purification and characterization of an elastolytic protease of vibrio vulnificus

8/3/2019 Purification and Characterization of an Elastolytic Protease of Vibrio Vulnificus

1/9

Journal of General M icrobiology (1987), 133, 1783-1791. Printed in Great Britain 1783

Purification and Characterization of an Elastolytic Protease ofVibrio vulnijkusBy M A H E N D R A H . K O T H A R Y A N D A R N O L D S . K R E G E R *

Department of Microbiology and immunology, The Bowman Gra y School of Medicine of WakeForest University, Winston-Salem, North Carolina 27103, U S A(Received I0 November 1986; revised 24 February 1987)

Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus wereobtained by sequential ammonium sulphate precipitation and hydrophobic interactionchromatography with phenyl-Sepharose CL-4B. The protease had an M , of about 50500(estimated by SDS-P AG E), a PI of 5.7, and a temperature optimum range of 5 5 to 60 "C. The pHoptimum and the results of inactivation studies suggested that the enzyme was a neutralmetalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-G lu-Phe-Gly. Th e purified protease was toxic for mice (about 1.5 mg kg-land 4.5 mg kg-l, intraperitoneal and intraveno us LD5, values, respectively), and sub cutaneou sinjection of the enzyme elicited rapid and extensive dermonecrosis.

I N T R O D U C T I O NThe halophilic bacterium Vibrio vulnificus is an oppo rtunistic pathogen that is an aetiologicalagent of rapidly developing, life-threatening wound infections and septicemia in humans(Wickboldt & Sanders, 1983; Tison & Kelly, 1984; Morris & Black, 1985). Putative virulencefactors produced by V . vulniJicus nclude ( a ) extracellular products, such as a lethal cytolytictoxin (Kreger & Lockwood, 1981 ; Gray & Kreger, 1985), phospholipases (T esta et al., 1984),siderophores (Andrus et al., 1983; Simpson & Oliver, 1983), and proteases (Carru thers & Kabat ,1981 : Poole et af . ,1982; Smith & Merkel, 1982; Kothary & Krege r, 1985b), and ( b ) a surfaceantigen(s) that confers resistance to phagocytosis and the bactericidal activity of normal serum(Kreger et al., 1981;Amako et al. , 1984: Yosh ida et al., 1985) and possesses protective a ntigenactivity (Kreger et al., 1984). Ou r interest in the extracellular proteases of the bacter ium was

prompted by the possibility that they may be responsible, at least in part, for the extensive localtissue necrosis often observed during wound infections caused by V .vulnificus.We ha ve reportedpreviously (Kothary & Kreger, 19856) that V . vulnijicus AT CC 29307 produces only oneextracellular protease in a medium composed of 2% (w/v) Proteose Peptone (D ifco) and 1.5%(w/v) sodium chloride, and that the protease is produced in large amounts an d is elastolytic. Th estudies in this paper ( a ) extend our previous studies by describing a simple procedure forobtaining large am ounts of highly purified elastase, an d ( b ) use the highly purified elastase toconfirm and advance ou r knowledge concerning the physicochemical and biological propertiesof the enzyme.METHODS

Assays. Protease activity against azocasein was determined as previously described (Ko thary & Kreger, 1985b) .Elastase activity of the fractions obtained by hydrophobic interaction chromatography was estimated by apreviously described plate diffusion procedure (K othary & Kreger, 19856).Elastase activity of the step 3 protease~~

Abbreviations : PBS-DM-AA, phosphate-bu ffered saline suppleme nted with nonfat dry milk and Antifoam A ;OPA, orthophenanthroline0001-3816 0 987 SG M


