Purification, characterization and Genetic Cloning of Cystatin from Crucian Carp (Carassius Auratus) Oocyte

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(2). . . . . . . . . . . . . .

(3). . . . Purification, characterization and Genetic Cloning of Cystatin from Crucian Carp (Carassius Auratus) Oocyte. . . . . . ! " #. $. +. % & '( ) *. ,. -. .. /. 0. 1. 2. 340. 5. 6.

(4) . Department of Biotechnology Chia-Nan University of Pharmacy and Science.

(5). . . Thesis for the Degree of Master. . . .

(6). . . . . . . . . . . Purification, characterization and Genetic Cloning of Cystatin from Crucian Carp (Carassius Auratus) Oocyte. . . . . . ! " #. $. %Dr. Shinn-Shuenn Tzeng&. ' ( )* + ,%Chien-An Liu&. -. .. /. 0. 1. 2. 3. 4. 562. 13, July 2007 . 7. 8.

(7) .

(8) .

(9) . . . 1. . 2 J. . . . . 3 4. E. o. . p. @. . r. 6. L. ^_ q. . 5. K. . t. . 8. * u. ". . #. . N. ( s. !. 7. M. '. . . 9. O. Q. a W. $.

(10) %. v. [. @. =. x. c. @. R. . $. %. T. 5 p. . . *. . 5. U g. &. j. ,. D. W i. . *. ? . +. C. V h. . A B. . f o. . . S. e z. ). ?. ). y. (. >. d . '. P. b w. &. : ;<. P. `. -. E X ;k. . .. F. /. 4. G. l. !. . Y. 0. H. Z . . m. [. I. ;. n. \ &. ].

(11) . CST-II %. &. '. -.. ( )

(12) /. ,. . . . . .

(13). . . . . CST-I 14 kDa- CST-II 7. . 1. 2. 9.2 kDa89:. 14 kDa)=. . 12258 Da )J. E. F. G. H. 2.0-11.0MN . ( . Z. [. O. CST-I *. . 1. 2. 4-70 oC Q R P. . . Jg. CST-II . GIPGGLVDAD q*r. h. >. ?. CST-I . N i. j. ]. ^. . . N lmno. . `. L. p. . cystatin smno. 6. .

(14). . . . t. k. u. v. w. 90%) | | }. . ~. . ". "

(15). . . . . . "". C. W. . > L. U ?. . L. c. D. X. . . Y. . d. e. ). AGIPGGLVDA *. p. *V. ab. . B. pH K . . . β-mercaptoethanol ;. U. _ . 98 * 112 +. @ CST-I #A . 1 S T ) . CST-I $ CST-II \. B *f . I. . SDS-PAGE 4 5 ( 6 3. CST-I $ CST-II 1 2 < (. . CST-I * CST-II +,. 22 % 20 %)0 . . "CM-Sepharose FF #$ Sephacryl S-100 HR !. . L. . (. x. y. z. {.

(16) Abstract Two cysteine proteinase inhibitors, designated as CST-I and -II, were purified from the oocyte of crucian carp. The purification included acidification, CM-Sepharose FF, and Sephacryl S-100 HR chromatographs. The purification fold and recovery of CST-I were 98, and 22%, while those of CST-II were 112 and 20% respectively. The molecular masses (M) of CST-I and II estimated on SDS-PAGE were 14 kDa and 9.2 kDa. However, both inhibitors were shown to be about 14 kDa on β-me-free SDS-PAGE. Furthermore, the CST-I analyzed on MALDI-TOF spectrometer had a M of 12258 Da. No significant loss in the inhibitory activity within pH 2.0 ~ 11.0, 40 oC and 1 hr of incubation at 4~70 oC. Both cystatins could effectively inhibit papain, but not trypsin and cathepsin B. The N-terminal sequences of CST-I and -II were identical, which were determined to be AGIPGGLVDA and GIPGGLVDAD. They shared a 90% identity of amino acid sequences cystatin of with common carp (Cyprinus carpio).. Key word: Crucian carp; oocyte; cysteine proteinase inhibitor; cystatin; purification; characterization. . .

