Pur if ica tion and Character iza tion of Ang ioten sin Converting Enzym e 3
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- Σωτήρης Βονόρτας
- 7 χρόνια πριν
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1 ISSN CN gQ 16 6 Ch in. J. B iochem. M o l. B io l V o l. 16 N o ( ) (angio ten sin converting enzym e A CE) A CE m o lgl 200 g m g A CE U gm g SD S2PA GE 180 kd (p I ) ph % A CE A CE A CE FaPGG ph ph 7. 6 Ea= Jgm o l Α2 A CE A CE Q 555 Pur if ica tion and Character iza tion of Ang ioten sin Converting Enzym e 3 L IU Hong CH EN L an2ying 3 3 (Ch inese A cad emy of M ed ical S ciences and P ek ing U nion M ed ical Colleg e Card iovascu lar Institu te and F u W ai H osp ital B eij ing Ch ina) ) Abstract In o rder to get a fu ll com p rehen sion on p roperties of hog angio ten sin converting en2 zym e (A CE) the research on the purification and characterization of hog A CE w as carried ou ṫ A f2 ter hog lung hom ogenate p rocessed w ith m o lgl amm on ium su lfate fractionation and so on m g angio ten sin converting enzym e (A CE) p ro tein w as purified from 200 g hog lung tissue u sing affin ity ch rom atography co lumm. T he purified A CE specific activity reached un itsg m g sodium dodecyl su lphate2po lyacrylam ide gel electropho resis (SD S2PA GE) detecting show ed a single band and gave an apparen t M r abou t 180 kd. T he isoelectric po in t (p I ) of A CE w as abou t ph 4. 5 and saccharide con ten t w as abou t 23. 6% am ino acid residues w ere found to con tain in an A CE m o lecu le concluding h igh level of acid am ino acid residues th rough am ino acid com po si2 tion analysiṡ T he op tim um ph of A CE w as bou t 7. 6 and the h istidine and Α2am ino am ino acid analogs w ere suppo sed to partake in the enzym e catalytic reaction by enzym e reaction k inetics ex2 perim en ts and calcu lated that the reaction activation energy of A CE catalyzing FaPGG w as Jgm o l. T he effects of am ino acid com po sition characterization and enzym ic p roperty of A CE estab lished a foundation fo r fu rther research. Key words A ngio ten sin converting enzym ea ffin ity ch rom atography Pu rification Enzym e p rop2 erty (angio ten sin converting en2 3 ( ) zym e A CE) g (angio ten sin g A ng 3 3 T el: Fax: E2m ail: lychen@m ail. sparkice. com. cn g ) : :
2 6 : 789 g (angio ten sin g A ng g ) 1. 5 m l 1 m o lgl N ac l 228 nm (b radyk in in BK) (U ) A CE (HHL ) 1 m o l [ 1 ] 10-3 U gm l( U gm l) (A CE inh ib ito ra CE I) [ 12 A CE ] : [ 2 ] A CE ( ) 2 m l 0. 5 m o lgl N a2 A CE C l FaPGG 0. 1 m o lgl A CE ph A CE Κ2 A CE nm [ 3 ] A CE 1 m in FaPGG 2 A CE 10-4 m o lgl Ε= m o l - 1 L A CE cm - 1 FaPGG m o lgl Ε= m o l - 1 L cm - 1 A CE V o : Sepharo se CL 24B L eamm li Pharm acia T ris E. M erck (h ippuryl2l 2h istidyl2l 2 leucine HHL ) (1 42bu tanedio l diglycidyl ether) (D isc IEF) [ 6 ] (N abh ) Sigm a L isinop ril SD S N N N N ACE [ 7 B io2r ad ] A CE (FaPGG) Serva : Κ214 (PE ) ; 2 m g 2 m l 6 m o lgl Innova ( Incubato r Shaker Innova Co. ) ; 110 (B io2r ad ) ; ph M eter (BECKM AN ) ; ( ) 112 ACE : [ 4 ] : 125 l 5. 0 mm o lgl HHL 0. 6 m o lgl N ac l 90 mm o lgl ph ACE 125 l 1 m o lgl HC l 200 g m l s m in 125 l 1 m o lgl HC l 750 l 5 m in (m o l - 1 L m in - 1 ) 113 L ow ry [ 5 ] BSA [6 ] 114 SD S- SD S2PA GE ( 7. 5% T 4. 0% T ) [ 8 ] 20 h HC l 2 m l m o lgl 100 l A CE 2 m l 10% T riton X m in 4 4 (6 000 g 20 m in) (1 000 rgm in 1. 5 m in) 500 l m o lgl
![5 25 A CE Κ2 A CE 14 328 nm [ 3 ] A CE 1 m in FaPGG 2 A CE 10-4 m o lgl Ε= - 2 300 m o l - 1 L A CE cm - 1 FaPGG 4 10-3 m o lgl Ε= - 520 m o l - 1 L cm - 1 A CE V o 1 111 11111 : Sepharo se CL 24B L](/docs-images/42/22828897/images/page_2.jpg "eamm li Pharm acia T ris E.")
