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The complete amino acid sequence of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of streptomyces albus G

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The Complete Amino Acid Sequence of the Zn^^-Containing

D-Alanyl-D-Alanine-Cleaving Carhoxypeptidase of Streptomyces albus G

Bernard .lORIS. Jo/cl" VAN BEEUMEN. Eabiaiiii CASAGRANDE. Charles GERDAY. .lean-Marie FRERE, and Jean-Marie GHUYSEN Sen'icc dc MK-vohiologic. Inslilut dc Chlmic. Dnivcrsile dc Liege au Siirt Tilman, Liege;

Laboratoruini voor Microbiologic en miLTohielc Genelica. Rijksuniversiieil-Getil. Gcnl; and Scrviee dc Biochimie musculaire. Instiiui dc Chitiiic, Universiic de Liege au Sari Tilman, Liege

(Received July 30/Sepieniber 2S. hJS:) - EJB 5847

The 22016-Af, Zn"^-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces ahuls G effectively catalyses the transfer of the A^^A^'-diacetyl-L-lysyl-D-alanyl electrophilic group o^ the standard tri-peptide substrme A^',A''-diacetyl-L-lysyl-D-alanyi-D-alanine to water. It also performs a weak /i-lactamase activity, hydrolysing penicilhn into penieilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyelization of the dipeptide Asn-Giy into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the i'-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[''^C]-acetamide. and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-ff^-3.6-endoxo-J'^-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn^"^-containing enzymes (earboxypeptidase A, thermolysin. carbonic an-hydrase B and alcohol dehydrogenase) and several completely or partially sequeneed. serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn^"^/serine-containing ^-lactamases.

The D-aianyl-D-alanine-cleaving peptidases (in short DD-peptidases) are enzymes involved in the bacterial wall pep-tidoglycan metabolism. They catalyse transfer of LAla-DGlu-(LRaa-DAla) (where DGIU is a /-glutamyl residue and Raa is a diamino acid or an amino acid grouping possessing a free o a m i n o group) from LAIa-DGIu(LRaa-DA!a-DAla) pen-tapeptide units either to water (carboxypeptidation) or to the oj-amino group at the L-Raa position of another peptide (transpeptidation). On the basis that the sequenee iRaa-DAla-DAIa is that part of the carbonyl donor mainly involved in substrate activity, the tripeptide A^'.A^'-diacetyl-L-lysyl-D-alanyl-D-alanine (Ac^LLys-DAla-DAla) has been used as a substrate analogue to isolate several DD-peptidases exhibiting, with varying efficiencies, carboxypeptidase activity (Aei-LLys-DAIa-DAla + H2O -^ DAla + AczLLys-DAla) and/or transpeptidase activity (Ae2LLys-DAla-DAla + a suitable amino compound NH2-X -» DAIa + Ac2LLys-DAla-C0NH-X) (for a recent review, see [1]). The R61 (from Streptomyces

Abbreviations. Cya, cysleic acid; CmCys, S-carboxymethyl-cysieine;

PeCys, S-[2-(4'-pyridyl)ethyl]cysteine; SCM-protein, protein treated with iodoaeetic acid giving rise to CmCys residues; SPE-protein. proiein Ireaied with 4-vinyl-pyridine giving rise to PeCys residues; DABITC, 4-.V.A^-diinethylaminoazobcnzene-4'-isolhiocyanate; PITC. phenyliso-thiocyanaie; dansyl and Dns. 5-dimethylaminonaphthalene-l-sulfony); Nbs2. 5.5'-dithio-bis(2-nitrobenzoic acid); ETPA. exo-m-3,6-endoxo-/l*-teirahydrophthalic anhydride; EtSH, 2-mercaptoethanol; Hse. homo-serine; MetO^. methionine sulfone; HPLC, high-performance liquid chromalography; SDS. sodium dodecyl sulfaie.

Enzvmcs. D-Alanyl-D-alanine-cleaving carboxypepiidase or

DD-pep-lidase (EC 3.4.17.8): Armi/laria mellea protease (EC 3.4.99.32); carboxy-peptidase Y (EC 3.4.16.1); chymotrypsin (EC 3.4.21.1); pepsin (EC 3.4.23.1); Staphylococcus attrcus V^ protease (EC 3.4.21.19); thcrmo)ysin (EC 3.4.24.4); trypsin (EC 3.4.21.4).

R61) and R39 (from Actinomadura R39) DD-peptidases are highly penicillin-sensitive, bifunctional carboxypeptidases/ transpeptidases. They operate by covalent catalysis via an active serine residue. In contrast, the G (from Streptomvces

alhus G) DD-peptidase is a highly penicillin-resistant,

mono-functional carboxypeptidase. It operates by liganding cata-lysis via a Zn^^ cofactor which is firmly bound to the apo-protein (A'A: 2 X 1 0 ' ' ' M " ' ) [2]. On the basis of X-ray crystallographic studies [3]. this G Zn^ "^ DD-carboxypeptidase is a two-domain protein, A cleft, with the Zn^"^ eofactor located in it. extends throughout the large domain and serves as binding site for the two enzyme competitive inhibitors, the dipeptide AcDAIa-DGlu and the /i-iactam compound p-iodo-7-^-phenyIacetylaminocephalosporanic aeid. The interpreta-tion of the 2.5-A (0.25-nm) electron density map of the G Zn^"^ DD-carboxypeptidase has been carried out simultane-ously with the establishment of its primary structure. This paper is the first report of the complete amino acid sequence of a member of this special class of bacterial DD-peptidases that characterize themselves by their ability specifically to attack peptide bonds extending between two D centres.

MATERIALS AND METHODS

Preparation of the DD-Peptidase (Native Protein)

The DD'peptidase was isolated and purified to protein homogeneity as described in [4].

i and Alkylated Protein

After unfolding the protein in 6 M guanidinium chloride and cleavage of the S-S bridges with dithiothreitol, alkylation

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ol" the s n groups in the raiuLxM protein \v;ts carried out hy trciitnicnl willi ioiloiicctic iicid. gi\ ing rise to .S'-carhoxy-iiietlul cvsicine ((. niC'ys) residues (SC'M-protein). |?I or 4-vin>l-pyi'idinc\ giving ri.se lo \-12-(4'-pyridyl)clIiyl]cysIcinc (PeC\s) residues (SPI-prolein) jd]. The elTieaey oleaeh olthe treatments \\;is cheekeci b\ annno aeid analysis lo ileleet eystie aeid (Cya) after further perforniie ;ieid oxidation [7] of the redueed and alkylated protein,

Protcidvlu lji:vnics

Viypsin (treated with L-l-tosylamldo-2-phenylethylchIoro-methsl ketone) and ehymotrypsin were from Worthington (Freehold. N,I. USA). Carhoxypeptidase Y and pepsin were from Boehringei" (Mannheim. FRG). Thcrmolysin was from Calbiochem (San Diego. CA. USA) and Siophylcicoccusaureus protease V8 from Miles (Slough. England). Annillaria mcllca protease was a gift from Dr V. Barkholt Pedersen (Copen-hagen. Denmark).