2/9

1784 M . H . K O T H AR Y A N D A . S . K R E G E Rpreparation and in the determination of pH optimum studies was estimated by assaying against elastin-Congo red(Sigma) as previously described (Kothary & Kreger, 19856), except that t he incubation period w as 20 min. O neunit of elastase activity is the amount of enzyme which hydrolyses 1 mg elastin-Congo red in 20 min at 37 "C.Protein was estimated by the method of Bradford (1976) with bovine gamma globulin as the standard. Thestandard and the assay reagent were obtained from Bio-Rad.Purification of protease. The culture supernatant fluids (step 1 preparation) from three Proteose Peptone brothcultures (about 450 ml of culture) of V .vulnifims ATC C 29307 (designated strain A8694 by the Centers for D iseaseControl, Atlanta, G a., U SA), and the amm onium sulphate-precipitated enzyme preparation (step 2 preparation)were obtained as previously described (Kothary & Krege r, 198 56). Th e step 2 prepara tion contained in 2.5 ml of100 mw amm onium bicarbonate (p H 7.8) was fractionated by hydrophobic interaction c hromatography withphenyl-Sepharose CL-4B (Pharmacia) as previously described (Kothary & Kreger, 19824, except the gel waswashed with 15 bed vols 10% (v/v) ethylene glycol in 100 m -a m m on iu m bicarbonate rather than with 7 bed volsof buffer before eluting the enzyme with 30% ethylene glycol (v/v) in 100 mw amm onium bicarbonate. Th e sixfractions comprising the peak of eluted activity were pooled (step 3 preparation) a nd were stored at -60 "C .Antisera. New Zealand White rabbits (2.5 kg) were injected subcutaneously with 0.5 ml w ater-in4 1 emulsioncontaining 10 mg dry wt (about 200 protease units) of a lyophilized step 2 protease preparation dissolved in waterand em ulsified with 2 vols complete Freund adjuvant. T he rab bits w ere injected subcutaneously 4 and 8 weekslater with a similar emulsion prepared w ith incomplete Freund adjuvant. Th e rabbits were exsanguinated about 3weeks after the last injection, and the anti-step 2 serum was lyophilized and stored at 4C.Antiserum against the purified step 3 protease preparation was prepared by a m odification of the procedured escrib ed by H a r k & Closs (1983), which involves vaccinating rabbits with an immunoprecipitate that isobtained by crossed imrnunoelectrophoresis and c ontains the desired antigen. Th ree samples (60 g total protein)of the step 3 protease were subjected to crossed immunoelectrophoresis with the anti-step 2 serum. The three gelswere washed and pressed, and each of the three precipitin arcs containing the protease w as cut out of the gels. Thethree pooled precipitin arcs were mixed with 0.6 ml PBS (20mM-Na2HP04, 150 mM-NaC1; adjusted to p H 7.4with HC l) and the mixture was sonically disrupted in an ice-bath with a Branson Sonifier (model 185) until theagarose gel had visibly been dispersed. A water-in-oil emulsion was prepared by emulsifying the sonicatedpreparation with 2 vols incomplete Freund adjuvant, and New Zealand White rabbits were injectedsubcutaneously with 1 ml of the emulsion. The rabbits were similarly vaccinated again 4 weeks later and wereexsanguinated about 3 weeks after the second injection. The serum was lyophilized and stored at 4C. Thespecificity of the anti-step 3 serum w as examined by crossed imm unoelectrophoresis as described below and in thelegend to Fig. 1. Briefly, samples (1 8 pl) containin g 200 pg and 20 pg of the step 2 and step 3 protease preparations,respectively, were electrophoresed in the first dimension without serum and were electrophoresed in the seconddimension against the anti-step 3 serum and the anti-step 2 serum. Th e specificity of the anti-step 3 serum wasdetermined by comparing the number of precipitin arcs obtained with the anti-step 3 Serum to those obtained withthe anti-step 2 serum.Crossed immunoelectrophoresis. This w as done w ith the LKB 21 17-401 immunoelectrophoresis kit. The generalmethodology describ ed by H eiby & Axelsen (1983) and in the L KB instruction m anual and application note 249(Wallenborg & Andersson, 1978) was followed.Results from preliminary crossed immunoelectrophoretic analyses of the step 2 preparation suggested that theprotease partially digested some of the precipitin arcs. Therefore, subsequent crossed imrnunoelectrophoresis ofthe step 2 preparation was done in the presence of a protease-inhibiting concentration ( 2 0 m ~ ) forthophenanthroline [OPA (Sigma)].SlabSDS-PAGE. This was done by a m odification (Gray & Kreg er, 1985) of the method described by Laem mli(1970). Two different conditions were used to dena ture the protease p reparations before electrophoresis. In oneseries of experiments, the enzym e specimens were denatured by heating w ith SDS before electrophoresis (Gray &Kreger, 1985) and, in a second series of experime nts, enzyme preparations were inactivated and denatured at 4 "Cby a modificatio n of the proced ure described by Ga rdi a nd L ungare lla (1984). Briefly, trichloroacetic acid a ndsulphosalicylic acid were added to a sample of the step 3 preparation (about 550 pg protein in 480 pl) to a finalconcentration of 11.5% and 3.5% (w/v), respectively. Th e solution was kept for 1 h at 4 "C, and the precipitatedprotease was recovered by centrifu gation (5100g, 20 min) and suspended in 10 ml 25% (v/v) ethanol and 8% (v/v)acetic acid. After 16 h at 4 "C, the precipitate was recovered by centrifugation a nd dissolved in 0.55 ml of therunning buffer (25 mM-Tris, 192 mM-glyCine, 0.1% SDS; pH 8-3)used for SDS-PAG E. The prepa ration was boiled