(17) . CST-I. Cystatin I. CST-II. Cystatin II. SDS-PAGE. Sodium. . . dodecyl. sulfate-Polyacrylamide. electrophoresis TCA. Trichloroacetic acid. Tris. Tris (hydroxymethyl) aminoethane. β-me. β-mercaptoethanol. . . gel.

(18) . . . . . . ……………………………………………………………………I . . . ……………………………………………………………………II . ………………………………………………………………………III . …………………………………………………………………………IV . ?. . ". ………………………………………………………………1 . 1-1. . ………………………………………………………………..1. 1-2. . .. …………………………………………………………..3 . 1-2-1.

(19). . . . . 1-2-2.

(20). . . . . 1-2-3.

(21). . 1-2-5. . . 1-2-6.

(22). . £. "¥. ¤ ¦. . . . . . ………..……………………….……6 . …………………………………………8 s. ……………………….……….11 . …………………………..………………………13 . . . . . . . s . ¡. ..……………………14. ……………………………………………………….....16 . *§. c. ……………………………………………………...17. 2-1. ¥ ¦. ……………………………………………………………….17. 2-2. C ¨. *©. 2-2-1. C . . 1-2-4.

(23). 1-3. ¢ . …………………..……………………….3. ¨. ª. ……………………………………………………….17 ………..………………………………………………17 .

(24) 2-2-2. © 2-3. §. ………..………………………....................................18 ª. ………………………………………………………………..19 c. 2-3-1. « ¬. . ®. s¯. °. ……..……………………………………19. 2-3-2. « ¬. . ®. . !. ………..…………………………………19. 2-3-3. . %. &. '. 2-3-4. . U. . (. 2-3-5. . ±. L. . µ. . ". ¶. *·. 3-2. . . ±. 3-2-1. 4.

(25). 5. 3-2-2. U L. . . 4. . n². ³. ´. …………………24. C. s. . …………………….28. (casein-SDS-PAGE)……………………….29. ………………………………………………….30. . ………….…..………….……………………..31 L. . . ………………………………………….….28. 5. L. . . (. (. . . N. º. . (. .………………………………..31. ....……………….……………………………..32. 3-2-7. N lmno. . …………..…………………………21 (. (SDS-PAGE)…..………………………………..29. Y. K. ¹ B. . . . (. . y. 3-2-5. ¸. . L. X. 3-2-4. R.

(26). . 3-2-3. pH K. 3-2-6. A. L. ……………………………………………………....28 . 3-1. . …………………………….……………….21. . 2-3-6. . ..…………………………………………20 (. p. ». ¼. ………………………………………32. .

(27) 3-2-8. .

(28). . . . . n². ³. ´. …………………33. ……………………………………………………………….34. . ½. . ". ¾. ¿. . . À. . ………………………………………………………………………….45. À. Á. ………………………………………………………………………….47. . . …………………………………………………………………….36. .

(29) 1-1.. (cysteine proteinase inhibitor).

(30) cystatin . (cysteine proteinase) . . (2). 4"#. (5). 6D E. !"# $)5. F. $% &'()*+,. $(3)647. &'8. 9. :*;. GH. (4). <. I J. -. .. =. >. K. /0 ?. L/M. VWXY. (bromelain)ST R. I(lysosome)%. cystatin _ G `. (enzyme-inhibitor complex) d&e =. >. \l>. z_t \}. (8). 6m. ng. . d472{| G. . }. o. p. q. f r s. ~ . ". g. {. nd 1. ^. N. (6). . P. . . D. aH. du. O. Z [. I-b. %hij t. C.

(31) J. H (cathepsin H). L (cathepsin L)\ S (cathepsin S)6]. B. (ficin)4L! U. (cathepsin)D7. B (cathepsin B). "

(32) . 23. A. (papain-like proteinase) 8 9 ( &'M N (papain)M (chymopapain)Q. (1). 1 @. N. . &e. vw. (7). c. . 4k)*. xy. cystatin . {. X. }. . . .

(33) 7{VW| ¤. ¥. ¦§¨. °. ±. 8. ¿E. F. À. É. ÊH. 9. p. (10). ©. Â. (11). ³ Ã. (9). g. D. \². ´. (12). Ì. ÍÎ. Á. ". £~ cystatin (. VW¢. ¶. !·. ¸ Æ. ¹. Á. Ï ÐÑ. ×. . ÒÓ. ¬. c. Ç. Á m. âã. ä. Ùå. ®. ». È. J. K. ¼½¾ Á. 6. À U. Á. (13). /ÙÚÛ. $Sæ í. ¯. nÕ. % ÐØ. àÜáÏ. . º. Ô. êë"/

(34) stefincystatinkininogen ì. 6é ð. µ. g.