Chcniical.s

Anahtieal grade reagents were used. Cyanogen bromide and 4-vinyl-pyridine were from Aldrieh-Europe (Beerse, Belgium). Mereaptoethanesuifonic aeid and anhydrous hydra-^ine were from Pierce Chemicals Co. (Rockford. IL. USA). Fluoreseamine was from Fluka (Buchs. Switzerland), 5.5'-dithio-bis(2-nitrobenzoic acid) (Nbsi) from Sigma (St Louis, MO. USA), and iodo['-^C]acetamide (50 Ci/mol) from New England Nueiear (Dreieieh. FRG). Exo-t/.v-3,6-endoxo-^'*-tetrahydrophtalic anhydride (ETPA) was synthesized as de-scribed by Riley et al. [8]. All the other chemicals were from Merck (Darmstadt. FRG).

Thin-Layer Sheets. Chromalography and Eleetrophoresis Papers

Polyamide-coated thin-layer sheets (F1700) were either from Pierce Chemicals Co. or from Sehleicher & Schiill {Dassel. FRG). Chromatography and eiectrophoresis papers were from Whatman (Maidstone, Kent, UK).

dithiothrcitol added to the chlorobutane to a final concen-tration of O.OOl";, was from Calbiochem. The acetonitrile used in the liPLC analysis of the phenyihydantoins was Lichrosolv grade from Merck. The HPLC water was from Alltech Assoc. (IL, USA).

Aiiiiiu) Acid Analysis

Protein samples (40 nmo!) were hydrolyzed under vacuum in 150 Ml 0^ 6 M HCl at I I O X for 24 h, 48 h and 72 h and analysed with a Beckman 120B analyser, Peptide samples (5 —30 nmol) were hydrolyzed for 24 h under the same conditions and analysed with a Beckman Multiehrom 4255 or a Dionex D300. Cysteine and methionine were determined us Cya and methionine sulfone (MetO2) after performic oxidation of the protein [7]. Tryptophan was estimated after hydrolysis with 3 M mercaptoethane sulfonie aeid for 96 h at n o C [9].

Determinaiion of the N-Terminal Residues

Dansyl chloride was used according to the procedure recommended for the proteins by Gray [10]. For peptide fragments, the determination of the N-terminal residues was carried out by the method of Hartley [U].

Determination ofthe C-Terminal Residues

Samples (1 mg) of SCM-protein and of peptide CB3 (obtained by cyanogen bromide cleavage ofthe protein) were treated with 0.5 ml anhydrous hydrazine for 24 h at 80T. Eaeh hydrazinolysate was dried under vacuum over concen-trated sulphuric acid, and the residue was dissolved in 0.5 ml H2O. The solution was adjusted to pH 3 with 6 M HCl and filtered in water on a column (0.9 x 11 cn:i) of Amberlite IRC50{H^ form). The first 15 ml of the eluent were collected and, after freeze-drying, the residue was submitted to an ad-ditional chromatography under the same eonditions as above (in order to remove all the hydrazides). The free amino acid residue released by hydrazinolysis was then identified with the amino acid autoanalyser.

Solvents and Reagents

for Manual Amino Acid Sequencing

l-DimethyIaminonaphtaiene-5-sulphonyl (dansyl) ehloride was from BDH (Poole. England). Phenylisothiocyanate (PITC). 4-A'..V-dimethyIaminoazobenzene-4'-isothiocyanate (DABITC) and trifluoroacetic acid were from Pierce Chemi-cals Co. Pyridine (analytical grade from Merck) was purified by two successive distillations, first over ninhydrin (20 g/i) and then over KOH pellets under nitrogen. Butyl acetate was distilled over ninhydrin (lOg/1). Heptane and ethyl acetate were spectroseopic grade reagents from Merck.

Solvetils and Reagenis

for Automatic Liquid Atnino Aeid Sequencing

and High-Pcrformatice Liquid Chromatography I HPLC) The solvents and reagents used in the sequenator were 'sequena! grade" and were not purified further. The compounds 1 M quadrol. «-propanoI (used to dilute the quadro! to a 0.3 M concentration), benzene, ethyl acetate and chlorobutane were from Merck, llexafluorobutyric acid was from Pieree and trifiuoroacetic acid was from Rathburn (Scotland). The

Sequence Determination

The SCM-protein (100 nmol) and large-size peptides (100 nmoi) were submitted to automatic liquid-phase Edman degradation [12] in a Socosi JIO sequenator (Saint-Maure. France), using 0.3 M quadrol as coupling buffer and 2 mg of polybrene [13] as earner. The thiazolinones were converted manually using 20"„ trifluoroacetie acid [14] for 25 min at 55 C. The phenylthiohydantoins were analysed by 'reversed-phase^ HPLC on a column (0.46x30 cm) of C18 HL RSil (RSL. Eke, Belgium) using either an isochratic [15] or a gradient [16] elution method with acetonitrile as the organic solvent.

Small-size peptides (5 — 50 nmol) were sequenced manually, following the micro dansyl-Edman [17] or the DABITO PITC [18] method. In some cases where the presence of amides or tryptophan residues was expected and when the initial degradation with the DABITC/PITC method resulted in a severe wash-out of the residual peptides, a combination of the two methods was used. In those eases, the first N-ter-minal residues were cleaved by the dansyl-Edman method without removing samples for dansylation and the following degradation steps were carried out by the DABITC/PlTC

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method. All the peptides were sequenced in I-ml conie vials closed with a teMon septum.

ofthe SH Grottps

Quantitative estimation was carried out spectropholo-metrically as described in [1^)). using NBs: as reagent. Either sodium dodecylsult\itc (SDS) (4",,; w/v) or a mixture o r 6 M guanidinium chloride and 2 mM EDTA were used as denaturing agents. Glutathione served as control.

Estimation of Sugars and S-Glyceryl-cysteine Thioethcr in the Native Ptoicin

The possible occurrence of a carbohydrate moiety in the protein was tested by using the method of Dubois [20] and that of 5-glyceryl-cysteine was tested as described in [21]. except that the quantities and volumes were divided by 10.

Cyanogen Bromide Cleavage of the SCM-Protein

A solution ofthe SCM-protein (10 mg/ml) made in 70",,

formic acid was supplemented with cyanogen bromide (40 mg ml. final concentration), left in the dark for 24 h at room temperature under nitrogen and finally freeze-dried.