for 2 m in with disruption solution containing SDS, l-mercaptoethanol, and glycerol as previously described (Gray& Kreger, 1985), and samples (30pg) w ere loaded onto the gel.The M, f the denatured a nd reduced step 3 protease was estimated by the relative mobility method of W eber etal. (1972), using the slab SD S-PAGE protocol described a bove. M, tandards were obtained from Pharmacia.Western blot analysis.Step 3 protease preparations w ere subjected to SDS-PAG E and the sepa rated protein bandswere e lectrophoretically transferred (0.3 A and 60V at 10"C or 4 h) t o nitrocellulose strips (Bio-Rad) using


3/9

Purification of V . vulnificus elastase 178525 mM-Tris buffer containing 192 mw glycin e and 20% (v/v) methano l, a s described by Towb in et ul. (1979). Afterelectrophoretic transfer, the nitrocellulose strips were gently washed with PBS. Some strips were stained withAm ido Black, and o ther strips were gently agitated for 2 h a t 25 "C in PBS containing 5 % (w/v) nonfat dry milkand 0.001%Antifoam A (PBS-DM-AA), washed three times ( 5 min each wash) with PBS, and incubated withprimary antibody (1 :30dilution of anti-stage 3 serum in PBS-DM -AA) for 4 h at 25 "C with gentle agitation. Thestrips were washed three times with PBS and incubated with secondary antibody [ I :lo00 dilution of goatantirabb it IgG-horseradish peroxidase conjugate (Bio-Rad)] in PBS-DM -AA for 1 h a t 25 "C w ith gen tleagitation. The strips were washed four times with PBS and imm ersed in Tris-buffered saline (50mM-Tris, 250 mM-Na Cl; adjusted to pH 7.5 with HCI) containing H 2 0 2 0-018%, v/v) and 4chloro-n-naphthol (0.06:& w/v; Bio-Rad) until colour developed at 25 "C ( 5 to 20min); the strips were then rinsed with water to stop colourdevelopment. Control strips were incubated either with the secondary antibody or substrate alone.Analytical isoelectric ocusing. Analytical thin-layer isoelectric focusing in p olyacrylamide gel was done w ith anLKB 21 17 Multiphor electrophoresis apparatus and commercial polyacrylamide gel plates (pH 3.5 to 9.5) asrecommended by the manufacturer.Amino acid analyses. A sam ple of the step 3 protease was dialysed against 50mM-m"nOniUm bicarbonate for16 h at 4 "C and was lyophilized. Another sample of the step 3 protease was concentrated and transferred into100 mM-ammonium bicarbonate with a Centricon-30 microconcentrator (Amicon). The amino acid compositionof the lyophilized preparation and the amino-term inal am ino acid sequence of the concentrated prepa ration weredetermined as previously described (G ray & Kreger, 1985) for the V . vulnificus cytolysin.DeterminationofpH optimum andstability. A sample of a step 3 protease prepa ration freed of ethylene glycol andtransferred into 100 mM-ammonium bicarbonate with a Centricon-30 microconc entrator was used to determinethe pH op timum of the enzym e, the stability of the enzym e at various pH values, and the sensitivity of the enzym eto heat and to v arious metal ions and enzym e inhibitors a s previously described for the partially purified enzyme(Kothary & Kreger, 19856).Determinutwnof temperatureoptimum. Samp les (20 to 100 pl) of a step 3 protease p reparatio n (2 units m1-I) freedof ethylene glycol were added to assay mixtures containing azocasein substrate solution (0.5 ml) and 1 ml of100 mM-Tris/HCI buffer (pH 7.9 , and activity w as assayed by incubating the mixture for 10 min at 25 ,30 ,37 ,40 ,45, 50, 55, 60, 5, 70 and 75 "C.Toxicity studies. The step 3 protease was concentrated and transferred into 100 mM-Tris/HCl buffer (pH 7-5)with a Centricon-30 microcon centrator. Gro ups of female, 6 to 8 weeks old (about 30 g), CD -1 strain, ran domlybred albino m ice (Charles River Lab oratories) (10 mice per g roup) were injected intraperitoneally or intravenouslywith portions (0.1 ml) of the protease preparation s diluted w ith buffer. The LD,, values were estimated by themethod of Reed & Mu ench (1938) after ob serving the mice for 3 d p ostinjection. Also, m ice were injectedsubcutaneously with portions (0.1ml) of the diluted protease preparations and were examined for dermonecrosis3 h postinjection.