(35) cystatin ÖF Þß. ¡.

(36) ÄÅ. I 7Ë. DÜÝ. . vx cystatin «. Dª:e. ² Á. }. ÜÊç. Äîì. D µ. è í. ï. cystatin superfamily(14)(15)Dstefin

(37) òó " ñ.

(38) 11 kDa ôõ ö. ÷Gøa S". Xã. ä. (intramolecular disulfide. bond)\æ ÜÊç è (glycosylation)(16)Dcystatin " ù ø/ú û". ü. cystatin þ. stefin. ( 13 kDa ý þ. (". % C (16)Dù. Xã. ä. (æ. ÜÊç. è 7_ stefin ò øò.

(39) kininogen ".

(40) 50-120 kDa æ. ÷. cystatin (cystatin like domains)Gøa(17)

(41) (. ì. /ú. 9 " Xã ä " ý. þ. . . ë% (domain I)Z. (h. (15)D. 2.

(42) Cystatin ù øý 7 "/aì í g . . DªÄ M. j &. '. ' (. (19) îH $. . (18). "^ $%. pH . ). .(/. I_ù. 0. 1. c. N. 5. ø(20)(21)(22) ;< M. 67. â4. 8. > =. `. ù 9. #. :. !. . (papain family) í. Ù(association constant, Kon) '. j. L+ cystatin \M ]. (. ï

(43) . ,(Õ. ù -. c. : cystatin ndu 2. ÞF. Ù(inhibition constant, Ki)

(44) nM ½ fM. Ù(dissociation constant, Koff)G+. z*'. N. Êé. Ú4. ndn

(45) /. ndD ë% 3. cystatin u 0. _ cystatin 7 N. 8. +. j. (wedge shape): ø N Gly9% Gln53. -Val55- Gly57 (first hairpin loop)L! C Trp104 (second hairpin loop) _h. g. ?@. A. ù B. c. 7îC. ù. c . ? D. E. FÓ. cystatin x%DHIJ cystatin / 0 _p j ý xÜÝ. g. Êé. LO. 1-2.. . . . J. &P %. . L. m. zM. cystatin g. ¼½R Q. . 1-2-1. T U 3. S. t. K. GG(. ;<. %J dD. N. V.

(46)

(47) yzxõ x(23)DªÄ. ö. ä. ®. Ie. vg. }. (23). O. _. |. K. . ndý.

(48) É. X ,. u. Y. ÊV °. Z. X"#. ±. k)*. I\ [. W. ¨. . v(24)6D. e ]. I òJ. ¯. vëû/hij ¢. H. LÞndu. ^. "

(49) õ. (endopeptidases)Do ` ëh. ö. (exopeptidases)\õ. ?aÜb. ÊÚd c. ö. X_. . êë[ ".

(50) eàá(serine proteinase)(cysteine proteinase)á (aspartic proteinase)L!f ñ f Ij E m. k. c. n. h%g

(51) (f ". ø`. l_c. Ê % o"p K. c. (viruses)u vg. í. *. Äf. ñ.

(52) ( eàá(serine acid)_q. (prokaryote) L. *. ª:d&h. H i. ?

(53) (7. Êa%g4eàá!. Ê%gDs. ö. ñ. Êa 7áh. àá(aspartic acid)Ln

(54) c. Ln

(55) J. _z. (metal proteinase)(25)D ñ. "

(56) y. t. w / "p. r. àá(cysteine acid). G(g Òx

(57) M. % 6². ³. /(26)D P N. í. (papain family). (calpain family)4Streptococcus WX4Clostripain ('. Ö Clostridium histolyticum)L!². (26)D ³. 4.

(58) M. (papain)' N. "]. I(model enzyme)DM H. 23.4 kDa ûg X ~. . . _c. M. N. . Üxù . c. (28). D. Êu. $.N. . /. ö. VzM. ^ û. K. |. ÷Gøa ".