3 m o lgl N ac l 20 mm o lgl T ris 2. 2 ACE ph % T riton X m o lgl N ac l 20 SD S2PA CE A CE mm o lgl T ris ph 7. 5 ( g 1 F ig. 2) 180 h 20 m lgh (1. 0 cm 2. 3 ACE (pi) 8 cm ) 40 m l 0. 3 m o lgl N ac l 0. 3% T ri2 ton X mm o lgl T ris ph 7. 5 T riton A CE (p I ) ph mm o lgl ph 9. 5 T ab le 1 SD S2PA GE 0. 2 m o lgl N aoh F ig. 1 T ab le 1 kd F ig. 3 A CE A CE F ig. 1 A ffinity ch rom atography p rofile of hog lung A CE g p ro tein; enzym e activity Table 1 Purification p rocedure of hog lung A CE Step To tal p ro teingm g To tal activitygu Specific activity gu m g - 1 Purification efficiency A ctivity recovery (% ) C rude extract mo lgl (N H 4) 2SO A ffinity co lum n ACE T ab le 2 A CE A CE 30 m in A CE 90% 2. 5% 93% 2. 5 ACE A CE A CE 23. 6% ( ) 2. 7 ACE 2. 6 ACE A CE A CE A CE A CE 11 lgk 1g (T ab le 2) T F ig. 4 A CE FaPGG
4 6 : 791 E a= Jgm o l Table 2 T he am ino acid component of A CE A CE F ig. 2 SD S2PA GE identification of A CE 1: C rude enzym e samp le; 2: Purified A CE; M : P ro tein mo lecular m ass m arker Am ino acid Θgm g L - 1 per mo le of enzym e N um ber of residues A SP THR SER GLU GL Y ALA CYS VAL M ET IL E L EU T YR PH E L YS H IS TRP A RG PRO To tal am ino acids Cals. M r F ig. 3 D isc IEF pho tography of A CE ph A CE FaPGG lgv m ph A CE ph A CE ph ph 7. 6 A CE F ig. 5 A CE p K es H is p K Α2 p K [ 9 ] p K F ig. 4 A rrhenius curve of temperature influence raction rate F ig. 5 lgv m 2pH curve of A CE k inetic reaction
5 A CE (References) A CE A CE D EA E2 (38. 8 [ 10 U gm g) ] (10. 2 U gm g) A CE A CE A CE 180 kd cdna A CE [ 11 ] 5 1 : A CE (p I 4. 5) L i J ian2w u X iao N eng2qing Yu Rei2yuan ed. 11 [ 12 ] SD S2PA GE (180 kd ) A CE A CE A CE 19. 5% 7: : A CE 23. 6% (D u J in2zhu Ru B ing2gengw ei X in2cheng ed. E n2 A CE A CE 170 kd 30% [ 13 N 2 ] A CE cdna A CE 17 N 2 13 [ 11 ] A CE 12 A CE A CE A CE A CE A CE A CE A CE A CE 1 Yo sip iv I V E l2d ah r S S D evelopm ental bio logy of angio tensin2 converting enzym e. P ed iatr N ep h rol : Zimm erm an Ben G D unham Earl W. T issue renin2angio tensin system: a site of drug action? A nn R ev P harm acol T ox icol : (Chen L an2ying T ian M in X ie Gui2fu. Purification of angio tensin converting enzym e from hog hung. Chin B iochem J ) : D as M Soffer R L. Pulmonary angio tensin2converting enzym e. Structural and catalytic p roperties. J B iol Chem ; 250: E xp erim ental M ethod s and T echniques in B iochem istry. Beijing: Pek ing U niversity P ress 1994: : (X ia Q i2chang ed. P rotein Chem ical Investigate T echniques and P rog resṡ Beijing: Scince P ress) B rew er J M. E xp erim ental T echniques in B iochem istry. N ew Jer2 sey: P rentice2h all Inc Englewood C liffs 1974: (Zhu J ing Yan Zi2zheng Zhang Shu2 zheng. T he am ino acid compo sition and chem ical modification of Α2galacto sidase from A bsidia Ramo sa. Ch in B iochem J ) 1991 zym e S tructure and M echanism. Beijing: Peking U niversity P ress) M eng Q C King S JB ranhan K E D elucas L JLorber B Opar2 il S. P reparative iso lation of angio tensin2converting enzym e from hum an lung. J Ch rom atog r ; 579: Soubrier F A lhenc2gelas F H ubert C A llegrini J John M T regear G Co rvo l P. Two putative active centers in hum an an2 gio tensin converting enzym e revealed by mo lecular cloning. P roc N atl A cad S ci U SA : (Chen L an2ying T ian M ing D u J ian2sheng. Changes in confo rm ation and activity of an2 gio tensin converting enzym e during guanidine denaturation. A c2 ta B ioch im B iop hy s S in) : T akada Y H iw ada K Kokubu T. Iso lation and characterization of angio tensin converting enzym e from hum an kidney. J B iochem :
![E n2 A CE A CE 170 kd 30% [ 13 N 2 ] A CE cdna A CE 17 N 2 13 [ 11 ] A CE 12 A CE A CE A CE A CE A CE A CE A CE A CE 1 Yo sip iv I V E l2d ah r S S D evelopm ental bio logy of angio tensin2](/docs-images/42/22828897/images/page_5.jpg "converting enzym e. P ed iatr N ep h rol 1998 12: 72 79 2 Zimm erm an Ben G D unham Earl W. T issue renin2angio tensin system: a site of drug action?")
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