Volume 266, number 1,2, 123-127 FEBS 08520 June 1990

Complete amino acid sequence of fetal bovine serum and its comparison in various regions with other

B.P. Doctor, T.C. Chapman, C.E. Christner, C.D. Deal, D.M. De La Hoz, M.K. Gentry, R.A. Ogert, R.S. Rush, K.K. Smyth and A.D. Wolfe

Division of Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA

Received 26 February 1990; revised version received 2 April 1990

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS ACHE) is presented. This has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, -6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human ACHE, 60% with T. californica ACHE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Acetylcholinesterase; Amino acid sequence; Sequence homology; Serine esterase

1. INTRODUCTION Primary structures of the catalytic subunit of AChE isolated from Torpedo californica, Torpedo marmorata Fetal bovine serum (FBS) contains relatively abun- and Drosophila melanogaster, and butyrylcholinester- dant amounts (0.3 mg/liter) of true acetylcholinesterase ase (EC 3.1.1.8, BChE) isolated from human serum (EC 3.1.1.7; ACHE) and thus is a good source for ob- have recently been reported [6-11], as have partial se- taining large quantities of purified mammalian AChE quences of AChEs isolated from human erythrocytes with relative ease [1,2]. We have attempted in the past [12,13], human brain [14], and bovine brain [15]. Phar- four years to (a) develop a procedure to obtain large macological studies and enzyme kinetic studies with amounts of a mammalian AChE in purified form, (b) these , including multiple molecular forms, elucidate its primary structure, and (c) explore the have demonstrated that they possess similar catalytic possibility of using this enzyme as a pretreatment drug properties. The similarities and any differences in their for protection against highly toxic organophosphate catalytic functions should be reflected in the correspon- anti-ChEs [3,4]. For determination of chemical struc- ding similarities and differences in the structural do- ture and for demonstrating in vivo efficacy of AChE to mains of their catalytic subunit(s). The esteratic regions sequester organophosphate anti-ChEs before they reach of several cholinesterases share a high degree of amino physiological targets, large quantities of purified en- acid sequence homology [6], while some other regions zyme are required. Using procainamide Sepharose 4B are markedly dissimilar. gel as an affinity ligand, we have developed a simplified In this report we present the complete amino acid se- batch extraction procedure for purification of large quence of fetal bovine serum ACHE, the first mam- quantities (up to 100 mg) of purified FBS AChE [5]. malian AChE to be sequenced. We have also compared The use of AChE as an in vivo scavenger for the amino acid sequences of several regions in the organophosphate anti-ChEs requires that it be (a) im- catalytic subunit of FBS AChE with reported sequences munocompatible, (b) physiologically compatible with in the corresponding regions of other cholinesterases. the species of animal, (c) not degraded or cleared rapid- ly (long half life), and (d) readily available in sufficient quantities, since slightly more than one mole of enzyme 2. MATERIALS AND METHODS will be needed to sequester one mole of organo- Acetylcholinesterase from fetal bovine serum (FBS) was purified as phosphate. previously described [5]. The specific activity of purified enzyme was 400 U/nmol, and 1 mg of enzyme contained 14 nmol of protein. Cyanogen bromide-cleaved peptides of the catalytic subunit Correspondence address: B.P. Doctor, Division of Biochemistry, of FBS AChE (approximately 200 nmol) were obtained by labeling the Walter Reed Army Institute of Research, Washington, DC active site serine with [3H]diisopropylphosphofluoridate (DFP), 20307-5100, USA followed by denaturation with 7 M guanidine-HCl, reduction with

Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies 123 Volume 266, number 1,2 FEBS LETTERS June 1990 dithiothreitol, and alkylation with [14C]N-methylmaleimide to label tained by digesting the peptide with trypsin, separating the reduced cysteine residues. The sample was exhaustively dialyzed the fragments, and analyzing each of these tryptic pep- against 10 mM NH4HCO3, lyophilized, resuspended (10 mg/ml) in tides. The rest of the overlaps were determined by com- 80o70 formic acid, and digested with cyanogen bromide. The cleaved peptides were separated first on a Sephadex G-100 (superfine) column parison of the amino acid sequence of this AChE with (1.2 x 50 cm) in 1.0% HOAC, followed by HPLC on ttBondapack the Torpedo AChE sequence. C4 and/or C~s columns using TFA/acetonitrile or phosphate/ The FBS-AChE sequence shares an overall sequence acetonitrile solvent gradients. The amino acid sequences of purified identity of 90°70 with mouse AChE (personal com- peptides were determined using a gas phase peptide/protein sequencer (ABI model 477A). PTH derivatives were identified on-line with munication, P. Taylor) and 60°70 with T. californica HPLC (ABI model 120A). AChE [7]. In the small disulfide loop located between Trypsin-digested peptides of FBS-AChE were obtained by digesting amino acids 257 and 272, there are four additional denatured, reduced, and alkylated enzyme before lyophilization, as residues in the FBS and murine enzymes. FBS AChE described above, with 1070CPTK trypsin (w/w) in 50 mM NI-I4HCO3, pH 8.2, for 16-20 h. The peptides were separated on a Sephadex G-75 also has a relatively high degree of identity (52070) with column (1.2 × 50 cm) followed by HPLC on/zBondapack C4 and Cls butyrylcholinesterase from human serum [10] and columns using TFA/acetonitrile solvent gradients. human fetal tissue BChE [14], but somewhat less identi- The Torpedo californica AChE sequence is from Schumacher et al. ty (36%) with Drosophila AChE [9], rabbit and rat liver [7]. The Torpedo marmorata sequence used here for comparison was carboxyesterases [16] (33°7o and 35%, respectively), reported by Bon et al. [15]. The amino acid sequence of human serum BChE was obtained from published reports by Lockridge et al. [10]. Drosophila esterase-6 [17] (29%), bovine thyroglobulin The sequence of AChE from Drosophila melanogaster was done by [18] (32%), and rat lysophospholipase [19] (32070). Hall and Spierer [9]. The partial amino acid sequences of human The amino terminus sequences of several ChEs were AChEs shown here were determined by Soreq and Prody (human compared by Bon et al. [15]. Fig. 2 updates their data brain) [14l, Chhajlani et al. (human erythrocyte) [13], and Haas and Rosenberry (human erythrocyte) [12]. for the first 12 amino acids with the addition of the FBS AChE sequence for this region. The amino acid se- quences of multiple molecular forms of enzyme from the same source appear to be identical, although little 3. RESULTS AND DISCUSSION homology appears among the ChEs isolated from Fig. 1 describes the complete amino acid sequence of various other species. The sequences of AChEs from the catalytic subunit of FBS ACHE, which consists of the two species of Torpedo differ at position 5 583 amino acids. For comparison, the amino acid se- (His/Asp, FBS AChE numbering) and at 12 other loca- quence of Torpedo californica AChE is shown, as well tions in the molecule (not shown). In the sequences as the strategy of the sequence determination. Gapping compared here, T. marmorata differs from T. califor- is introduced in these sequences to maximize amino acid nica at positions 171,199, and 517 (M vs V, L vs I, I vs sequence homology: at amino acid 113 in Torpedo M, respectively, FBS AChE numbering system). AChE (to accommodate one extra amino acid in FBS Since multiple molecular forms of AChE isolated ACHE), at 258-261 in Torpedo (to accommodate four from various species possess similar catalytic proper- extra amino acids in FBS ACHE), at 495 in Torpedo (to ties, the active site serine-containing region (Ser-203) accommodate one extra amino acid in FBS), and at 575 might be expected to have structural similarities. This (to accommodate one additional amino acid in human was shown to be true by MacPhee-Quigley et al. [6], serum BChE, sequence not shown here). The calculated who compared the amino acid sequences of Torpedo molecular mass for the FBS enzyme is 64 238, and ther~ AChE with human serum BChE in this region, and are five apparent N-linked glycosylation sites (under. Soreq and Prody [14], who added human brain AChE lined in the figure). The amino acid sequence of the to the comparison. Similarities are now further revealed amino-terminus region was obtained by subjecting the with our elucidation of the amino acid sequence of intact protein to direct sequence analysis. The analysis mammalian AChE from fetal bovine serum, as shown of nearly all of the trypsin-digested peptides and the in Fig. 3C. There is complete identity among 21 amino partial analysis of CNBr-cleaved peptides were per- acids between bovine and human AChEs, and matching formed several times. Most of the overlaps could be ob- of 17 of 21 with human serum BChE, 16 of 21 with tained by these analyses. One CNBr fragment consisted Torpedo AChE and 12 of 21 with Drosophila AChE of 232 amino acids. The overlaps in this region were ob- The sequence PXXXTXFGESAGXXSV is common t,

