

>2.1 r-

0

a1)C.)

C

('S

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0C,)en

EC

co

Elution with0% EG in 0.1 M AB

Elution with50% EG in

2 mM Glycine-NaOH( pH 9.8 )

0 30 60 90 120 150 210 240 270 300 330 360 390 420 450

ml of Effluent

FIG. 3. Hydrophobic interaction chromatography of an elastolytic protease preparation obtained from culture supernatant fluids of V.vulnificus ATCC 29307 by ammonium sulfate precipitation. Fractions were assayed for caseinolytic and elastase activities and for A280. EG,Ethylene glycol.

ing in broad and shallow pH gradients, and the enzyme hadan isoelectric point (pl) of ca. 5.8 (Fig. 4). Zymogramanalysis of the thin-layer isoelectric focusing gel showedonly one band of caseinolytic activity that coincided with aband of elastase activity and a Coomassie blue-staining bandhaving a pl of ca. 5.7 (data not shown), which is similar tothe pl determined for the elastolytic protease by isoelectricfocusing in the sucrose density gradients (Fig. 4). Themolecular weight of the Coomassie blue-staining band was

50,500 by SDS-PAGE (Fig. 5). Our data support the ideasthat V. vulnificus ATCC 29307 produces only one extracel-lular caseinolytic protease under the growth conditionsdescribed in this paper and that the caseinolytic and

A14 210

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elastolytic activities in culture supernatant fluids of thebacterium are functions of the same enzyme.

In addition, our data indicate that the V. vulnificus elastaseis a neutral metalloprotease that can be differentiated from (i)a V. cholerae soluble hemagglutinin-protease that is also aneutral metalloenzyme but has three distinct pH isotypes(pls of 6.3, 5.3, and 4.7) and exists as a polymer of subunits,each having a molecular weight of 32,000 as determined bySDS-PAGE (3, 6); (ii) three V. cholerae proteases describedby Young and Broadbent (31), two of which also have acidicpIs but are insensitive to EDTA and a third which has a pl of8.9 to 9.2; (iii) the V. cholerae mucinase that is an alkalineprotease unaffected by EDTA (21); (iv) five V. alginolyticus

B14 14e

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12- 10-E E

10 100

8- 80 c0( c

-r6 ~~60c c

6 12 18 24 30Fraction

FIG. 4. Isoelectric focusing of an elastolytic protease preparation obtained from culture supernatant fluids of V. vulnificus ATCC 29307by sequential ammonium sulfate precipitation and gel filtration. The pH of each fraction was determined at 4°C, and the fractions were assayedfor caseinolytic and elastase activities. (A) Results obtained with a pH 3.5 to 10 gradient. (B) Results obtained with a pH 5 to 7 gradient.

250 0 jE E

200 . %fia

a)

150 c:C C

100 CI)* CDoC.- _

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V. VULNIFICUS ELASTASE 539

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30 r

0. lK

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FIG. 5. SDS-PAGE of a Coomassie blue-staining band associ-ated with caseinolytic and elastase activities on a PAG plate used foranalytical thin-layer isoelectric focusing. Cathode at top of gel. Theband was denatured and reduced with a disruption buffer containingSDS, 3-mercaptoethanol, and glycerol (8). Lane 1, Molecularweight markers (Pharmacia); lane 2, band from a PAG plate used foranalytical isoelectric focusing. The gel was stained with Coomassiebrilliant blue R-250.

proteases that are sensitive to PMSF (9); and (v) the VibrioB-30 collagenase that has a molecular weight of ca. 55,600 bySDS-PAGE (17).

In conclusion, this paper contains new information on theproduction and some of the physicochemical properties of anextracellular elastolytic protease produced by V. vulnificusATCC 29307. The sensitivity of the enzyme to EDTA and1,10-orthophenanthroline and its neutral pH optimum sug-

gest that it is the same protease as that previously reportedto be produced by V. vulnificus A8694 (Carruthers andKabat, Abstr. Annu. Meet. Am. Soc. Microbiol. 1981). Apartially purified elastase preparation obtained from a dif-ferent strain of V. vulnificus (B3547) has been reported to belethal and to produce tissue necrosis in mice, to possess

permeability factor activity, to be active against albumin andcomplement components, and to protect a weakly virulentstrain of V. vulnificus (A1402) against the in vitro bacteri-cidal activity of human serum (Poole et al., Abstr. Annu.Meet. Am. Soc. Microbiol. 1982). Studies are in progress inour laboratory to further purify and characterize theelastase, so that we can evaluate its possible role(s) in thepathogenesis of disease caused by V. vulnificus.

ACKNOWLEDGMENT

This investigation was supported by Public Health Service grantAI-18184 from the National Institute of Allergy and InfectiousDiseases.

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15. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

16. Lyerly, D., and A. Kreger. 1979. Purification and characteriza-tion of a Serratia marcescens metalloprotease. Infect. Immun.24:411-421.

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