Purification ofthe Cvatwgen Bromide Peptide Fragments CB2, CB3. CB4, CB5 and CB6

Two procedures were used (see flow sheet in Fig. I). In a first experiment and as shown in Eig. 1 M (M refers to the miniprint section), the soluble peptides originating from 5 |imol of SCM-protein (and separated from an insoluble

core) were fractionated by filtration on a column (2.5 x 150 cm) of Sephadex G-50, fine, in 0.2 M NH4HCO3 bufTer pH 8.6 (flow rate 35 ml/h; volume of the fractions 5.3 ml; manual detection at 2K0 and 214 nm), Fraction CBl was the un-cleaved protein. Peptide CB2 (115 residues) and peptide CB3 (58 residues) were obtained in pure form. Peptides CB5 and CB6 (5 and 4 residues, respectively) were recovered in the salt volume and further isolated from each other by paper eiectro-phoresis at pH 3.5. In a second experiment and as shown in Fig. 2 M. all the peptide fragments originating from 0.8 (jmol of SCM-protein were solubilized in 50 mM ammonium formate buffer pH 3.5 supplemented with 2 M guanidinium chloride. Filtration of the solution on a column (1.25 x 150 cm) of Sephadex G-50 fine in 50 mM ammonium formate buffer pH 3.5 (fiow rate 15 ml/h; volume of the fractions 1.2 ml; detection at 230 nm) resulted in the isolation of an additional peptide CB4 (29 residues) which coeluted with peptide CB3. Complete separation of CB3 and CB4 from each other was achieved by paper eiectrophoresis at pH 6.5 (pyridine/glacial acetic acid/water; 100/4/900; v/v/v).

Etizymatic Cleavage of the Cyanogen Brotnide Peptide Fragments CB2., CB3 and CB4, and Isolation

of the Digested Peptides

CB2, CB3 and CB4 were digested as indicated in the flow sheet (Fig. 1) and under the conditions given in Table 1. The digested samples were freeze-dried. The large peptides (> 30 residues) were first filtered on Sephadex using, depending on the samples, columns of varying sizes ( l x 100 cm, or I X 150 cm). Sephadex G-25 fine or Sephadex G-50 fine, and 5 % formic acid or 50 mM ammonium formate buffer, pH 3.5.

NATIVE DQ-PEPTIDASE Unfolding and reduction

lodoacetic acid treatment SCM-PROTEIN CNBr cleavage 125 mg Sephadex G50 (pH8.6) 20 mg Sephadex G50 (pH 3S 1 Trypsm cleavage Methionine (abeUmg with iodo C'^"] ace t am ide

CB2 CBS CB6 CB2 Ion exchange [procedure A) CB3 CBi CB5 CB6 Radioaclive

fracttons Non radioactivefractions

Paper eiectrophoresis [on exchange

H65 5 Methionine Paper regeneration eiectrophoresis (pH3S) Bio-Gel P6 (procedure B) o radioactive fractions 4-vinyl-pyridtne treatment SPE-PROTEIN Lysine modification With ETPA

1

Trypsm cleavage

I

Amrno group regeneration

I Bio-Gel P6

1

Paper eiectrophoresis and chromatography Methtonme regeneration CB3 CB/. CB5 CB6 Sephadex G25 Subdigestion with I I I T T. C. C, TH. AM. SA. Bio-Gel P2 Paper electro phoresis

Fig. 1. Fragmentation oj the G Zn^'' DD-peptidase, Flow sheet, (a) T = trypsin; C = ehymotrypsin; AM = .4rmillaria tnellca protease; TH = thermolysin; SA — S'/ap/iy/ococcw.v aureus protease

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I .)b)i- ) I nrvimtlii diiicsttott ol dte pepttdes ohtuitted alter ('NBr ol the SC.\f-protein ( ; i | 0 . 2 M . u n n i o n i u n i ;iLvt.ilc p l U . S : ( b ) 0 . 1 M ,V-L>thyI-i n c a c c u ,V-L>thyI-i t c p l l S I ) , ( c l 0 , 1 M a m m u n ,V-L>thyI-i u m b ,V-L>thyI-i c m b o n ; ,V-L>thyI-i ,V-L>thyI-i e p l ! 7 . S ^ t i i M 1 n i A rioicasc Vr>psin C'li\niotr\psin Therniohsin •S itui\u.s protease .^. tuellcu protease IVplKlc concen-tration lui: ml 10 10 10 1.3 lo:..^ 7 Protease' pep! idc (u w) 1 411 ! 40 1:100 I 50 I 3000 Huller a a b c b

Duialion ol' ilie incubation ;il 37 C (unless otherwise staled) h • > 1 or 2 1 (50 C) 4 or 16 b.5

as eluants (fiow rate 20 ml h; size ofthe fractions 1.5 ml). Detection was made with a Uvicord 11 at 280 nm (in the presence o( formic aeid) or manually at 230 nm (in the pres-ence of ammonium formate). In all cases, samples of the collected fractions u'ere spotted along the same base line (0.5 cm for each fraction) on a Whatman 3MM paper and submitted to eleetrophoresis at pH 6.5. Detection of the peptides was made by successive staining with fluoreseamine (O.OOl "^ in dry acetone) [22], ninhydrin/acetie acid/acetone (0.25 1 99; w/v/v) [23] and Pauiy's reagent as described in [24]. On the basis ofthe maps thus obtained, the relevant fractions were pooled and then purified by preparative paper eiectro-phoresis at the same pH 6.5. Checking the purity of the peptides thus obtained and. when necessary, further purifi-cation of the peptides were carried out by paper eiectro-phoresis at pH 3.5 (pyridine/glacial acetic acid/water; 1/10/89; V V V) and finally, by descending paper chromatography in butane-I-ol acetic acid./pyridine/water (15/3/10/12; v/v/v/v). Purity tests also involved N-terminal group and amino acid composition determinations.

For small peptides ( < 30 residues), the same techniques as above were used except that the gel filtration was omitted.

Trvpsin Cleavage of the SCM-protein. Subsequent Radioactive Labelling of the Methionine Residues and Idetitifieation of the Methionine-Containifjg Peptide Fragments

The SCM-protein (0.8 ^moi) was treated with trypsin and. in a second step, the methionine residues were labelled with iodo['*C]acetamide as described in [25]. Two techniques were then used (see flow sheet in Fig. 1).

Procedure A. The mixture of radioactive and

non-radio-active peptides was chromatographed on a column (0.8x24.5 cm) of Technicon chromatobeads P (a cationie exchanger) using a pH gradient from pH 2.4 (50 mM pyridine/ acetic acid) to pH 5 (2 M pyridine/acetic acid) (flow rate 16.3 ml/h; volume ofthe fractions 3.6 ml). Samples (0.1 ml) of each of the fractions were analysed with respect to their radioactivity and ability to react with fluorescamine after alkaline hydrolysis [26]. The radioactive fractions were freeze-dried. treated with 2-mercaptoethanoI (EtSH) as de-scribed in [25] to regenerate the methionine residues, and re-chromatographed under the same conditions as above.