(59). a }þ. (l. VWxY. c . . j +. L (cathepsin L)\. g. ® X. ¯. I%. ?(Cys25 in papain)L!. Ê. D. Y. G(conserved). ,âÓ. B (cathepsin B). ë V"

(60) . H (cathepsin H) 6]. Êu. ôõ. ?(His159 in papain) . `. N. J. (27). àÜáÚú K. c E. ID. H Úú. ý. d{. S (cathepsin S) ± 4k)* B . °. IK. l_ plasminogen activator hÊ\ collagenase w 7 . ¡. . · . "#. ¸. V. W. (30). D. !. ¡. VW¢ . . u. % í. (31). *. B H. £(29)4. L J. %x"z[. 8. "/_M. (z. . /ú. N. 9. g1 0 í. 1. Ä . 6. _hÊ % 5. j. K. . . ù . u. ç. è. %$ p. òý c. . VWXx. û l_. $Û. D. hÊ(d. I dLyz. L!,. ñ. ð. . ÊhDÄ/H. j. nd. z. ö. . IÞhÊG.

(61) z. *. â "a7 . 5 mM z. * 100 µM z Ø. hÊz. *. hÊz. *. II (calpain II) û ö. I (calpain I)! ö. I (Ca.

(62) z. 3 . proteinase I)\z II (Ca proteinase II)(32)(33)Då \ z ö G® .. ¯. / Xz. ¤. ° , ö. . ± . ¥. (glutamate)ù c ¤. ¬. ô T. \ z. ö. ¦. _. Y. |. &. 4. Ùå. ?© . ý. '. E. . K k. . A . ¡. ¢. £. . . ,. (34)(35)(36). c. DÍù. L ¬. , . U . ¤. ª. g . X. . . (hippocampus synaptic). band 1 double protein Z¦§¤ §. ù. (¿Ù. í. ö. « z. ç. è. ¨àá. _VW (37). D. 1-2-2. c Êu G((cysteine proteinase)yz $&ÖH ®. /¯ ° *. (15)(25). D LdM. I± àÜá8. ù 9. Ih%g. N. (Thiolate/Imidazolium ion pair4Cys-S – /His-Im+)c

(63) "]. ø²hÊ. ?`. &z2. a³7´. µ. c ¶. Êndu. 7øaëh. : Cys25His159 ! Asn175(15)(25) )·¸G¹(25)D. 6. Ê. H ?J. K.

(64) Û . ºyzÜxx. H. aä. (enzyme-substrate complex)g c. _ His159 ¿. ÷Üb. His159 Ã. Â. c. Ê. ¯. °. À. . ä. ù. (15)(25). ö. ä. ¾ I Gln19 _ Cys25. (hydrogen bond)ù c ²H I-Üx % C=O. ÷Üb. (imidazole ring)X –NH nd Lä Á. ¼ 7 Cys25 ¿. D7²õ. ?» Är j. aDr Asn175 ¿. Cys25 - S– x ¢ £½Üxõ ö a. ¶. nd 7²; Gln19 ¼ Cys25 n£½ . \Üxx C C=O ` b. Iµ. %Ç. S– Ä. ÷Üb. vÅ. ÷ –NHR Z_õ `. aÈ 7. É. Ê Ë. ö. Æ. ². ÷Ç. `. %(tetrahedral.

(65) intermediate)(15)(25) ÄÊ Ì Z. ¢. Äê. £½Ï. . ¢. Ð GL Õ.

(66) . . ¢. aÜ. ö. . c. ä. Üxõ . Asn175 o®. °. _Ö Ç. Þ. Ñ. . C=O ã ä ù Î. È. %\ ý À . u. ¸. Â. Ø. . %}ý É. Ò. "Û. c. Ò. . * His-159 Ô. È ù. . øë%. ¾. %@ É. ×Ê. ÄL. a. Ø. Ih. H. ö. ä. . ù. His159 Ã. . ? ÊH. z*,& rhÊ ²h LÚ ß. ·à. À. 7 His159 %¯ §. aD. Cys25 –S J. /ý. C Â ö. xõ ¯. . I. E F_ëá. Cys25 Lã. . "Ú x C Ù. ? Cys25 –S Ý. Gly19 . Í. I(acyl enzyme)(15)(25)D

(67) :²;LhÊ ÄÜ. ÊH. Êyzu. . I. ¯. I[×yzaêÜx . Ñ. . –NHR’ §Ó ä. ¿ õ. Î. DÐ. ö. _Z. £7Í. xÜx.| `. u. £½õ L.

