ISSN 100727626 CN 1123870ΠQ 2002 2 Chinese Journal of Biochemistry and Molecular Biology 18 (1) :44 49 1) 2) 1) 1) 3,,, ( 1), 100850 ; 2) 0, 100039) cdna pcdna3 1, pcdna2 AChE 293, rhache rhache AChE rhache K m 137 molπl ; huperzine A eserine ( IC 50 215 10-8 molπl 110 10-7 molπl) ; HI2 6 (10-4 molπl) sarin (10-6 molπl 10-7 molπl) rhache,4 h 86 % 97 % rhache 3,, Q55,Q78 Gene Expression and Biotoxicological Properties of Recombinant Human Brain Acetylcholinesterase LI Qian 1), LIU Wei 2), LI Feng2zhen 1), SUN Man2ji 1) 3 ( 1) Beijing Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences, Beijing 100850, China ; 2) Clinical Laboratory, 304 Hospital of Chinese People s Liberation Army, Beijing 100039, China) Abstract The encoding sequence of human brain acetylcholinesterase was subcloned into an eukaryotic ex2 pression vector pcdna 3 1 and then transfected into human embryonic kidney cell line 293 for expression of recombinant human acetylcholinesterase The biotoxicological properties of recombinant acetylcholinesterase and native acetylcholinesterase were very much alike The K m value of rhache was 137 molπl ; rhache ex2 hibited the excess substrate inhibition Inhibitor huperzine A and eserine could inhibit rhache with IC 50 of 215 10-8 molπl and 1 10-7 molπl respectively HI26 (10-4 molπl) reactivated the sarin (10-6 molπl and 10-7 molπl)2inhibited rhache with reactivation rates of 86 % and 97 % The rhache could stand repeated freezing and thawing for three times without loss of enzyme activity Key words human brain acetylcholinesterase, biotoxicological properties, eukaryotic expression (acetylcholinesterase, AChE), AChE,, Ach, 1990 Soreq [1 ] AChE, AChE Velan [2 ] Kronman [3 ] AChE, :2001203208, :2001206204 AChE (962Z2016) 3 Tel : (010) 66930691,Fax : (010) 68211656 AChE E2mail :sunmj @nic bmi ac cn AChE,,1970,, AChE Received :March 8,2001 ;Accepted :June 4,2001 AChE, AChE cdna CMV2 E6, pcdna2ache Supported by the 9th Five2Years2Plan Medical Research Key Program of PLA (No 962Z2016) 3 Corresponding author Tel : (010) 66930691,Fax : (010) 68211656 E2mail :sunmj @nic bmi ac cn 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
1 : 45 1 111 [ 5] CMV2E6 : AChE cdna 118, Hebrew University Hermona Sorgq ;pcdna 311 ( + ) Invitrogen, (25 mmolπl Tris, 10 % ) 3, DNA2 37 10 h, 1 ml 2, 8 14 h 211 pcdna2ache 24 72 h [ 4] 117 DTNB AChE PB (1Π15 molπl, ph 712) 180 AChE, AChE cdna l, 10 mmolπl 90 l CMV2E6 5 Hind 3 Xba, 25 1 h, 1 molπl HCl 30 l, 20 min 0103 % DT2 NB 600 l, 300 l 96, 414 nm SDS2PAGE, 15 min, (25 112 293 mmolπl Tris, 011 molπl Gly, 10 % ),, (013 molπl Tris, 10 % ) 3 2 3 2 113 2 2, 118 Xba Hind T4 DNA maπcm 2 1h, Promega ; DNA (Wiz2 1 % BSA 1 h AChE 1 50 ard R plus miniprep DNA purification system), 2 h, TBST 3, Promeag ; DNA, 5 min HRP IgM 1 100 1 h,tbst 3, 114 6 mg DAB 9 ml 011 molπl Tris2HCl, Sigma ;5,5 2 ph 716 1 ml CoCl 2 (0103 %), 10 l 222 (DTNB) H 2 O 2 HI26 sarin [ 6] 119 IgG( 1 1000),, 9 %, IgM ( 1 1000) ; 215 % 50 mmolπl Tris, 011 AChE AChE molπl Gly, 1 10, 100 V, 115 DNA (01065 molπl PBS,pH 610, 5 mmolπl,3 mmolπl CuSO 4, 015 molπl KFe (CN) 3,0105 % 116 293 ),37 30 min 37 18 24 h, 50 % 80 % [ 7] 1110, A (1 2 g DNA 100 l ) PBS 2, 4 % Π014 B (2 25 l 100 l ) %, 15 min PBS A B, 15 45 min,, (01065 molπl PBS ph 610, 5 DNA2, 018 ml mmolπl, 3 mmolπl CuSO 4, 015 mmolπl, 2 KFe (CN) 3,0105 % ),37 2 h 2 21111 pcdna2ache CMV2 E6 AChE cdna,, pcdna311 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