Fig. 1. Amino acid sequence of FBS AChE and strategy of sequence determination. The amino acid sequence was determined by automated Edman degradation of (i) denatured, reduced, and alkylated intact enzyme (39 amino acid residues from amino terminus end), (ii) complete sequence of 4 CNBr-cleaved peptides (1,2,7, and 9), (iii) partial sequence of the remaining 5 CNBr-cleaved peptides, (iv) complete sequence of 44 tryptic pep- tides, and (v) partial sequence of one tryptic peptide (T14). CNBr peptide 6 was further digested with trypsin, the peptides were fractionated and sequenced (Ta-Ts). Cysteine residues were identified by ~4C label. The disulfide loops shown are for Torpedo ACHE. The active site serine (@) was identified by 3H-labeling. The asterisk over Asp-95 and the pound sign over His-447 denote residues which, along with Ser-203, may be revolved in charge relay. The presumed N-linked glycosylation sites in Torpedo and FBS AChE are underlined. Gaps have been introduced into the Torpedo sequence to accommodate additional amino acids in the FBS sequence; gapping has been introduced into both sequences at position 575 to accom- modate an extra residue in human serum BChE (not shown here) [10].

124 Volume 266, number 1,2 FEBS LETTERS June 1990

A

1 i0 20 30 40 50 60 ] 80 90 * I i00 ii0 120 Torpedo AChE DD HS E LLVNTKSGKVMGTRVPVL S S H I SAF LGI PFAEPPVGNM~RFRRP E P KKPW SGVWNAS TYPNNCQQYVD EQFPGF SG S EMWNPNREMS EDC Ly LN IWVP S pRp KS TT -VMVW I yG

FBS-AChE EGP E D P E L LVMVRGGE L RGLRLMAP R GPVSAF LGI P F AEP PVGPRRF LP P E P K RPWPGVLNATAFQSVCYQyVDTLY PGFEGT E MWNPNREL S EDCLY L NVWT P YPRP S S PT PVLVW I YG

Intact Protein EGP EDP E LLVMVRGGE LRG LRLMAP RGPVSAF LG I PFAE

I cilia-l-- I c:lBn-2 I I csax-3- I I c'mB]t-4 I EGPEDPELLVM APRGPVSAFLGIPFAEPPVGPRRFLPPEPKRPWPGVL ~ WNPNRELSEDCLYLNVWTPypRP S SPT VRGGELRGLRLM

IT1 I IT2--1131 IT4--1 ITS I ClTT--I IT8 I IT9 I IT10 I EGPEDPELLVMVR GLR GPVSAFLGIPFAEPPVGPR FLPPEPK ELSEDCLYLNVWTPYPR GGELR LMAPR RPWPGVLNATAFQ SVCY QYUDT LY PGF EGTEMWNPNR PSSPTPVLVWIYG B

121 130 140 150 160 170 180 190 200 @ 210 220 230 240 Torpedo AChE GGF Y S G S S T LDVYNGKY LAY T E EW LVS L S Y RVGAFGF LA L HG S Q E APGNVGL LDQRMALQWVHDN I Q FFGGDP K TVT I F GE SAGGASVGMH I L S P GSRD LFRRA I LQSGS P NCPWASV S

FBS-AChE G GF Y S GAS S L DVYDGRFLVQAEGTVLV SMNY RVGAFGFLA LP G S REAP GNVGL LDQRLALQ SVQENVAAF G GDP T SVTLFGE S AGAASVGMH LL S P P SRGLFHRAVLQSGAP N GPWATVG

c~Bn-4 I I cnm-5.-- I I cwsrt-6 I NYRVGAFGFLALPGSREAPGNVGLLDQRLALQSVQEN HLLS PP SRGLFHRAVLQSGAPNGPWATVG

T--'t0 I I'ell IIT12 I I'e13 IIT14 I ITlSl IT1( I FLVQAEGTVLVSMNYR EAPGNVGLLDQR GLFHR GGFYSGAS SLDVYDGR VGAFGFLALPGSR LALQSVQENVAAFGGD P T SVT LFGE SAGAASVGMH L ~ AVLQSGAPNGPWATVG

