PUR IF ICATION AND CHARACTER IZATION OF EXTRACELL ULAR PROTEASE ECPASE 54 FROM A EROM ONAS H YD RO PH ILA
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1 , 1 9 ( 3 ) : J ou rna l of N anj ing A g ricu ltu ra l U n iversity ECPase 54 Ξ 3 3 (, ) (A h) J21 56 (ED TA ) (PM SF), (ECPase), A h J 21 SD S2PA GE, 37,, 5 A h J21 D EA E2 Sephadex G2200, 22, , 22, ,, ED TA, PM SF (T SM P), ECPase 54 ph 715, (Km ) 1177 m ggm l, V ero, ; ; ; S PUR IF ICATION AND CHARACTER IZATION OF EXTRACELL ULAR PROTEASE ECPASE 54 FROM A EROM ONAS H YD RO PH ILA L i H uan rong, Chen H uaiqing and L u Chengp ing (Co llege of V eterinary M edicine, N an jing A gric U n iv, N an jing ) ABSTRACT A t least five extracellular p ro teases (ECPase) in A erom onas hy d rop h ila strain J 21 culture su2 pernatant w ere assayed by in situ p ro teo lytic activity on SD S2PA GE containing 0. 1% casein. O ne of the ECPases from the supernatan t w as pu rified by amm on ium su lfate p recip itation, an ion2exchange ch ro2 m atography on D EA E2cellulo se and Sephadex G2200 gel filtration ch rom atography. SD S2PA GE of the pu2 rified p ro tease indicated that 22M E reduced samp le m igrated at , w h ile non2reduced samp le m igrated at T h is p ro tease w as acco rdingly designated ECPase 54. T he activity of ECPase 54 w as inh ibited by ED TA, but no t by PM SF o r by heating at 56 fo r 30 m in. mo stable m etallop ro tease (T SM P). p ro tyo lytic and cyto toxic activitieṡ It show ed that the ECPase 54 w as a ther2 T he ECPase 54 w ith op tim um ph 7. 5 and Km m ggm l po ssessed It also elicited lethal toxicity to m ice. Key words A erom onas hy d rop h ila; extracellular p ro tease; purification; characterization, 1989,,, [ 1 ] Ξ 3 3 Co rrespondence autho r :
2 3 : ECPase 54 89, S [ 2, 3 S, ] (Ex tracellu lar p ro tease, ECPase),, (A h) J21 ( ) ECPase, [ 1 A h J21 ] 112 ECPa se [ ] A h J 10 ggl, h, (A zocasein) A llan Steven son [ 4 ] ph m o lgl T ris2hc l (1. 15 m l) (0105 m l) A zoca2 sein (100 ggl, m l),, 37 1 h, (100 ggl, m l) ; 30 m in,,, 1 m o lgl N aoh, OD m in A zocasein OD SD S2PA GE [ 5 ], 1 gg L, A h J21 SD S2PA GE, PA G T riton X2100, 37 1 h, R 250, 113 ECPa se h A h J21, 25 2, 4, 8, 12, 20, 28, 35, 45, 52, 68, 72, 84 h, 4,,, %, 40%, 50%, 60%, 70%, 80%, 90% 100%,, 114 ECPa se 24 h A h J21, 25 (300 rgm in) 48 h, g,, 70%, 4, ( g), T ris2hc l,, ECPase 115 ECPa se D EA E2 ECPase D EA E2 (216 cm 30 cm, ph m o lgl T ris2hc l ), 0 1 m o lgl N ac l, 0105 m o lgl T ris2hc l, 280 nm, 5 m l (cm 2 ) - 1 h - 1, 5 m l,, PEG, T ris Sephadex G2200 5%,
3 90 19 (116 cm 100 cm ), 0105 m o lgl T ris2hc l (ph 7. 8), 6 m l (cm 2 ) - 1 h - 1, 5 m l,,, PEG T ris, 116 ECPa se SD S2PA GE g ( 100 ggl 22 ) g ( 22 ) g 5 m in, L aemm li SD S2PA GE, (50 ggl ), (125 ggl ), 100 V, 200 V,,, ECPase 48 h ECPase m in, ECPase 37 2, 10, 20, 30,, 120 m in, ECPase 0 20, 30, 37, 50, 56, 60, 70, m in, ph ECPase ph , ph , ph 8. 0 T ris2hc l ph 215, 3, 4, 5, 6, 615, 7, 715, 8, 9, 10, 11, ECPase, mm o lgl (ED TA ) 100 mm o lgl (PM SF) ECPase ECPase 22 ECPase, 15 m in, ECPase ECPase, 15 m in,, m in,, [ ECPase ] ECPase, 50 Λl, 50 Λl T ris (ph 7. 8) 1115 m l, 0, 3, 6, 9, 12, 15, 20, 25, 30, 40, m in, 100 ggl 1125 m l 1 m o lgl N aoh, OD 440,, ECPase, 50 Λl, ( 1),, 100 ggl 1125 m l V [S ] 1 m o lgl N aoh, OD 440 : Τ= Km + [S ], Τ, V, [S ] (m ggm l), Km 1 [S ] Τ, 1gΤ 1g[S ], - [S ] = 1 Km, Km 117 ECPase, 96, V ero, M EM V ero