R E S U L T S A N D D I S C U S S I O NPurification of pro tease

The quantitative results of the purification scheme are summarized in Table 1 . T hehydrophobic interaction chromatography step was similar to the procedure described earlier(Kothary & Kreger, 19856), except that the gel was washed w ith 15 bed vols 10% (v/v) ethyleneglycol in 100 mM-ammonium bicarbonate rather than with 7 bed vols of buffer. Comparison offused rocket immunoelectrophoresis patterns of the fractions obtained by the two proceduresindicated th at the protease p reparation was homogeneous only when the gel was washed with 15bed vols of buffer (data not shown). The specific activity against azocasein and the recovery ofthe protease in three different step 3 preparations ranged from 237 to 250 units mg-l and 45 toSO%, respectively. In addition, the step 3 protease had about 120 units of elastase activity mg-' .The specific activity of the step 3 protease preparation was only about 50% more than thespecific activity of the culture supernatant fluids (step 1 preparation). However, the results ofstability studies indicated tha t the re latively small increase observed in specific activity w as realand did not reflect significant inactivation of the enzyme during purification. T hus, the moreplausible explanation for the small apparent increase in specific activity during enzymepurification is that the step 1 preparation contained relatively small amo unts of contaminatingproteins capable of reacting with the Bradford reagent and, therefore, their removal did notcause a large increase In specific activity during enzyme isolation. A similar situation has beenobserved during the purification of a Serrutia m arcescens protease (Lyerly & Kreger, 1979) and aVibrio damselu cytolysin (Kothary & Kreger, 1985~ ) .


4/9

1786 M . H . K O T H A R Y AND A . S . K R E G E RTable 1. Purification of extracellular elastolytic protease produced by V . vulnificus

Total SpecificVolume Total protein activity YieldFractionation step (ml) units* (mg) (uni tsm g-') (%)

fluidsprecipitationchromatography

1 . Culture supernatant 410 12710 80 159 1002. Ammonium sulphate 3.4 11900 57.5 207 943. Hydrophobic interaction 23 6210 26.2 237 49