Trypsin digested peptides of CNBR-6 ~ ]TL I ITD--I ITo I HLLSPPSR AVLQSGAPNGPWATVG GLFHR C 241 250 1260 270 ] 280 290 300 310 320 330 340 350 360 Torpedo AChE VAEGRRRAVELGRNLNC .... NLNSDEELIH•LREKKPQ•LID•EWNVLPFDSIFRFSFvP•IDGEFFPTSLESMLNSGNFKKTQILLGvNKDEGSFFLLYGAPGFSKDSE•KIS•EDFM

FBS-AChE VGEARRRATLLARLVGCPPGGAGGNDTELVACLRARPAQDLVDHEWRVLPQEHVFRFsFVPVVDGDFLSDTPEALINAGDFVGLQVLVGVVKDEGSYFLVYGAPGFSKDNESLISRAQFL

Iemm-~ [ VGEARRRAT LLARLVGCP P ~ I

ITIGI JT19-11T20 IIT21 IIT22 IIT23 IIT24 IIT25--11T26 ATLLAR ARPAQDLVDHEWR FSFVpWDGDFLSDTPEALINAGDFVGLQVLVGWK DNESLISR VGEAR LVGCPPGGAGGNDTELVACLR VLPQEHVFR DEGSYFLVYGAPGFSK AQFL

Trypsin Digested Peptides of CNBR-6 ~ I ITa--I ITf--IITg IITh IITl IITJ IITk' 1IT1 IITI] VGEAR LVGCPPGGAGGNDTELVACLR VLPQEHVFR DEGSYFLVYGAPGFSK AQFL ATLLAR ARPAQDLVDHEWR FSFVPWDGDFLSDTPEALINAGDFVGLQVLVGVVK DNESLISR

D 361 370 380 390 400 [ 420 430 440 #450 460 470 480 Torpedo AChE SGVKLSVPHANDLGLDAVTLQYTDW~DNNGIKNRDGLDDIVGDHNVICPLMHFVNKYTKFGNGTYLYFFNHRASNLVWPEWMGVIHGYEIEFVFGLPLVKELNYTAEEEALSRRIMHY~

FBS-AChE AGVRVGVPQASDLAAEAVVLHYTDWLHPEDPARWREALSDwGDHNVVCPVAQLAGRLAAQGARVYAYIFEHRASTL•WPLWMGVPHGYEIEFIFGLPLEPSLNYTIEERTFAQRLMRYW

I cmut-i IlCamn-7 I1-1 GVPHGYEIEFIFGLPLEPSLNYTIEERTFAQRLM RYW i2111T2? 1281T29 IIT30--11T31 IIT32 IIT331341 AGVR WR LAAQGAR ASTLSWPLWMGVPHGYEIEFIFGLPLEPSLNYTIEER LMR VGVPQASDLAAEAWLHYTDWLHPEDPAR EALSDVVGDHNVVCPVAQLAGR VYAYIFEHR TFAQR YW

Trypsin Digested Peptides of CNBR-6 ~ l I TatlITa I I Tp IITq--IITr liT- I AGVR LAAQGAR ASTLSWPLW VGVPQASDLAAEAVVLHYTDWLHPEDPAR EALSDVVGDHNVVCPVAQLAGR VYAYIFEMR E

481 490 500 510 520 ] 540 550 570 580 Torpedo AChE ATFAKTGNPNEPHS-QESKWPLFTTKEQKFIDLNTEPMKV•QRLRVQMCVFWNQFLPKLLNATATID•AERQWKTEFHRWSSYMMHWKNQFDHY-SRHESCAEL