46 18 MCS Hind Xba, AChE 212 21211 rhache K m rhache nache, rhache K m Lineweaver2 Burk, ( Fig 1) K m,rhache Linewaver2Burk Fig 1, K m 137 molπl Fig 2 Substrate inhibition curve of rhache rhache was assayed with the indicated concentrations of acetylthio2 choline AChE eserine huperzine A 100 %, (Fig 3) huperzine A IC 50 215 10-8 molπ L,eserine IC 50 110 10-7 molπl1 Fig 1 Kinetic parameters of rhache:lineweaver2burk double2 reciprocal plot K m was calculated from the Lineweaver2Burk plot 21212 rhache,,, AChE,, AChE 0105 Fig 3 Effects of selective inhibitors on rhache activity rhache was assayed in the presence of eserine ( ),and huperzine A ( g) at the indicated concentrations Acetylthiocholine ( 10 mmolπl) was the substrate in all cases 30 mmolπl, DTNB 21214 HI26 sarin rhache rhache, 293, AChE rhache, Fig 2, 011 molπl (ph 714) 21213 Huperzine A eserine rhache PB sarin 211 ml,sarin 10-5 molπl,37 1 h 100 l, AChE, 10-5 molπl eserine Huperzine 2 ml 2, 1 ml, 1 A AChE 20 l 800 l PB sarin PB eserine huperzine, 1 600 l HI26 200 l PB (HI26 180 l, 1 1 10-4 molπl), HI26 sarin 1 37 1 PB, h,, 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
1 : 47 HI26,, 37 (Table 1) 10-6 molπl 10-7 molπ 180 l, L sarin rhache HI26 (10-4 molπl,37 = ( EIR ( EΠER - EI)Π( E - EI) ph 714), 86 % 97 %(Ta2 100 % HI26 sarin rhache ble 1) Table 1 Reactivation of sarin2inhibited rhache by HI26 Sarin concentration Enzyme activity E EI EIR ER Reactivation( %) by HI26 gx s, n = 3 10-6 molπl 01810 01042 01177 01018 01907 01045 11016 01116 86 10-7 molπl 01810 01042 01187 01021 01991 01092 11016 01116 97 E: normal enzyme activity ; EI : inhibited2rhache activity ; EIR : inhibited2rhache reactivated by HI26 ; ER : HI26 control 21215 rhache 293 hh11,western2 ( Fig 4), - 20 3, 30 l,rhache, 67 kd 3, (Table 2) Table 2 Stability of enzyme activity of rhache during repeated freezing2thawing Store at 4 Freezing2thawing Once Twice Thrice 01695 01022 01750 01013 01746 01009 01786 01030 gx s, n = 3 21216 rhache 6 ml,, PEG 20 000 1 ml, 011 molπl PB (ph 714) 20 l SDS2PAGE, rhache, PVDF, AChE rhache, 21217 293,, 37 30 min Fig 5 rhache 2 4 2 Fig 5 Nondenaturing gel stained for AChE enzyme activity A,B,C: Purified torpedo AChE D,E: rhache Fig 4 Western2blotting of recombinant rhache A : Protein marker B : Concentrated cell supernatant of transfected 293 cells 21218 pcdna2ache 293, 72 h, 293 Fig 6 293 AChE,, 293, 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