* Determined with azocasein substrate

The step 3 protease preparation was hom ogenous by crossed immunoelectrophoresis (Fig. 1 a,b) and analytical thin-layer isolectric focusing (Fig. 2). The weak mobility of the purified step 3enzyme during crossed imm unoelectrophoresis (Fig. 1b) most likely was caused by interaction ofthe enzyme with the agarose gel, and the enhanced mobility of the enzyme in the step 2preparation (Fig. 1 Q, c) could have resulted from the contam inants in the preparation interferingwith the enzyme-agarose interaction. In that regard, we have observed (data not shown) thatwhen the standard, electrophoresis grade agarose (Bio-Rad) used to p repare the gel shown inFig. 1 (b) was replaced with ultra-pure, DN A grade agarose (Bio-Rad), the mobility of the step 3protease was markedly enhanced and was similar to that observed in Fig. 1(a , c) . The elastaseproduced by Pseudomonas aeruginosa and the cytolytic toxin of V. vulnificus also interact w ithand exhibit weak mobility in agarose gels during immunoelectrophoresis (Kreger & Gray, 1978;Gray & Kreger, 1985). Also, we observed previously (Kothary & Kreger, 1985b) that the V .vulnificus elastase interacts with and exhibits zone broadening during gel filtration withSephadex G-100, a dextran-based gel.

Results of SDS-PAGE analysis of the step 3 protease preparation showed that theelectrophoretic patterns of the preparation were influenced by the conditions used to denaturethe enzym e before electrophoresis (Fig. 3a ) . Preparations that were inactivated and denaturedat 4 "C before heating with SDS and 8-m ercaptoethanol showed only one band having a M , ofabout 50500, but preparations that were not inactivated at 4 C before heating andelectrophoresis exhibited two m ajor bands w ith M , values of about 50500 and 42000 and aminor band with an Mr of about 9000. In addition, step 2 preparations tha t were inactivated anddenatured at 4 "C before heating with SD S and /I-mercaptoethanol showed the band with a n M ,of about 50500 but did not show the two bands with M, alues of 42000 and 9000 (data notshown). Our observations suggested that the 42000 and 9000 M , components were fragmentsgenerated by autodigestion of the enzyme. In that regard, Western blot analysis of the threebands observed with a step 3 protease preparation that was not inactivated at 4 C beforeheating with SDS and /I-mercaptoethanol showed that all three bands reacted with rabb it serumraised against the step 3 protease preparation (Fig. 3b). A comparison of the precipitin patternsobtained by crossed immunoelectrophoresis of the step 2 and step 3 protease preparations w iththe anti-step 2 and anti-step 3 sera indicated that the anti-step 3 serum was specific for theenzyme. Multiple precipitin arcs were observed when the anti-step 2 serum was tested with thestep 2 protease preparation (Fig. 1a ) ;however, only one precipitin arc was visible when the anti-step 3 serum was tested with the step 2 (Fig. lc) and step 3 protease preparations (data notshown). Thus, the results of the Western blot analysis support the idea that the 42000 and9000 M, omponents are fragments of the enzyme molecule rather than contaminants.The Western blot analysis of the step 3 protease p reparation also revealed two faint bandswith Mr values >50 500 (Fig. 3b, lane 2). The M , valuesof these bands are identical to those oftwo Coomassie-blue-staining bands observed during SDS -PAG E analysis of a step 3 proteasepreparation that was not denatured by heating or by acid treatment at 4 "C (data not shown).Thus, we believe that the faint bands ( M , >50500) in the immunoblot are minute am ounts ofprotease aggregates present because of insufficient heating of the protease preparation beforeSDS-PAG E analysis.


5/9

PuriJication of V . vulniJicus elastase 1787

Fig. 1. Crossed immunoelectrophoresis of V .uulnificus elastolytic protease preparations.First dimension : samples (18pl) were placedinto wells (4 mm diameter) cut in gels com-posed of 1.2% (w/v) agarose (Bio-Rad) inTris/barbital buffer (pH 8.6) and were elec-trophoresed (anode to right) at 8 to 10 V cm-1for 60min at 12 "C. Second dimension: theupper part of each gel (about 61 cm 2) wascomposed of a 1.2% agarose gel (1 1 ml)containing either 0.5 ml anti-step 2 serum (a,b) or 1 ml anti-step 3 serum (c). The gels usedto analyse the step 2 preparation also con-tained 20 mM-OPA. Electrophoresis (anode a ttop) was done at 2 V cm-* for 18 h a t 15 "C(a)Step 2, 200 pg; b) step 3,20 pg; (c) step 2,200 pg. Th e gels were stained with Coom assiebrilliant blue R-250.