Procedure B. Filtration of Ihe mixture of radioactive and

nonradiouctive peptides on a column (I.15x 140cm) of Bio-Gcl P6(20() mesh) in 50 mM ammonium formate buflfer, pH 3.5 (flow rale 9.8ml/h; volume of the fractions 1.1 ml) yielded two radioactive fractions. Bach of these fractions was freeze-dried, treated with r-tSfi and, depending on the size ofthe peplidcs to be purilied. liltered in 50 mM ammonium bi-carbonate bufl'er pH 8 either on Sephadex G-25 fine (I X 140 cm; 10 nil/h; volume of the fractions 0.8 ml) or on Bio-Gel P2 (100-200 mesh; 1 x 110 cm; 10 ml/h; volume of the fractions 0.2 ml). In ail cases, the peptides were purified by paper eiectrophoresis and/or paper chromatography as described above.

Limited Tryptic Digestion

of the SPE'Protein after Blocking the Lysine Residues

The following procedure was used (see flow sheet in Fig. I). The SPF-protein (36 mg in 3.6 mi 0. J5 M sodium te-traborate bufTer pH 8.5) was treated with three suecessive additions of 6.5 mg of ETPA as described in [8]. The solution was dialysed at 4 C against 50 mM NH4HC0^ buffer pH 8.0 and after freeze-drying. the residue was submitted to tryptic digestion as described in [4]. Subsequently, the amino groups were generated by dissolving the digested material in 2 ml

10"n acetic acid and gently stirring the solution at room tem-perature for 16 h. The solution was then filtered on Bio-Gel P6 in 50 mM ammonium formate buffer pH 3.5 and the peptides were purified by paper eiectrophoresis and paper chromatog-raphy as described above.

Pepsin Cleavage of the Native Protein

The native protein (5.7 mg in 0.3 ml 10 mM Tris/HCl pH 8.3 containing 2 mM MgCb) was dialysed against 5% formic acid (v/v) and then supplemented with 1 °o (w/w) pepsin). The solution (0.5 mi) was incubated for 8 h at 37 C and then freeze-dried.

Carhoxypeptidase Y Cleavage of Peptides

The selected peptide (5 — 50 nmol), an equimolar amount of norleucine, and carboxypeptidase Y (protease/pep-tide: 1/30; w/w) were incubated together at 37 'C in 15-50 ^1 of 50 mM pyridine/acetic acid buffer pH 5.5. Samples were removed after increasing times and the reaction was stopped by addition of 0.2 M sodium citrate buffer pH 2.2.

Peptide Nomenclature

Capital letters refer to the methods used for the cleavage of the protein into peptide fragments. CB - cyanogen bromide; T — trypsin; TL — trypsin digestion limited at the Arg sites (after blocking the amino groups of lysine); TMI and TMS ^ trypsin digestion followed by radioactive labelling of the methionine residues and purification by either ion ex-change (TMI) or molecular seiving (TMS); C = ehymotryp-sin; TH = thermolyehymotryp-sin; SA = S. aureus protease; AM = A.

mellea protease; P = pepsin. Arabic numerals refer to the

fractions (in the order of increasing elulion volumes) collected by column chromatography and containing the relevant peptides. Small letters refer to further purification steps carried out on paper: letters in normal print = paper eiectro-phoresis at pH6.5; letters underlined = paper eiectropho-resis at pH 3.5; letters followed by an asterisk =

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descend-ing po per chromatogruphy. In each case, letters a, b. c. etc. indicate the relative position (from anode to cathode clurinji eiectrophoresis or on the basis of increasing Ri values during chromatography) of ihe relevant peptide when compared to the other peptides present in the same preparation. Tims for example, peptido CB2Tleeb* was a pcplidc gcneraled by cyanogen bromide cleavage of the SCM-protein and recov-ered in fraction 2 after column chromatography. Following further cleavage with trypsin, the peptide was recovered in iVaction I after column chromatography. Finally, it was purified by paper eiectrophoresis at pH 6.5. foUowecJ by paper eiectrophoresis at pH 3.5, and paper chioniatography. The peptide was in position e during the first and the second pteparation methods, and in position b during the third one.

Allocation of the Disulfide Bridges

The diagonal technique of Brown and Hartley [27] was applied to the pepsin digest of the native protein. After the first eiectrophoresis at pH 6.5 and subsequent treatment with performic acid, the second eiectrophoresis. carried out in a direction perpendicular to the first one, gave rise to three groups of peptides located outside the diagonal.

Amide Assignment

The amide groups were assigned both by identifying the phenylthiohydantoin and 4-iV,Af-dimethylaminoa2obenzene-thiohydantoin derivatives and on the basis of the electro-phoretic mobilities ofthe peptides at pH 6.5 [28].

Search for Sequence Homology and Repetitive Fragments

The search for homology between different sequences or repetitive fragments in the same sequence was carried out by comparing all possible spans of n (3 to 25) contiguous amino acids [29]. The amino acids were compared on the basis of a structural test which made use of the relative frequencies of substitution found in different families of homologous pro-teins [30].

RFSULTS

The amino acid sequence of the DD-peptidase is shown in Fig. 2. It was established as described in the ensuing and miniprint sections.

Intact Protein and Cyanogen Bromide Fragments

Table 2 presents the amino acid composition of the pro-tein as found after acid hydrolysis and as deduced from the primary structure. The value of 22076 thus obtained for the

Mr was 20"^ larger than that determined by polyacrylamide

gel eiectrophoresis in the presence of SDS. Asp (or Asn) was at the N terminus and lie at the C terminus of the protein, as shown by dansylation and hydrazinolysis experiments, res-pectively. Nbs2 (EUman's reagent) failed to detect any free SH group (even after denaturation of the protein). Neither S-glyceryl-cysteine thioether (as found in the Braun's lipo-protein of Escherichia eoli [21]), nor sugar residues were detected. Fragmentation of the SCM-protein with cyanogen bromide yielded five fragments designated, in the order of decreasing sizes. CB2, CB3, CB4, CB5 and CB6 (see Materials and Methods, and Table 3).