(68) S– . ä Zå ±. ¼°. æ. x. °. K À. ndvg ½ â. L%\. . . ¾. ò». D. 1-2-3. ª:}¿Ù) %Dg. 6u. vg. . /Ùg. %/

(69) ç. 8. ¬. xV W. !Úd. g X. e. . vn.

(70) d Ðè g. (} k. %Xg. v ê. s. ®. ë. ¯. ì. í. î. g @. DÄ . vòw. Ihe. ±. °. D. h cystatin superfamily . ÞàÜáÏ a

(71) ì. Û. é. í. â". ã. ä. Ùå. \æ. ÜÊç. è. 6". ù. øé. L. ". stefin (family 1)cystatin (family 2)kininogen (family. 4"p. 3) ëé "Í)·D Stefin (family 1)* Stefin ôõ ö. ÷(single polypeptide chain)ù. øï 100 àÜ. áGøa ".

(72) 11 kDa ý. S". cystatin superfamily %òó(38)DStefin. áó. Xã . |. ä. â!ôÒ. þ. ï M. (ð. N. ñ. æ. ÜÊY. !. áÊç ò. è . ?. õ $(Õ. D . " stefin g. Y ö. C. g X. Üá stefin 8. X` À. ÷ 9. aý. þ. hã. (dimer) u. z*ahô(monomer)D.<à :.. / stefin ABC ø. ,. . (porcine leukocyte cystein proteinase inhibitor, PLCPI)L!ù. α _ βDStefin A J K. ". i3. v,. 9. . _û. stefins. ü. ú ý. þ. ¢. VW.

(73) (lymphoid follicular dendritic cell)(39) stefin B o "VW! % (40) stefin C \ PLCPI (41)(42). . v,. !ø. VW%p . j. D. Cystatin (family 2) ". ù. ø_ stefin . ï( 115 " Êç. è. "Úú.

(74) ôõ. ö. ÷ù. ø àÜáÙ. ï

(75) 13 kDa cystatin Sð. 2 ". Xã. ä . æ ñ. ÜÊY. %x C ò Ô. á ò p. j. (43)D í. ( CDSSN \ SA y. . pI ï

(76) 8.0-9.5 " . 6(15) s t. . } 8. .. . $(15)(46)(47)}. (Õ. . 3. ]. ^. ë% cystatin C . × . 1. ó

(77). VWÇ. . \$%(44)DCystatin D o. . $%(45) 7 SSN \ SA 6]. 9. Ø. ë pI ï

(78) 4.7(15) J ^. %(48)DCystatin þ. $5. $_ . Y. I. BH L !M. ( ! N. K. . a. 6. %(49)D. Kininogen (family 3) Ùx xl_,. P. $%ï

(79) ( kininogen ª:h. /, $O. \p . a(50)DKininogen

(80) ôõ 10. ö. ÷,. -. .

(81) ". ò ï

(82) 70–120 kDa Dù. (heavy chain)\. ÷(light chain)D÷\. (signal peptide)¢ . ø "

(83) ÷. (ï 18 àÜá. äD? N ÷ [V"

(84) ì. »§. õ . ö. . cystatin ù ø

(85) kininogen hJ K &'D)·¸G¹(15). /ú. (7. þ. Üç. æ. àÜáGøa,. è . . ? 7. I(kinin)A. »D". "p.

(86) 2 \ì. ý. h(15) þ. 1-2-4. ù + cystatin ù. 5 Þ. . ¢. 9. sheet)L! §ý | Ù. ä. ". ì. %. 3 D ä . . ÷D. I. ø. × X . (turn) α- . Xã. ( ã. ? Ä. ï 10 . ì. . È. zM . (helix) \ 5 . α- . ,xJ a¼. ¬. K. . β!. Gøa ;<ë% β- ! 11.

(87) (. (pleated 8 ". .

