ISSN 10072762 CN 1123870gQ 16 6 Ch in. J. B iochem. M o l. B io l. 2000 V o l. 16 N o. 6 788 792 2000 12 3 3 3 ( 100037) (angio ten sin converting enzym e A CE) A CE 1. 6 2. 6 m o lgl 200 g 0. 79 m g A CE 38. 8 U gm g SD S2PA GE 180 kd (p I ) ph 4. 5 23. 6% A CE 1 346 A CE A CE FaPGG ph ph 7. 6 Ea= 4. 37 10 4 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 100037 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 1. 6-2. 6 m o lgl amm on ium su lfate fractionation and so on 0. 79 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 38. 8 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%. 1 346 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 4. 37 10 4 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 (962901205285) zym e A CE) g (angio ten sin g A ng 3 3 T el: 010-68314466- 8068 Fax: 010-68313012 E2m ail: lychen@m ail. sparkice. com. cn g ) 1971 10 : 2000201227 : 2000204211
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 1. 2. 2 ] : [ 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 7. 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 eamm li Pharm acia T ris E. M erck (h ippuryl2l 2h istidyl2l 2 leucine HHL ) 1 42 222 115 (1 42bu tanedio l diglycidyl ether) (D isc IEF) [ 6 ] (N abh ) Sigm a L isinop ril SD S N N N N 2 116 ACE [ 7 B io2r ad ] A CE (FaPGG) Serva 117 1. 1. 2 : Κ214 (PE ) ; 2 m g 2 m l 6 m o lgl Innova ( Incubato r Shaker Innova Co. ) ; 110 (B io2r ad ) ; 5 271 ph M eter (BECKM AN ) ; ( ) 112 ACE 1. 2. 1 : [ 4 ] : 125 l 5. 0 mm o lgl HHL 0. 6 m o lgl N ac l 90 mm o lgl ph 8. 0 211 ACE 125 l 1 m o lgl HC l 200 g 37 600 m l 10 20 s 15 30 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 0. 02 m o lgl 100 l 835250 A CE 2 m l 10% T riton X2100 30 m in 4 4 (6 000 g 20 m in) (1 000 rgm in 1. 5 m in) 500 l 1. 6 2. 6 m o lgl
790 16 0. 3 m o lgl N ac l 20 mm o lgl T ris 2. 2 ACE ph 7. 5 0. 3% T riton X2100 0. 3 m o lgl N ac l 20 SD S2PA CE A CE mm o lgl T ris ph 7. 5 ( 50 000 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 X2100 20 mm o lgl T ris ph 7. 5 T riton A CE (p I ) ph 4. 5 50 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 5 340 257. 9 0. 048 1 100 1. 6-2. 6 mo lgl (N H 4) 2SO 4 864 127. 0 0. 147 3. 0 49. 4 A ffinity co lum n 0. 79 30. 7 38. 81 808 11. 9 2. 4 ACE T ab le 2 A CE A CE 30 m in 1 346 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
6 : 791 E a= 4. 37 10 4 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 35. 7 134 THR 15. 3 64 SER 40. 4 192 GLU 72. 3 246 GL Y 26. 8 178 ALA 10. 2 57 CYS 2. 71 11 VAL 16. 1 69 M ET 3. 80 13 IL E 9. 22 35 L EU 26. 2 100 T YR 4. 23 12 PH E 15. 4 47 L YS 12. 7 44 H IS 10. 2 33 TRP 3 13. 7 34 A RG 15. 4 45 PRO 7. 44 32 To tal am ino acids Cals. M r 1 346 144 900 F ig. 3 D isc IEF pho tography of A CE ph A CE FaPGG lgv m ph A CE ph A CE ph 5. 5 10. 0 ph 7. 6 A CE F ig. 5 A CE p K es 6. 6 7. 8 20 H is p K 5. 5 7. 0 Α2 p K 7. 5 8. 0 [ 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
3 792 16 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 1 346 cdna A CE 1 306 [ 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: 142 146 9 : 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 1998 12: 72 79 2 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 1997 37: 53 69 3 1 (Chen L an2ying T ian M in X ie Gui2fu. Purification of angio tensin converting enzym e from hog hung. Chin B iochem J ) 1988 4: 345 351 4 D as M Soffer R L. Pulmonary angio tensin2converting enzym e. Structural and catalytic p roperties. J B iol Chem 1975 10; 250: 6762 6768 E xp erim ental M ethod s and T echniques in B iochem istry. Beijing: Pek ing U niversity P ress 1994: 168 170 6 : (X ia Q i2chang ed. P rotein Chem ical Investigate T echniques and P rog resṡ Beijing: Scince P ress) 1997 99 122 7 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: 128 159 8 (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) 1991 167 171 10 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 1992 7; 579: 63 71 11 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 1988 85: 9386 9390 (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) 1989 21: 421 430 13 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 1981 90: 1309 1319