Table 2. Amino m-id rontpo.sition ol the jicrltirmie-aeid-oxidizedprotrtn (lie (Irst tlii'cc c-okimiis jiive Ihc iuiiounls ul' ;imino acid icsiducs us ob-Uiiiic'd iirici" Jiicrcjsing pcridds oC hydrolysis wilh 6 M IIC! iil I (0 C. T r p wiisdcicrmincd on a separate sample or the native prolein after hydrolysis Inr ')(S h al 1 t o C wilh 3 M mcrcaplocthane sulfonic acid

Amino acid Lys His Arg Cya Asp Thr Ser Glu Pro Gly Ala Val MetO2 lie Leu Tvr Phe Trp Amount 24 h nmoi/mg 0.233 0.32;^ 0.459 0.261 1.074 0.618 0.682 0.728 0.360 t.487 1.427 0.459 0.166 0.336 0.519 0.237 0.385 4.82 after 48 h 0.242 0.362 0.462 0.265 LtOO 0.619 0.683 0.743 0.392 1.485 1.462 0.466 0.175 0.336 0.530 0,239 0.392 72 h 0.256 0.341 0.471 0.233 1.063 0.619 0.677 0.713 0.417 1.520 1.417 0.462 0.152 0.336 0.543 0,278 0.40f^ mean 0.244 0.344 0.464 0.253 1.079 0.619 0.681 0.728 0.389 1.497 1.435 0.462 0.164 0,336 0.531 0.251 0.395

Number o\r residues as calculated from hydrolysis 5.07 7.14 9.64 5.25 22.41 12.85 14.14 t 5 . ] 2 f^.O8 31.09 29.80 9.59 3.41 6.99 11.03 5.21 8.20 4.82 sequence 5 7 10 6 22 13 15 15 8 31 29 10 4 7 11 7 8 4

Table 3. Amino aeid eomposition of the peptides produced hy CNBr

fragtvetitation ofthe SC M-protein

Number of residues as obtained after acid hydrolysis with 6 M HCl for 24h at 110"'C. The figures in parentheses give the relevant numbers as obtained by sequencing Residue Lys His Arg CmCys Asp Thr Ser Glu Pro Gly Ala Val lie Leu Tyr Phe Trp Hse Total Amount in CB2 3.25 5.31 2 29 (3) (5) (3) 12.98 (12) 8.41 6,86 10.92 3.98 (9) (8) 01) (4) 14.16(14) 16.53 5.31 3.39 6.42 4.28 3.91 •1 0.99 115 (17) (6) (4) (7) (5) (4) (2) (1) fragmen' CB3 4.49 t.95 2.31 5.90 2.11 2.93 2.69 3.20 10.98 1 7.56 1,30 t.68 2.93 1.71 3.05 58 t (5) (2) (2) (5) (2) (3) (3) (3) (12) (9) (!) (2) (3) (2) (3) (I) CB4 0,94(1) 0.68 (i) 1.69 (2) 0.19(1) 5.10(5) 1.74(2) 3.60 (4) 0.93 (1) 4,90 (5) 1.23(1) 2.83(3) 0.84(1) 0.90 (U 0.54(1) 29 CB5 0.87 0.97 0.99 1.17 7 0.56 6 (1) (1) (1) (1) (1) (1) CB6 1.10 0.91 1.32 0.95 4 (1) (I) (i) (1)

The CB2 Peptide Fragment (115 Residues)

Automatic sequencing revealed that the first six residues were Asn-Gly-Xaa-Tyr-Thr-Trp. The yield of phenylthio-hydantoins was very low (2.5%) although, as controlled with whale sperm myoglobin. the sequenator was in perfect

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ope-10

15

20

25

ASN-GLY-CYS-TYR-THR-TRP-SER~GLY-THR-LEU-SER-GLU-GLY-SER-SER-GLY-GLU-AU-VAL-ARG-GLN-LEU-GLN-ILE-ARG-Uao

-©7 -6-7 -&r -e-7 - e r -frr -Ov -QT -Q-r -Q-7 -6-7 ~&7

I CB?Tlbca='

CB2

C B 2 T 2 f c " -Q-r -6-, |— CB2C2a —j CB2Clh CB2Clfe CB2Cln CB2AM2b 0 / 0/ TMr3c TL3a 5 1 - - - 5 5 - - - - 6 0 - - - - 6 5 - - - - 7 0 - " " ' 7 5 GLN-ARG-PHE-GLN-SER-ALA-TYR-6LY-LEU-ALA-ALA-ASP-GLY-1LE-ALA-GLY-PR0-THR-PHE-ASN-LYS-ILE-TYR-GLN-LEU-C B 2 T l i I -e-7 -e-? CB2T3b CB2C3n-|— CB2C3d I — C B 2 C 3 i c " — I

CB2

C B 2 T l e e b " C B 2 C l f b C62AM2d 1— C82Tlb£b" — = ^ ^ ^y" = 7 I— CB2Tlb^b== — CB2C21 —j CB2Clb CB2AH2a CB2AH3 CB2AH2aSAa — TMI4c TLI T M I 5 C TL5 THI3£ T L l T c T L 4 a c

Fig. 2. Amino aeid .sequence ofthe G Zn' * DD-earhoxypeptidaxe. The CNBr peptide fragments ofthe SCM-protein are represented by heavy lines between hydrolysis and sequencing gave rise lo identical or at least consistent data. The melhods used for sequencing are represented by the following symbols'. . : carboxypeptidase Y

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2 6 • • - 3 0 - - - - 3 5 - - - - i O • - - - i 5 • ' " ' 5 0 VAL-ALA-GLY-TYR-PRO-GLY-THR-GLY-ALA-GLN-LEU-ALA-ILE-ASP-GLY-GLN-PHE-GLY-PRO-ALA-THR-LYS-ALA-ALA'VAL--6-7 -&» -©* -*T> -e-? - e ^ -0-7

CB2

C B 2 C l n C B 2 T l e e a " CB2C2f I CB2C3e — CB2TM ~v7 "G 7 I — CB2T2g C B 2 C l q c - ' 7 = 5 ' CB2C1S

h

-97 -&y CB2AM2d — CB2AM3 — TLI TLlTa TLlTc 76 - - • 80 - - - - 85 - • - - 90 - - - - 95 - - - - 100

GLN-ASP-ASP-ASP-CYS-THR-PRO'VAL-ASN-PHE-THR-TYR-ALA-GLU-LEU-ASN-ARG-CYS-ASN-SER-ASP-TRP-SER-GLY-GLY-3 -J

CB2

C B 2 T I b c b - ' C B 2 n b d b -• 0 / ~O 7 ~O~7 0 / ~©T 0 / 0 7 © / I CB2T2CC U / O ^ O ./ O y CB2C4e|- CB2C2C \ C B 2 C l j a = CB2C3b CB2AM2a CB2AM2aSAa 0 / 0 7 O ^ ~O 7 "O 7 O / r- CB2AM2aSAb 0 7 —©7 Ov "O 7 Pb — Peb

triangles (T • ) . The other full lines (| i) refer to all the other peptides analysed. In all cases, the amino aeid composition as delermined by acid — : manual dansyl-Edman degradation; - O ^ : DABITC/PITC melhod;=^; automatic method (phenylthiohydantoins): *•—: hydrazinolysis;