(88) #. α- . £. (helix) . Ê . DÄA. ù B. ø% Fj. ?(inhibitory reactive sit)DîC. a. "zÜxc. (51). . ¬. A. 4 B. =. ù. c. $. Cys25 Ð cystatin ý & Üx' yz ( Ó cystatin * j Ó. ý. u. ndñ. ?s. _. Ic. *H. ø|. Gù. _. ). Ê%g. DÄ. (15)D. . &' cystatin àÜá p. G "p. %. . ,ì. N Gly9 % +. j. 53Gln-Val-Val-Ala-Gly57 \ C . ,. Íì Ñ. . (Iâ. (hairpin loop) . . " 103Pro-Trp104 ( ) · ¸ G. ¹)(15)(21)D. Cystatin %ã. ä. "p . ) β-me (β-mercaptoethanol)À h. i. u. (±. ù. ø70 ì. Cys71-Cys81 \ Cys95-Cys115 ù ÷. · .. hD. 12. Î. ã. ä. ù. §:. ² cystatin S/.

(89) 1-2-5.u cystatin _@ H. IyzÜxc. ój 4. 6. ½ S2 7. c. GLc. 5. M. N. Üxù. ÊÜxyzndÄ| Ê. (Leu7-Ala10)L/ú Üx9. c Ê. Ala136 \ Ala137 6ì. Trp177 _ Trp181 ù. ù. 4 =. (15)(16)(21). c. p B. ø` 3. 2. Ê%g Cys25. D. ,. þ 3. yù. c. Zù 7. (âÓ. D. L. (indole ring) _M N. N. c. M. =. ä. ù. >. c. ²H. N. . GàÜá Gly23 . + Ñ. 3. %. (Ú¿. ÷. DÄ cystatin nd&Ö_M c. _. ,. N. I_`. aA B. D. Cystatin G` IS/. ,. c. Ò1. :. c. L!ù. N. *M. ¼ Cys25 Z_M. ½. Trp104 GÑ ¿ ÷; < À. %. (elephant trunk)ø8 Æ. Ì. +. : 2. % cystatin N L Gly9

(90) % g Ì. Å. nd(21) 71. c =. ? S1’ _ S2’ 7 N Gly9 o . ? (15) 7 + p. Gln53-Gly57 ù. ø`. c. ? cystatin $. g. 4. ?D? cystatin _ì. Ê. ndp. g. a±. (c. ?. @. 2. A. `. :M. Ê()·¸G¹)(15)D. 13. N. _Üxù. c ÐH.

(91) 1-2-6. @ Q. d. Cystatin " Ô ó d&C l cystatin Ü½G O PQ. À. (52). 4ªR S. H _!m. I. J. §. d«. n². Á. À. ó D. «. D. 14. K . L. E M. å. Ê. . Î. Á. F Ä ¢. ¼½N . o. ¡. ¶ È. y Á. dÊx.

(92) GÁ J Q Y. K w. Z. r[. /. 0. W. C 2R ]. S. ^. ª Ð]. g. §ôa`. Ê. d. vXg. g. §½q. ù. aU 2-. ]. /(59)o. aÝ g. ( . U. t. Å. § -. Õ. U. ,. . é. (E. coli)\H z ´. xX S. _T Q. "_R. ]. U. Ï. U. Xg S. x . r §ô 50-70. (IB. L MHC

(93) J Î. G`. âH·`D

(94) :¦§2/R S. ac. a` d _. Ã i. k. aU. (55). . (. s. §Lg @. w. ø. xt. h. a| R. d . ) S. b. Y. -. D /. l. Lr. . _. ù. h\nd ²x` F. V. r. nd(modori) 78. z. P. (56)+(57)A m. â )U. "*q. ÊL. Ú¦§DxÜ¢ Ï. R. na ]. U. . % Í Ì. òT. p ¦( 4. S. Ì. øDÄ ê/E. % ú j. ;ÒJ. (Õ è. m. ÷(myosin heavy chain, MHC)7ý O. U. e. 2/R. (54) j. "zR. U. Ë K. yznd!@. 7G;Ò]. f. @. dDÛ. p. a.

(95) û . t. _. U. "z(autolysis)w. Ö# S. Gh(53) ý Ó. :2/§V \. R. DL2/x

(96) k /×Ê X. ÞF. o. ). p. )v. ¶. Ø. LÔ q. (58). n. &-. p; ý ½ug. B. K. % Lt. § r t. dug. 2{w(60)!+ cystatin x y. ´. (Pichia pastoris)%ij s a k D7{ 15. !.