M , and PIThe M , and PI of the step 3 protease (about 50500 and 5.7, respectively) were the same as thevalues previously determined (Kothary & Kreger, 19856) with the partially purified protease.

Amino acid analysesAcidic, basic and nonpolar hydrophobic am ino acid residues accounted for about 21, 9 and33% of the total residues, respectively (Table 2). The presence of four half-cystine residuessuggests that the protease has two intra-chain disulphide bonds. Based on the M , and the amino


6/9

1788 M . H . K O TH A RY A N D A . S. K R E G E R

Fig. 2. Analytical thin-layer isoelectric focusing of V. ufnificuselastolytic protease preparations. The cathode was at the top ofthe gel. Lane 1 , PI standards; lane 2, step 2 preparation(40 g);lane 3, step 3 preparation (1Opg). The gel was stained withCoomassie brilliant blue R-250.

acid concentrations, the enzyme has about 429 amino acid residues. The first 20 amino-terminalresidues of the protease are Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gl~Arg-Tyr-Glu-Phe-Gly.Determinationof p H and temperature optima

The pH optimum for caseinolytic and elastase activities was pH 7 to 8, which is the same asthe value determined (Kothary & Kreger, 1985b) with the partially purified enzyme. Thetemperature optimum for caseinolytic activity was 55 to 60C (data not shown). Althoughactivity was 3-fold low er at 37 "C than a t 55 to 60 "C, the protease was assayed routinely a t 37 "Cbecause activity was more stable at that temperature.Stability and inactivation studies

The results of the stability and inac tivation studies with the step 3 protease were very similarto those previously observed (Ko thary & Kreger, 19856) with the partially purified enzyme, and ,together with the pH optimum data, they suggest that the enzyme is a neutral m etalloprotease.Also, the neutral pH optimum and the sensitivity to EDTA and OPA of our purified enzymesuggest that it is the same protease as that previously reported to be produced by V . vuln$cusA8694 (Carruthers & Kabat, 1981).Toxic activity

The LDS0values for the step 3 protease by the intraperitoneal and intravenous routes were 11units per mouse (about 1.5 mg kg-l) and 32 units per mouse (about 4.5 mg kg-I), respectively.Subcutaneous injection of 10units (40 pg) caused ex tensive dermonecrosis by 3 h postinjection.The activity of the enzyme in mice is consistent with the extensive dermonecrosis observed innaturally occuring and experimentally induced wound infections caused by V . vuZn$cus. Apartially purified elastase preparation obtained from a different strain of V . vulnificus (B3547)


7/9

Purification of V . vulnificus elastase 1789

Fig. 3. SDS-PAG E and Western blot analysis of V. ulnifcus elastolytic protease pre parations. (a )SDS-PA GE analysis. The cathode was at the top of the gel. Preparations in lanes 1 and 2 were denatured byheating with SDS before electrophoresis, and the pre paration in lane 3 was inactivated and denaturedat 4 "C with trichloroacetic acid and sulphosalicylic acid before heating with SDS nd electrophoresis.All preparations w ere reduced with p-mercaptoethanol. Lane 1, step 2 (100 pg); lane 2, step 3 (30 pg);lane 3, step 3 (30pg). The gels were stained with Coomassie brilliant blue R-250. The estimated M,values of the components in the stained bands in lanes 2 an d 3 (as determined by the relative m obilitymethod of W eber et al., 1972) are indicated. (b) Western blot analysis of a step 3 protease preparation( 1 5 pg) that w as not inactivated a t 4 "C before heating with SDSand b-mercaptoethanol. Lane 1, bandsvisible after staining with Am ido black; lane 2, bands visible after sequential probing with rabbit anti-step 3 serum, goat antirabbit IgG-horseradish peroxidase conjugate, and peroxidase substrate. Thethree bands observed by SDS-PAGE analysis (a) do not coincide precisely with those observed byWestern blot analysis (b) because of uneven gel-swelling caused by th e solvent used during the stainingand destaining of the 4 to 30% (w/v) polyacrylamide gradient gel.