48 18 Fig 6 Enzyme activity staining for human embryonic kidney cells (A) Before transfection (B) After transfection 3, AChE [14 ],, AChE [8 ] AChE AChE, AChE [2 ] rhache,,,, 293 AChE HeLa AChE, ( References), AChE 293 AChE,, 293 AChE AChE, AChE 0 18 UΠml [9 ] AChE, 293, AChE, AChE ( 60 ) 156 56 1 h, Soman Soman AChE, IC 50 10-8 molπl 10-6 molπl Soman,,37 24 h 293 AChE 5d, 293 AChE 01045 IU 10-6 24 h 293 rhache, Ach rhache, AChE Huperzine A AChE,eserine, huperzine A eserine rhache Huperzin A IC 50 215 10-8 molπl, eserine IC 50 110 10-7 molπl Sarin AChE HI26 10-4 molπl HI26 10-6 molπl 10-7 molπl sarin rhache, 4 h 86 % 97 % [13 ] 293 rhache, 3, rhache AChE 1 Soreq H, Ben2Aziz R, Prody C A,Seidman S, Gnatt A,Neville L,Lie2 man2hurwitz J,Lev2Lehman E, Ginzberg D,Lapidot2Lifson Y,Zakut H Molecular cloning and construction of the coding region for human AChE reveals a G + C2rich attenuating structure Proc Natl Acad Sci USA, 1990, 87 : 9688 9692 2 Velan B, Kronman H, Grosfeld H, Leitner M, Gozes Y, Flashner Y, Sery T, Cohen S, Ben2Aziz R, Seidman S, Shafferman A, Soreq H Recombinant human AChE is secreted from transiently transfected 293 cells as a soluble globular enzyme Cell Mol Neurobiol, 1991, 11 : 143 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet
1 : 49 3 Kronman C, Velan B, Gozes Y,Leitner M,Flashner Y,Lazar A,Marcus D,Sery T, Papier Y, Grosfeld H,Cohen S, Shafferman A Production and secretion of high levels of recombinant human AChE in cultured cell lines : micro heterogeneity of the catalytic subunit Gene, 1992, 121 : 295 304 4 DTNB enzyme J Biol Chem, 1985, 260 : 4312 4318 (Dong Li2chun A method for microassay of ChE activity with DT2 NB Bull Milit Med Sci ), 1987, 11(6) : 480 483 5 Karnoviky M J, Root L A Direct2coloring thiocholine method for cholinesterase J Histochem Cyto Chem, 1964, 12 : 219 221 6 Jonathan M G, George E P Protein blotting : principles and applica2 tions Anal Biochem, 1983, 131 : 1 15 7 Duval N, Massoulie J, Bon S H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms J Cell Biol, 1992, 118 : 641 653 8 Chen C, Okayama H High2efficiency transformation of mammalian cells by plasmid DNA Mol Cell Biol, 1987, 7 : 2745 2752 9 De La Hoz D, Doctor B P, Ralston J S,Rush R S wolf A D A simpli2 fied procedure for the purification of large quantities of mammalian ace2 tylcholinesterase Life Sci, 1986, 39 : 195 199 10 Gnagey A L, Forte M, Rosenberry TL Isolation and characterization of acetylcholinesterase from Drosophila J Biol Chem, 1987, 262 : 1140 1145 11 Ralston J C, Rush R S, Doctor B P, Wolfe A D Acetylcholinesterase from fetal bovine serum, purification and characterization of soluble G4 12,,, (Wei Wan2li, Zhang Han, Qin Jun2chuan, Sun Man2ji Characteristics of recombinant human butyrylcholinesterase expressed in Bombyx mori expression sys2 tem Chin J Biochem Mol Biol),1999,15 (5) :797 801 13,,, (Luo Chun2yuan, Sun Man2ji, Yang Jin2sheng, Zhu Mei2cai In2 hibition of human brain acetylcholinesterase activity by nerve agents and reactivation by oximes 152 Chin Pharmacol Toxicol), 1994, 8 (2),151 14 Ashani Y, Shapira S Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poising in mice Biochem Pharmacol, 1991, 41 : 37 41 2001,, 2001 ( ),1942 7, 1964,, ; A B : (1),, DsbC DsbA,, (2), GroEL, (3), A B, 100, SCI, 1994-2006 China Academic Journal Electronic Publishing House All rights reserved http://wwwcnkinet