(97) } |. :î/ E. ý. 7FI¦§2-. 1-3.. . . w (60)(64) \. é. øé. g. ( . ¬. ]. ^. xÚú. é. . Z"M. :. âD (. ÊÜ¢ q. 8 . (61). &'o(~. $L. U. cystatin .. L!ù. 6. ]. F. x. j. (_E. þ. 9. . :; (62). . ¶. _ij. <. (4). . A (63). 23 (2) &Öy@

(98) J. K. . 7". . / . Å 0 . . DÄ / . . h"M. cDNA Ü¥ ¶. D. 16. y. B. (5). l>. (8)(13). /(1)(3) 6Dªv. 2{. n. . g. 0. =. >. " cystatin. . êLg. Ï. . %2n

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(188) . . . . 1. Green GD, Kembhavi AA, Davies ME, et al. Cystatin-like cysteine proteinase inhibitors from human liver. Biochem J. 1984;218(3):939-46. 2. Synnes M. Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L). Comp Biochem Physiol B. 1998;121(3):257-64. 3. Abrahamson M, Barrett AJ, Salvesen G, et al. Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem. 1986;261(24):11282-9. 4. Wu J, Haard NF. Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants. Comp Biochem Physiol C. 2000;127(2):209-20. 5. Rodis P, Hoff JE. Naturally Occurring Protein Crystals in the Potato: Inhibitor. of. Papain,. Chymopapain,. and. Ficin.. Plant. Physiol.. 1984;74(4):907-911. 6. Aranishi F, Hara K, Ishihara T. Purification and characterization of cathepsin H from hepatopancreas of carp Cyprinus carpio. Comp Biochem Physiol B. 1992;102(3):499-505. 7. Berri M, Venien A, Levieux D, et al. Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle. Comp Biochem Physiol B. 1996;113(2):275-9. 8. Kondo H, Abe K, Nishimura I, et al. Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular 36.

(189) cloning, expression, and biochemical studies on oryzacystatin-II. J Biol Chem. 1990;265(26):15832-7. 9. 0 1 2 Dv 2{q r |. }. u. /. àá"z Cystatin q 0. Ê_. D

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(198) i . i 1. 2 2. . !. 2. {. . . Table 1. Summary of the carp body weight and oocyte weight Body weight (g). Oocyte weight (g). Oocyte weight/Body weight. 1. 121.12. 31.44. 25.90%. 2. 103.37. 23. 22.20%. 3. 105.63. 30. 28.40%. 4. 133.5. 34.3. 25.60%. 5. 116. 33.7. 29%. 6. 116.9. 26.5. 22%. Average. 116.08. 29.82. 25.51%. 45.

(199) i 2.2 {. V. W. . . . . . . . . q. Ê. Table 2. Summary of the purification of cystatin from crucian carp oocyte Total protein Total activaty Specific activaty Procedure. (mg). (U). (U/mg). yield(%). purity(fold). Carp oocyte. 2920. 675.7. 0.23. 100. 1. 952. 458.3. 0.48. 67. 2. 145. 308.5. 2.1. 45. 9. 6.79. 154.7. 22.7. 22. 98. 5.24. 136.4. 25.9. 20. 112. Acid denaturation (pH 4.0) CM sepharose FF Sephacryl S-100 HR (CST-I) Sephacryl S-100 HR (CST-II). 46.

(200) . ¸ 1. 2 {. V. W. ¸. cystatin CM-Sepharose FF . M. ¸. Figure 1. CM-Sepharose FF chromatography of crucian carp oocyte cystatin. The oocyte extract was applied to a CM-Sepharose FF column (2.6 x 18 cm) equilibrated with 20 mM Tris-HCl buffer, pH 8.8. Flow rate: 2 ml/min; collection: 5.6 ml/fraction; elution: linear gradient of 0–0.5 M NaCl.. 47.

(201) ¸ 2. 2 {. V. W. cystatin Sephacryl S-100 HR M. ¸. Figure 2. Sephacryl S-100 HR chromatography. The inhibitor fractions from CM-Sepharose FF column were applied to a Sephacryl S-100 HR column (1.6 x 90 cm) equilibrated with 20 mM Tris-HCl buffer, pH 8.0, at a flow rate of 0.5 ml/min; 2mL/tube was collected.. 48.