FBS-AChE ANFARTGDPNDPRAPKAPQWPPYTAGAQQYVSLNLRPLGVPQASRAQACAFWNRFLPKLLNATDTLDEAERQWKAEFHRWSSYMVHWKNQFDHY-SKQDRCSDL

ICZ~-| liCit an-9 I VHWKNQFDHY-SKQDRCSDL ANFARTGDPNDPRAPKApQWPPYTAGAQQYVSLNLRPLGV

IT351[T31--13?--IT38 IIT39--11T40 114111T42 14311T4411T45 IIT4i 14711T40 TGDPNDPR APQWPPYTAGAQQYVSLNLR AQACAFWNR LLNATDTLDEAER AEFHR NQFDHY-SK CSDL ANFAR APK PLGVPQASR FLPK QWK WSSYMVHWK QDR

125 Volume 266, number 1,2 FEBS LETTERS June 1990

isI ~6J FBS AChe" EGPEDPELLVMV A: FBS AChe° YRVGAFGFLAL Bovine brain ACheb m/~PEDPELLVMV Human brain ACheh YRVGAFGFLAL Human erythrocyte AChec E/BG~ED~ t. californica AChe° YRVGAFGFLAL T. marmorata AChea ~jSELLV~ Drosophila AChE ~ YRVGAFGFLmL ,. ca, ,orn,ca AChe" ~~LbV~ Human serum BuChE° YRVGAIGFLAL Drosophila AChe f Human serum BuChE Q ~~ ~)o leo Fig. 2. Comparison of the first 12 amino acids of the N-terminal se- B: FBS AChem NVGLLDQRLAL quence of the catalytic subunit of FBS AChE with cholinesterases Human brain ACheh NVGLLDORLAL from other species and sources. Numbering corresponds to the amino T. californica AChe° NVGLLDQ~AL acid sequence of FBS ACHE; non-matching amino acids are shaded; Drosophila AChe f NVGLIIDQIWLA| the identity of the first amino acids in (b) and (c) has not been resolv- Human serum BuChEe NfGL~DQmLAE ed. aFBS AChE sequence reported here; hsequence from bovine brain AChE obtained by Bon et al. [15]; %equence of human erythrocyte AChE from Haas and Rosenberry [12]; asequence determined by Bon et al. for T. marmorata AChE [15]; %equence reported for T. califor- C: FBS AChe" DPTSVTLFGESAGAASVGMHL nica by MacPhee-Quigley et al. [6] and Schumacher et al. [7]; fse- Human brain ACheh DPTSVTLFGESAGAASVGMHL quence for Drosophila AChE by Hall and Spierer [9]; and ~sequence t. californica AChe" DP~VT|FGESAG|ASVGMH| Drosophila AChe' IIPIIilRTLFGESAGIlUSVIIImiL of human serum BChE by Lockridge et al. [10l. Human serum BuChEQ IIPIISVTLFGESAGAASVlUJIHL all 5 cholinesterases. It should be pointed out that the folding pattern of this active site domain may be pivotal D: FBS AChe" ~VLQSGAPNGPWATVGVG' Human erythrocyte AChe ~ AVLQSGAPNGPWATVGII(3_ in conferring on these molecules the specificity of T. californics AChe" AJiLOSGIIPN|PWAIIV~ catalytic function. Using polyclonal and monoclonal Drosophila AChe f IlIIJIQSGmN|P~ Human serum SuChE~ ASLQSG~IIPWAWlIIIlt antibodies it has recently been shown that this region is located in a pocket-like conformation [20]. Soreq and Prody [14] have reported partial amino E: FBS AChe° ~RPAQDLVDHEWRVLPQEHV~ acid sequences, deduced from a cDNA clone of fetal Human erythrocyte AChe i |RPXO|LVXHEUlVLPQEUVF T. californica AChe° human brain ACHE, which represent 4 regions of the Drosophila AChe f molecule which are potential candidates for catalytic Human serum BuChE g F vlI INI sites. We have compared the amino acid sequence of FBS AChE with the sequences of five other ChEs