Amino acidAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineValineMethionine

Table 2. Amino acid composition of V . oulnificus elastolytic proteaseNo. of residues No. of residuesConcn found per molecule Concn found per molecule(mol%) of protease* Amino acid (mol%) of protease*

15.47 68 Isoleucine 2.40 107.64 33 Leucine 4.56 209-98 42 Tyrosine 6.91 315-42 24 Phenylalanine 4.60 213.12 13 Lysine 4.86 228.89 36 Arginine 2.48 1 17.14 31 Half-cystine 0.88 41-49 7 Tryptophan 1-17 5

10-80 41 Histidine 2.17 10

* Based on an M, f 50500

than we used also has been reported to be lethal and to produce derm onecros is in mice (Poole etal . , 1982).In conclusion, the results of the studies described in this paper advance our knowledgeconcerning the elastolytic protease of I/. vulnificus in three ways. First, we describe a simplescheme for obtaining large amounts of V. vulnificus elastase of rigorously documented purity.The availability of large amounts of the highly purified enzyme should help investigators


8/9

1790 M . H . K O T H A R Y A N D A . S . K R E G E Rexamine the possible importance of the enzyme in the pathogenesis of diseases caused by V .vulnificus. Second, using the highly purified enzyme preparation, we confirmed the M , , PI, pHoptimum and stability data previously obtained (Kothary & Kreger, 19856) with a partiallypurified preparation of the enzyme. Contaminants can sometimes influence the physicochemi-cal and pharmacological properties of biologically active proteins (Gray & Kreger, 1985;Kothary & Kreger, 1985a), so initial results obtained with preparations of undocumentedhomogeneity always should be re-examined using rigorously purified preparations. Third, weextended the information in our earlier publication (Kothary & Kreger, 1985b)by reporting theamino acid composition and partial amino-terminal amino acid sequence of the enzyme, thetemperature optimum of the enzyme, and some of the biological activities of the enzyme inmice.

We thank Dr Mark Lively (Protein Sequencing Laboratory, Oncology Research Center of the Bowman GraySchool of M edicine ) and D r Lowell Ericsson (AAA Laboratory, M ercer Island, Wash.) for am ino acid analyses.This investigation was supported by Public Health Service grant AI-18184 from the National Institute ofAllergy and Infectious Diseases.R E F E R E N C E S

AMAK O, ., OKADA, . & MIAKE, . (1984). Evidencefor the presence of a capsule in Vibrio vulnificus.Journal of General Microbiology 130, 2741 -2743.ANDRUS, . R., WALTER,M., CROSA,. H. & PAYNE,S.M. (1983). Synthesis of siderophores by pathogenicVibrio species. Current Microbiology 9, 209-21 4.BRA DFO RD, . M . (1976). A rapid and sensitivemethod for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dyebinding. Analytical Biochemistry 12, 248-254.CARRUTHERS,. M. & KABAT,W . J. (1981). Isolation,purification and characterization of protease fromBeneckea vulnifica (lactose-positive vibrio, Vibriouulnijicus). Abstracts of the Annual Meeting of theAmerican Society for Microbiology 81, 24.GARDI,C . & LUNGARELLA,. (1984). Detection ofelastase activity with a zymogram method afterisoelectric focusing in polyacrylamide gel. AnalyticalBiochemistry 140, 472477.GRAY, .D. & KREGER, . S. (1985). Purification andcharacterization of an extracellular cytolysin pro-duced by Vibrio vulnificus. Infection and Imm unity48,HARBOE, M. & C L ~ S S , . (1983). Immunization withprecipitates obtained by crossed immunoelectro-phoresis. In Handbook of Immunoprecipitation-in-GelTechniques, pp. 353-359. Edited by N . H. xelsen.Oxford : Blackwell Scientific Publications.H0 I BY, N . & AXELSEN,N . H. (1983). Crossedimmun oelectrophoresis as modified for quantitativepurposes. In Handbook of Immunoprecipitation-in-GelTechniques, pp. 125-134. Edited by N . H. Axelsen.Oxford : Blackwell Scientific Publications.KOTHARY, . H. & KREGER, . S. ( 1 9 8 5 ~ )urificationand characterization of an extracellular cytolysinproduced by Vibrio damsela. Infection and ImmunityKOTHARY,. H . & KREGER, . S. (1985b). Productionand partial characterization of an elastolytic pro-tease of Vibrio vulnijicus. Infection and Immunity50,534-540.KREGER, . S. & GRAY, . D. (1978). Purification ofPseudomonas aeruginosa proteases and microscopic

62-72.