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101 - - - 105 110 115 120

125

LYS-VAL-SER-ALA-ALA-THR-ALA-ARG-ALA-ASN-ALA-LEU-VAL-THR-MET-TRP-LYS-LEU-GLN-ALA-MET-ARG-HIS-ALA-MET-O / 0 ' "LYS-VAL-SER-ALA-ALA-THR-ALA-ARG-ALA-ASN-ALA-LEU-VAL-THR-MET-TRP-LYS-LEU-GLN-ALA-MET-ARG-HIS-ALA-MET-O / 0 / 01 0 7

CB2

CB5

«-

CB6

•»

C B 2 T l h b ' ' C B 2 C l j a - - \ - C B 2 C l t a " — | C B 2 C l q b - 1 C B 2 C l r g -C62AM2e Peb I TMS2,2c • T M S 2 , 2 d TH 110 , 1 151 - - - 155 160 165 170 - 175

SER-ARG-HIS-MET-TYR-GLY-H1S-ALA-ALA-ASP-LEU-GLY-ALA-GLY-SER-GLN-6LY-PHE-CYS-ALA-LEU-ALA-GLN-ALA-ALA-U

210

- CBA J-

CB3

— CB^Cd C B 3 T l h C B 3 T l d T H S l , 2 c I | - CB3T2e CB3TH4ed" 1- CB3TH3ba-CB3TH2e - | - C B 3 T H 4 e f " - | CB3TH21 CB3SAla CB3SA3a CB3SA2ea = CB3SA2d Fig.2e-h CB3SA2eb" TL3gd

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126 - - - 130 - - • - 135 - - " - UO - - - - U5 - - - - 150

GLY-ASP-LYS-PRO-ILE-THR-VAL-ASN-GLY-GLY-PHE-ARG-SER-VAL-THR-CYS-ASN-SER-ASN-VAL-GLY'GLY-ALA-SER-ASN-93 J

-©-7

CB4Cb Cfl4Cd

-e-? -&7 -©-7 - e v -e-^ -&7 - e ? - & T -e-? TMSl,2c Pea TL2fb-= 176 - - - 180 - - - - 185 - - - - 190 - - - - 195 - - - - 200

ARG'ASN-HIS-GLY-PHE-THR-GLU-lLE-LEU-GLY-PRO-GLY-TYR-PRO-GLY-HIS-ASN-ASP-HlS-THR-HiS-VAL-ALA-GLY-GLY-CB 3

CB3Tlh I C B 3 T l i |- CB3T1J — | _ C B 3 T l j C h _ j C B 3 T l j C f j — C 8 3 T l j C d — — CB3TH21 —I CB3TH1C f— CB3TH3cf ~ —O ^ ©T* O / - O 7 O / • 0 7 O / I CB3TH5b I CB3THle

-e-7 -&7 -&7 -©7 -©-7 -9-7 -©7 -©-7-©-7 -&7 "07 -©"7

C B 3 S A l a 1 ' CB35Albe-=

CB3SA2ea"

TMI8b

TL2g

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ASP-GLY-ARG-PHE-TRP-SER-ALA-PRO-SER-CYS-GLY-ILE 169

CB3

C B 3 T l i CB3T1J C 0 3 T ^ a C B 3 T 2 b CB3TI jCd — I CB3TH3c£ — ] CB3TH3ad= Pc CB3SA2ab-TMI8b MZg -f-TM14b TL4b

trypsin treatment with a yield of 7.4%. indicating some chymotryptic activity of the (rypsin (despite treatment to remove it). Note that the same bond was also cleaved during tryplic digestion of the SCM-protein (yield 11.4%) and during limited Iryptic digestion of the SEP-protein (yield up to 24%).

The CB3 Peptide (5H Residues)

Automatic sequencing of CB3 gave the first 22 residues with a good yield. CB3 was exhaustively digested by trypsin (2 nmol peptide). thermolysin (1 ^mol) and the S. aureus protease (1 jimol), respectively, and the peptides listed in Tables 6 - 8 M were isolated and sequeneed. Peptide CB3T1J (177-203) was digested by chymotrypsin (Table 9M) per-mitting elucidation of the compiete sequence of CB2.

The following aspecific cleavage sites in CB3 were observed. Trypsin cleaved the His'"-Ala'^^ bond with a yield of 2.4% [generating peptide CB3T2e (155-157)] and the bond Ala'^'^-Leu'^' with a yield of 6.2% [generating peptide CB3Tld (155-170)]. The 5". aureus protease used seemed to be contaminated by a thermolysin-like protease and gave rise to several parasite peptides (Table 8 M). Finally, dan-sylation of peptide CB3SAIbe*, followed by 6 M HCl hydrolysis for 8 h, yielded both Dns-Ile and a parasite com-pound probably Dns-Ile-Leu, migrating close to Dns-Phe. Dns-Ile alone was obtained after prolonged (72-h) hydrolysis.

TL2h

ration conditions. This suggested a partial cyelization of the dipeptide Asn-Gly in the form of the anhydro-aspartylglycine imide [31].

CB2 was exhaustively digested by trypsin, chymotrypsin

and Armillaria mellea protease, using 2, 2 and 1.2 |imol of CB2. respectively. The tryptic and chymotryptic digests on the one hand and the A, mellea digest on the other were fractio-nated by filtration on Sephadex G-25 and Sephadex G-50, respectively. The tryptic digest resulted in three fractions (CB2T1 —3), the chymotryptic digest in five fractions (CB2C1 —5), and the A, mellea digest in three fractions (CB2AM1—3). Further purification of each of these fractions by paper electrophoresis and paper chromatography permitted isolation, final purification and sequencing of the peptide fragments listed in Tables 1 - 3 M . Onthe basis of their amino acid composition, these peptides covered the whole sequence of the CB2 peptide fragment.

Two overlaps at positions 85 — 86 and 90 — 91 were elucidated thanks to the peptide CB2AM2a (71-100). The

Staphvlococeus aureus protease cleaved this peptide (75 nmol)

into two fragments (Table 4M). The N-termina! fragment was digested with carboxypeptidase Y (Table 5M) permitting the ordering of the chymotryptic peptides CB2C2c (88 — 90) and CB2C4e (86-87). In turn, the C-terminal portion was sequeneed, yielding a good overlap between the chymotryptic peptides CB2C2C (88-90) and CB2C3fa (91-97). At this stage, two peptide regions were weakly established. One in-volved the residues 69 — 71 and the other the residues 37 — 53. This latter region included the overlap between CB2C2f (37-42) and CB2Clqc* (43-5)). These ambiguities were elucidated by sequencing TMl3g (58-71) and TLl (26-52), respectively.