(202) ¸ 3. 2 {. V. W. cystatin ¥ " M. ¸. Figure 3. SDS-PAGE pattern of cystatin (CST-I and CST-II) purified from crucian carp oocyte. The CST-I and CST-II were analyzed on 15% acrylamide. gel in the absence (A) and presence (B) of β-mercaptoethanol. Lanes 1 and 3, CST-I; lanes 2 and 4, CST-II.. 49.

(203) ¸ 4. 2 {. V. W. cystatin h ¶ ± ". M. Figure 4. Zymogram of crucian carp oocyte cystatin. The CST-I and CST-II were electrophorezed on 15% acrylamide gel containing 0.1% (w:v) casein. The cystatin proteins were renatured by soaking the gel in Brij 35 solution for 30 min and deionized water for 10 min. The casein in gel were proteolyzed by papain at 40 °C for 1hr. The reaction was stopped by immersing the gel in 12.5% TCA solution for 10 min and staining with Coomassie Brilliant blue G-250.. 50.

(204) ¸ 5. CST-I _ CST-II pH Ò . . Ô. Figure 5. Comparison of pH stability of CST-I and CST-II. Cystatins in pH buffers (20 mM citric buffer, pH 2.0-4.0; 20 mM phosphate buffer, pH 4.0-7.0; 20 mM Tris-HCl buffer, pH 7.0-10.0; 20 mM borate buffer, pH 10.0-13.0) were incubated at 40 °C for 1hr, and then an equal volume of 0.3 M phosphate buffer (pH 7.0) was added.. 51.

(205) ¸ 6. CST-I _ CST-II â. Ò. . . Ô. Figure 6. Comparison of thermal stability of CST-I and CST-II. Cystatins in 20 mM phosphate buffer pH 6.5 were incubated at 4, 30, 40, 50, 60, 70 and 80°C for 1hr. After being cooled in ice bath, the residual inhibition of both cystatins were assayed.. 52.

(206) ¸ 7. CST-I _ CST-II ý . . . . . . h. . Ô. Figure 7. Inhibition of various proteinases by CST-I and CST-II. Proteinases reaction with various concentrations of cystatins were reacted at 40 °C for 10 min.. 53.

(207) 12258.0000 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0. 1.216e4. 1.218e4. 1.220e4. 1.222e4. 1.224e4. 1.226e4. ¸ 8. CST-I MALDI-TOF x. 1.228e4. ð. . 1.230e4. ". 1.232e4. 1.234e4. M. Figure 8. Analysis of CST-I on MALDI-TOF spectrometer.. 54. 1.236e4.

(208) Source. Amino acid sequences. Crucian carp CST-I. A G I P G G L V DA. References*. This study. G I P G G L V DAD. CST-II Common carp. Identity (%). T G I P G G L V DA. 90%. This study. 90%. L23572. Chum salmon. G L I G G P MDAN. 50%. D86628. Rainbow trout. M KC G G T A E AK. 30%. U33555. Chicken. A G AR G C V V L L. 40%. J05077. Human C. P R L VG G P MDA. 40%. X52255. *Accession number in Genbank. ¸9. Cystatin N àÜá . Ô. Figure 9. Comparison of N-terminal sequences of crucian carp oocyte CST-I and -II with family 2 cystatins. The amino acid residues identical to crucian carp CST-I and CST-II were blocked. The Gly5 was conserved among family and marked with boldface.. 55.

(209) M ¸10. 2 {. V. W. . . . . . . . 2. . 3. PCR @ ¥. " M. ¸. Figure 10. Agarose gel pattern of PCR product amplified for cystatin. The first strand cDNA was reverse transcription from total RNA of crucian carp oocyte. Polymerase chain reaction (PCR) was conducted as follow: 94 oC for 10 min and 35 cycles of 94 oC for 30 sec, 55 oC for 30 sec and 68 oC for 50 sec, final extension of PCR product was 68 oC for 10 min. Product was analyzed on a 1.5% agarose gel. M, 100 bp DNA marker; lane 2, PCR product amplified from first strand cDNA; lane 3, control (Without PCR product).. 56.