4 3 : ECPase gλl A zocasein T risgm l (ph 7. 8) 1 ECPase 54 Table 1 D eterm ination of Km of the pur if ied ECPase 54 T ube N o ECPase, 011 m l, 100 Λg, PBS ECPa se,a h J 21 ECPase SD S2PA GE, ECPase, (20 68 h), 5 ECPase ( 1) 212 ECPa se A h J21 ECPase,, 68 h, h ECPase,, h A h J21 48 h,, OD 440 Incubation tim e: A : 8 h; B: 12 h; C: 70%, 20 h; D: 28 h; E: 35 h; F: 45 h; G: 55 h; H: 68, 70% h; I: 84 h 1 SD S2PA GE A h J21 ECPase 213 ECPa se F ig. 1 D etection of the p ro teo lytic activity of the ECPase of A h J 21 culture supernant by ECPase SD S2PA EG in situ 415% 85% D EA E2 Sephadex G2200,, Sephadex G2200 ( 2, 3), 214 ECPa se SD S2PA GE ECPase 22, SD S2PA GE,,
5 92 19, , ECPase SD S2PA GE, 4 P ro tein contents; N ac l p ro tein contents; 22 N ac l concentration; - - P ro tease ac2 P ro tease activity tivity 3 ECPase Sephadex G ECPase D EA E2F ig. 3 Sephadex G2200 gel filtration of the EC2 F ig. 2 D EA E2cellulo se ch rom atography of the ECPase Pase ECPase A h J h ECPase m in, 4617%, ECPase 54 ECPase m in, ( 5) ph ECPase ph, 37 1 h,, 30 m in, OD 440, ph ( 6) ECPase 54 ph 715, ECPase A. M arker ECPase 50 mm o lgl B. ECPase (22M E ) 22M E reduced puri2 fied ECPase ED TA 25 mm o lgl PM SF C. ECPase ( 22M E ) non 22M E re2 ECPase ECPase 54 duced pu rified ECPase D. ECPase ( 22M E ) 22M E reduced ED TA crude ECPase 7119%, ECPase 7613%, ECPase E. ECPase ( 22M E ) non 22M E re2 duced crude ECPase % PM SF 4 ECPase SD S2PA GE 30%, ECPase 15%, F ig. 4 A nalysis of crude and purified ECPaseon SD S2PA GE ECPase ECPase 54
6 3 : ECPase Purified ECPase C rude ECPase 5 ECPase Purified ECPase C rude ECP 6 ph ECPase F ig. 5 Effect of temperature on activities of ECPase F ig. 6 Effect of ph on activity of ECPase ECPase ECPase EC2 Pase 54 ; ; ECPase 54 62%, ECPase 3811% ; (3211%, 2514% ) ( 2) 2 ECPase Table 2 Effects of m etal an ion on activ ity of ECPase Enzym e activity (% ) Enzym e activity (% ) M etal anion cgmmo l L - 1 ECPase 54 ECPase Purified ECPase 54C rude ECPase M etal anion cgmmo l L - 1 ECPase 54 ECPase Purified ECPase 54C rude ECPase T ris2hc l ph Zn Ca M g M g Cu Ba M n H g Co Fe (m in), OD 440,, 6 m in, [S ] Τ, 1gΤ 1g[S ],,, - 1g[ S ] , Km 1177 m ggm l 215 ECPase 54, V ero,, ( 7) OD ECPase 54 V ero 01540, V ero F ig. 7 Cytopath ic effect of ECPase 54 on V ero V ero cells 216 ECPase m in,,,,, 4 h
7 94 19,, 3 ECPase, [ 5 ] N ieto A h ECPase,A h ECPase [ 7 ] L e2 ung Steven son 71 A h 4, (T SM P) (TL SP) [ 8 ] Chabo t T hune A h 3 (P 1 P 2 P 3), P 1, P 2, P 3 [ 9 ] A h J 21 ECPase, A h J21 5, m in, ECPase L eung ECPase A h J21 ECPase, SD S2PA GE, 22,, , 22, 56, ED TA, PM SF,, (T SM P), ECPase 54 ECPase,,,, T hune A h, ECPase [ 5,, ECPase ] ECPase 54,,, A h J 21 H EC [ 2 ], 1, 1 1, 1991, 14 (4): , 1 1, 1992, 32: ,, 1 S 1, 1996, 36 (2): A llan B J, Stevenson R M W. Extracellular virulence facto rs of A erom onas hy d rop h ila in fish infec2 tionṡ Canadian Journal of M icrobio logy, 1981, 27: T hune R L, Graham T E, R iddle L M, et al. Extracellular p ro tease from A erom onas hy d rop h ila: par2 tial pu rification and effects on age20 channel catfish. T ran saction of the Am erican F isheries Society, 1982, 111: :, N ieto T P, E llis A E. Characterization of extracellular m etallo2and serine2p ro teases of A erom onas hy 2 d rop h ila strain B 51. Journal of GeneralM icrobio logy, 1986, 132: L eung K Y, Stevenson R M W. Characteristics and distribution of extracellular p ro tease from A erom onas hy d rop h ila. Journal of GeneralM icrobio logy, 1988, 134: Chabo t D J, T hune R L. P ro tease of the A erom onas hy d rop h ila comp lex: identification, characteriza2 tion and relation to viru lence in channel catfish, Ictalu rus p unctatus. Jou rnal of F ish D iseases, 1991, 14: ( )
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