The CB4 Peptide Fragment (29 Residues)

Manual sequencing of CB4 gave the first 14 residues. The complete sequence was elucidated after subdigestion of CB4 with chymotrypsin (Table lOM) and by purifying TMS1.2c (138 —152), a tryptic peptide of the SCM-protein (see further).

The CB5 and CB6 Fragments (6 and 4 Residues, Respectively)

Sequencing of both these peptides was carried out manu-ally.

Assembly of the CB2. CB3, CB4, CB5 and CB6 Peptide fragments

CB2 was the only CNBr fragment which possessed an N-terminal Asn. It thus represented the N-terminal portion of the protein. In turn. CB3 was the only fragment from which He was released hy hydrazinoiysis. It thus represented the C-terminal portion of the protein. The correct aligrunent of the other cyanogen bromide fragments rested upon the following three series of experiments.

The limited tryptic digestion of the SEP-protein yielded several interesting peptide fragments (Table l l M ) . In parti-cular, peptide TL2fb* (123 — 137) provided the necessary overlap between CB6 and CB4 and peptide TL3gd (153-157) which resulted from a side cleavage of trypsin after His'-\ provided the necessary overlap between CB4 and CB3. The presence of the bond Lys'*''-Ala"*'^ (which is normally sensitive to trypsin) in peptide TLl (26-52) demonstrated the effi-ciency of the procedures used initially to block the amino groups of the iysine residues. Peptide TLl was subsequently digested by trypsin and the two expected fragments were isolated (Table 12M).

The exhaustive tryptic digestion of the SCM-protein followed by radioactive labelling of the methionine residues yielded, after ion-exchange chromatography (procedure A),

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throe viKiioactivc fviicvions (TMU). TMllO and ) (Fig.3M.ul and nine uonriidioiiclive fraclions (TMII -TMIS and T M l l l l (Fig.3M,b and Table I3M). Aflcr re-generation o\' the melhionitie residues and an additional ion-exchange chromatography. peptide TMIIO yielded peplidc TMIKll (IIS-122) (Fig.4M and Table 13M) whieh pro-vided the necessary overlap between CBS and CB6,

Finally, fraetionation of lhe same radioaetively labelled digest as iibove on Bio-Gel P6 (procedure B) followed by rcgeneriUioti o\' the methionine residues and filtration on suitable Sephadex columns yielded several peptides. Those possessing a His (on the basis of Pauly's test) or a Ser. Ala or Leu ill the N terminus were further investigated. No His-conialning peplide could be purified but three peptides TMS1.2C (138-152). TMS2.2c (109-117) and TMS2,2d (118- 122) wore obtained in pure fortn (Table 14M). Peplide TMS2.2d (1 IS— 122) contained one methionine residue. Pep-tide TMSI.2C (L^8- 152) was a tryptie pepPep-tide of CB4 and was helpful in completing the sequence of CB4. Peptide TMS2.2c provided the necessary overlap between CB2 and CB5.

Allocation of the Disulfide Bridges

The technique of diagonal paper eleetrophoresis at pH 6.5 allowed several peptides to be detected (Fig.5M). After performic oxidation, two acidic peptides became more acidic (peptides Pa and Pb). two neutral peptides became aeidie (peptides Pc and Pd) and two basic peptides became neutral. These latter peptides. Pea and Peb, were further separated by paper electrophoresis at pH 3.5. The analyses of all the peptides are shown in Table 15M. The couple Pea and Peb represented the sequences 134—150 and 91 — 106. re-spectively, thus demonstrating the occurrence of a S^^^-S^"*' disulfide bridge. The couple Pd-Pc represented the sequences

168 — 174 and 204 — 211. respectively, thus demonstrating the occurrence of a S'^'^-S"'^ disulfide bridge. The peptides Pa and Pb. however, differed from eaeh other only by the presence of an additional residue in Pb; they represented the sequences 78 —84 and 78 — 85. respectively. The procedure thus provided only one of the two peptide partner possibly involved in a third disulfide bridge. However, the fact that the protein (either native or denatured by guanidinium chloride or SDS) had no detectable SH group, carbohydrate moiety nor S-glycerol-eysteine thioether left little doubt on the occurrence of a third disulfide bridge extending between Cys-' and Cys^°. This conclusion was confirmed by crystallographic data [3].

Search for Sequence Homology

No homology was found by comparing the DD-peptidase with (a) the Zn^^-containing enzymes carboxypeptidase A [31], thermolysin [32]. carbonic anhydrase B [33] and alcohol dehydrogenase [34]; (b) the partially sequeneed serine DD-ear-boxypeptidase of Bacillus suhtilis and Bacillus

stearothermo-philus [35]: (c) the Zn^^ /?-lactamase 11 of Bacillus cereus

(Ambler; personal communication); and (d) other serine i5-lactamases [36,37]. Using the same computer program, internal homology in the DD-peptidase was found only in a few peptide regions containing at the most 10 amino acid residues (Table 4).

DISCUSSION

There is an excellent agreement between the amino acid composition of the DD-peptidase shown in Table 2 and the

T;ihlc 4. Inti-rtial .\C(/itcnce identities In the t>i)-/ycptidase

5 7 - 6 6 . 155-164

I l l s P'^-J'^ -Aki-CilyLeu Cjly

2H-.Ti. 1S7-19() Gly-Tyr-Pro-Giy (w 8. 96-98 Trp-Ser-Gly

52-56.203-207 Arg-Phe-^'"-Ser-Ala Trp 93-95, 141-143 Cys-Asn-Ser

amino acid sequence proposed in Fig. 2. However, a few peptide bonds were only proved weakly. The Arg^'^^-Phe"'^* bond was established on the basis of peptide CB3SAlbe* (182-212) only and the Met'^*-Tyr'^^ bond on the basis of peptide TL3gd (153 — 157) resulting from a tryptic side re-action of poor yield (3.5%). Deamidation and cyelization of Asn in the Asn'-Gly" sequence probably explains the diffi-culties encountered in attempts to sequence both the SCM-protein (C. Duez, personal communication) and the CNBr peptide fragment CB2. Reopening of the cyclic imide with generation of a mixture of a and /?-aspartyl peptides [38] probably explains why Asp, instead of Asn, has been located at the N terminus of the peptides CB2C3a, CB2T1bra* and TMI3c. Usually, a and ^-aspartyl peptides can be separated by electrophoresis at pH 3.5 [39] and peptide CB2C3a was indeed found to be heterogenous under these conditions.