in the F: FBS AChe• ~GVPQASDLAAEAVVLHYTDWLHPEDP same regions (Fig. 3A-C,G). Additionally, Soreq and Human erythrocyte AChe i VGVPO|SDLAAEAVVL|YTDWLXPXDP Prody have suggested residue combinations for the T. californica AChee MVP|A|DLDAVlL|YTDW~ Drosophila AChe~ ~iEIYTaW~i charge relay system [14]. Asp-170 (Fig. 3B, Asp-175 in Human serum BuChE o ~P~S~E~HYTDWHP the FBS AChE numbering scheme) is central to all their combinations. The sequence in this region is highly con- 4~ served, and there is complete homology between human G: FBS ACHE" ~RASTLSWPLWMGVPHGYEIEFIFG and bovine AChEs. However, based on results of the Human brain AChe" HRASTLSW--WMGVPHGYEIEF~FG Human erythrocyte AChe i XXTLIIIIPLXMGVPXGY binding of inhibitory monoclonal antibody AE-2 to r. californics AChe" HRASILIWPIWMGVIHGYEIEF|FG FBS ACHE, elucidation of its epitope, location, and the Drosophila AChef HR|S~ilI~W~WMGVIIHGalEIEIUlFG Human serum BuChE~ HRISLIWPIWMGV~GYEIEF,~FG sequence homology in the region surrounding Asp-95 [21], we feel that this amino acid, Asp-95, is a better candidate than Asp-170. Fig. 3G shows the amino acid H: FBS AChe" APQWPPYTAGAOQYVSLNLRPLGV sequence following His-432 and around His-447, both Human erythrocyte AChe i ~WPE_Y_YTAGAOOYVSLIILRPL~IV of which are conserved in all ChEs, and which are sug- T. ca]ifornica AChe"~P~ Drosophila AChE f gested by Soreq and Prody to be involved (His-423 and Human serum SuChE ° ~WP~QIIYILN~ His-438 in their numbering scheme). We believe that the higher degree of sequence homology surrounding Fig. 3. Comparisonof amino acid sequences in various regions of FBS His-447 makes this amino acid a more likely candidate AChE with other cholinesterases. Sources of sequences are as noted for involvement than His-432. Finally, the sequence for Fig. 2; amino acids are numbered according to FBS ACHE. (A) hsequence of human brain ACHE, Arg-147 region as determined by presented in Fig. 3A includes Arg-152, which is propos- Soreq and Prody [14]. (B) hAsp-170 region in [14]. (C) The gapping ed by Soreq and Prody as an alternate to histidine introduced in (h) between sites 6 and 7 by Soreq and Prody [14] to residues (Fig. 3G) in the charge relay system. Our pro- align this region with serine has been eliminated; the active posal is that since both His-447 and Asp-95 are located site serine is marked with an asterisk. (D) Sequence for human in disulfide loops [6] and since Asp-95 is located next to erythrocyte AChE (i) is reported by Chhajlani et al. [13]. (E,F) Xs denote unidentified amino acids; gapping is introduced into the Cys-96, which may affect its net charge, His-447 and Drosophila sequence for alignment in these regions. (G) hHis-423 and Asp-95 are the most likely candidates for involvement His-438 regions are merged with gapping between amino acids 439 with Ser-203 in the charge relay system. and 442 for alignment with FBS ACHE.