49, 25-3 1.

characterization of pseudomonal protease-inducedrabbit corneal damage. Infection and Immunity 19,KREGER, . & LOCKW OOD, . (1981). Detection ofextracellular toxin(s) produced by Vibrio vulnifcus.Infection and Immunity 33 , 583-590.KRE GER , . , DECHATELET,. & SHIRLE Y, . (1981).Interaction of Vibrio vulnificuswith human polymor-phonuclear leukocytes : association of virulence withresistance to phagocytosis. Journal of InfectiousDiseases 144, 244-248.KREGER,A. S., GRAY,L. D. & TESTA, . (1984).Protection of mice against Vibrio vulnifcusdisease byvaccination with surface antigen preparations andanti-surface antigen antise a. Infection and Imm unity45, 537-543.LAEMMLI,. K . (1970). Cleavage of s tructural proteinsduring the assemb ly of the head of bacteriophage T4.Narure, London 221, 680-685.LYERLY, . & KREGER, . (1979). Purification andcharacterization of a Serratia murcescens metalio-protease. Infection and Immunitya,1 142 1.MORRIS,. G ., J R & BLACK, . . (1985). Cholera andother vibrioses in the United States. New EnglandJournal of Medicine 312, 343-350.POOLE,M. D., BOWDRE,. H. & KLA PPER, . (1982).Elastase produ ced by Vibriooulnificus: in vitro and invivo effects. Abstracts of the Annual Meeting of theAmerican Society for Microbiology 82, 43 .REED, . J. & MUENCH, . (1938). A simple method ofestimating fifty percent end points.American Journalof Hygiene 21, 493497.SIMPSON,. M. OLIVER,. D. (1983). Siderophoreproduction by Vibrio vulnificus.Infection and Immun-ity 41, 644-649.SMITH, . C. & MERKEL,. R. (1982). Collagenolyticactivity of Vibrio vulnificus:potential contribution toits invasiveness. Infection and Immunity 35 , 1 155-1156.TESTA,., DANIEL,L. W. & KREGER,A. S. (1984).Extracellular phospholipase At and lysophospho-lipase produced by Vibrw oulnijicus. Infection andImmunity 45, 458-463.

630-648.


9/9

PuriJicationof V .TISON,D. L. & KELLY,M. T. (1984). Vibrio species ofmedical importance. Diagnostic Microbiology andInfectious Diseases 2 , 263-276.TOWBIN, . , STAEHLIN, . & GORDON, J. (1979).Electrophoretic transfer of proteins from polyacryl-amide gels to nitrocellulose sheets : procedures andsome applications. Proceedings of the NationalAcademy of Sciences o the United States of America76, 435W354 .WALLENBORG,B. & ANDERSON , U.-B. (1978).Immunoelectrophoretic techniques with the LKB21 17 Multiphor. LKB Application Note 249.Bromma: LKB Produkter AB.

vulniJicus elastase 1791WEBER,K. , PRINGLE, . R. & %BORN, M. (1972).Measu rement of molecular weights by electrophore-sis on SDS-acrylamide gel. Methods in EnzymologyWICKBOLDT, . G . & SANDERS, . V . (1983). Vibriovulnificus infection. Case report and update since1970. Journal o the American Academ y of Dermatol-YOSHIDA, .-I., OGAWA,M. & MIZUGUCH I, . (1985).Relation of capsular materials and colony opacity tovirulence of Vibrio vulnificus. Infection and Imm unity47, 4 4 6 4 5 1.

26, 3-27.

og y 9, 243-25 1 .

purification and characterization of an elastolytic protease of vibrio vulnificus

Documents