126 Volume 266, number 1,2 FEBS LETTERS June 1990

[2] Ralston, J.S., Rush, R.S., Doctor, B.P. and Wolfe, A.D. (1985) F~S ACHE" -I"VGCPPGGAGGNDTELVACLR J. Biol. Chem. 260, 4312-4318. Z. californica AChE~ BL-ICI .... DIEL~CLR [3] Wolfe, A.D., Rush, R.S., Doctor, B.P., Koplovitz, I. and Drosophila AChE --~IC~jRBIAmlI~TU-~.A. CIIR Jones, D. (1987) Fund. Appl. Toxicol. 9, 266-270. Human serum BuChE~ -L~GC~ .... INMTEIIICLR [4] Raveh, L., Ashani, Y., Levy, D., De La Hoz, D., Wolfe, A.D. and Doctor, B.P. (1989) Biochem. Pharmacol. 38, 529-534. Fig. 4. The comparison of an amino acid sequence of FBS AChE with [5] De La Hoz, D., Doctor, B.P., Ralston, J.S., Rush, R.S. and other cholinesterases in the region of a small disulfide loop (dashed Wolfe, A.D. (1986) Life Sci. 39, 195-199. line). [6] MacPhee-Quigley, K., Taylor, P. and Taylor, S. (1985) J. Biol. Chem. 260, 12183-12189. [7] Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPhee- Chhajlani et al. [13] reported the amino acid se- Quigley, K., Taylor, S.S., Friedmann, T. and Taylor, P. (1986) quences of 5 tryptic peptides of human erythrocyte Nature 319, 407-409. [8] Sikorav, J.L., Krejci, E. and Massoulie, J. (1987) EMBO J. 6, ACHE, shown in Fig. 3D-H. These authors observed 1865-1873. little or no homology when comparing the sequences [9] Hall, L.M.C. and Spierer, P. (1986) EMBO J. 5, 2949-2954. with Torpedo AChE and human serum BChE. [10] Lockridge, O., Bartels, C.F., Vaughn, T.A., Wong, C.K., However, when these sequences are compared to other Norton, S.E. and Johnson, L.L. (1987) J. Biol. Chem. 262, mammalian AChEs, approximately 88070 sequence 549-557. [11] Prody, C.A., Zevin-Sonkin, D., Gnatt, A., Goldberg, O. and identity is observed (85 of 97 amino acid residues). Soreq, H. (1987) Proc. Natl. Acad. Sci. USA 84, 3555-3559. Finally, the comparison of amino acid sequences of [12] Haas, R. and Rosenberry, T. (1985) Anal. Biochem. 148, FBS AChE with T. californica and Drosophila AChEs 154-162. and human serum BChE in the region of a small [13] Chhajlani, V., Derr, D., Earles, B., Schmell, E. and August, T. (1989) FEBS Lett. 247,279-282. disulfide loop (amino acids 254-275, Fig. 4) shows that [14] Soreq, H. and Prody, C.A. (1989) in." Progress in Clinical and FBS AChE has four extra amino acids in the loop. The Biological Research, Vol. 289 (Rein, R. and Golombek, A. eds.) presence of these additional amino acids in the loop in Liss, New York, pp. 347-359. mammalian AChEs would result in a different confor- [15] Bon, S., Chang, J.-Y. and Strosberg, A.D. (1986) FEBS Lett. mation in this region. 209, 206-212. [16] Long, R.M., Satoh, H., Martin, B.M., Kimura, S., Gonzalez, Among the sequences compared here, there is 90% F.J. and Pohl, L.R. (1988) Biochem. Biophys. Res. Commun. amino acid sequence homology between FBS AChE 156, 866-873. and human AChEs, 60°7o with T. californica ACHE, [17] Oakeshott, J.G., Collet, C., Phillis, R.W., Nielsen, K.M., 39% with Drosophila ACHE, and 50°70 with human Russell, R.J., Chambers, G.K., Ross, V. and Richmond, R.C. (1987) Proc. Natl. Acad. Sci. USA 84, 3359-3363. serum BChE, values equivalent to those for the [18] Mercken, L., Simons, M.-J., Swillens, S., Massaer, M. and homologies of the catalytic subunits. In general we con- Vassart, G. (1985) Nature 316, 647-651. clude that, not unexpectedly, mammalian AChEs ap- [19] Han, J.H., Stratowa, C. and Rutter, W.J. (1987) Biochemistry pear to have structures more in common with each 26, 1617-1625. other than with Torpedo AChE or human serum BChE. [20] Ogert, R.A., Gentry, M.K., Richardson, E.C., Deal, C.D., Abramson, S.N., Alving, C.R., Taylor, P. and Doctor, B.P. (1990) J. Neurol. Chem., in press. [21] Doctor, B.P., Smyth, K.K., Gentry, M.K., Ashani, Y., REFERENCES Christner, C.E., De La Hoz, D.M., Ogert, R.A. and Smith, S.W. (1989) in: Progress in Clinical and Biological Research, [1] Chubb, I.W. and Smith, A.D. (1975) Proc. R. Soc. Lond. B. Vol. 289 (Rein, R. and Golombek, A. eds) pp. 305-316 Liss, Biol. Sci. 191,263-269. New York.

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