Among the 212 residues whieh eonstitute the DD-peptidase. 14 are aeidie (4 Giu and 10 Asp) and 15 are basic (5 Lys and 10 Arg), providing a ratio of uncharged residues to charged residues of 7.31. The protein possesses 11 segments, each containing from 8 up to 15 uncharged residues in sequence. These segments which altogether comprise 113 residues, are as follows (the hydrophobicity index calculated according to Segrest [40] is given into parenthesis): 1 — 11 (1.2). 26 — 38 (1.5), 53-61 (1.6), 6 3 - 7 0 (1.4), 8 0 - 8 8 (L6), 1 0 9 - U6 (2.1), 129-136(1.7), 138-151 (0.1), 161-175(1.0), 183-192(1.6) and 204 — 212 (2.0). Only two fragments have a high hydro-phobic character and one of them occurs at the C terminus of the protein. The His and Met residues fall exclusively in the C-terminal half of the sequence. The cyanogen peptide frag-ment CB3 (155 — 212) contains five His residues out of the seven found in the complete polypeptide chain. Out of the 15 Glx present, 11 occur as Gin.

The DD-peptidase shows very few internal repetitive sequences and no homology is found by comparing the se-quence of Fig. 2 with those of other mechanistically and/or functionally related enzyraes. In agreement with this latter conclusion, the interpretation of the 2.5-A (0.25-nm) electron density map of the DD-peptidase has revealed a secondary and tertiary structure not found in any other protein [3]. Con-sidering that a full characterization must await final fitting of the protein molecule in the Riehard's box and crystallo-graphic refinements, the structure proposed on the basis of these X-ray studies [3] is in very good agreement with the amino acid sequence shown in Fig. 2. The protein consists of an N-terminal domain (1-76) and a C-tertninal domain (81 —212) which are connected via a single peptide sequence Leu"'^-Gln-Asp-Asp-Asp-Cys^^ (itself involved in a disulfide bridge with Cys^). The catalytic cavity is located in the large domain. Using the present numbering, important residues are ^'-\ His"* and His^'^^ (Zn-"^ ligands) and, probably,

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(prolon donor?) and Arg' carhoxytaic siihsUatc',').

(charge pairing with the

1 lie w o i k in 1 iCfic w . i s s u p p o i l c i ! in p;irt hy llic A'OMI/V tie la Recherche Siienntit/ii<' .Mcdtcah; H i i i s s e l s ( y n i i i l 1,4>(II.7V). (IK- I V I g u m <it>vc(niiiCTil

( l(/i.'/i eoneertcc 7^) 84-11) iind the i'.S. Natitmtl Institulcs ot Health. Ik-thcsdii i g r a m 2 ROl LV1(i4-05) .1 V, H, is indchlcd lo ihc Natiotud Fothh voor ]\chen.seltappetiik Otulcrzoek. lirusscls (Aic(/(V <((«) Navarser,\ 5 : s-AM.(-^)8).Tlii;skiirLil ;issisiancc ol" Mr O. Klein (Licgc; purHicLHion of ihc nn-pcplid;isc). Mr ,1. V;in Oamnic (Gcnl; phcnyUhiohydanloin aiKihscs) and Mr S. Collin (LiOgc; amino acid analyses) is greatly ap-preciated. We thank Prolessor ti. Hamoir (Lahoraloirc de Biochimie nniscnUnre, tjogc) and Professor J. Dc Ley (Lahoralorium voor Miero-hrologic, Gent) for ilicir liospi(atit\' and iiUercsl, This pii(icr is from a diNscriaiion lo he snbmiued by li, J. in parlinl l\illilnicnl of lhe ixquirenicnls for a degree of Dr en sciences etiintiques al ihc University o\'

. Belgium,

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LS. Van Bceiimcn. .1.. Van D a m m e . J., T c m p s l . P. & Dc Ley. J. (1980) in Melhods in Peplide atid Protein Sequence Analysis (Birr. C , ed.) pp. 503-506, Elsevier/Norlh-Holland. Amsterdam.

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IK, Wiumann-Liebold. B. & Lchmann. A. (1980) In Method,^ in Peptide

and Protein Sei/tienee Anatysis (Birr, C . ed.) pp. 49-72. Elscvier/

Norlh-Holland. Amslerdam.

19. Habccb. A. F. S. A. (1972) Mettiods Enzymot, 25. 457-464. 20. Dubois, A. S. (1956) Anal. Chem. 28. 350.

21. Hantke. K. & Braun. V. (1973) Eur. J, Biochem, 34, 284-296.

22. Vandekerekhove, J. & Van Montagu, M. (1974) Eur. J. Biochem. 44,

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23. Toennies. G. & Kolb. J. J. (\'-)i\) Anal. Chem. 23, 823-826. 24. Mann,T, & Leone. E, (1953) Biochem, J. 53, 140-148. 25. Degen. J, & Kyte. J. (1978) Anal. Bioehem. 89, 529-539. 26. Lai. Y. (1977) Methods Enzymat. 47, 236-243.

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31. Titani. K.. Ericsson. L. H.. Walsh. K, A. & Neurath. H. (1975)

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32. Tilani, K.. Hermodson. M. A.. Ericsson, L. H., Walsh. K. A. & Neurath. H. (1972) Nature (Lond.) 238, 35-37.

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34. Jornvall, H. (1970) Eur. J. Biochem. 16, 25-40.

35. Waxman, D. J.. Yocum, R. R. & Strominger. J, L. (1980) Phil

Traits. R. Soe. Lond. B2S9. 257-275.

36. Ambler, R. P. (1980) Phd. Trans. R. Soc. Lond. B289, 321-331. 37. Jaurin. B. & Grundslrom, M. T, (1981) Proe. Natl Acad. Sci. USA.

7A\ 4897-4901.

38. Borslein. P. & Balian. G. (1977) Methods Enzymol. 47. 132- 145. 39. Ambler. R. ?. {\915) Biochem. J. 151. 197-218.

40. Segrest. J. P. & Feldmann. R. J. (1974) J. Mol. Biol. 87, 853-858.

B. Joris, J. M, Frere. and J. M. Ghuysen. Laboratoire de Microbioiogie, Faculte de Medecinc de I'L/niversite de Liege. Instilut de Chimic Organique et de Biochimie, Universile de Liege au Sart-Tilman.

4000 Liege, Belgium

F. Casagrande and C, Gerday,

Laboratoire de Biochimie Muscuiaire, Institut de Chimie Organique et de Biochimie, Universite de Liege au Sart-Titman. B-4000 Liege. Belgium

J. Van Beeumen. Laboratorium voor Microbioiogie en Microbiele Genetica, FacuUeit der Wetenschappen, Rijksuniverslteit Gent, K.. L. Ledeganckstraat. 35, B-9000 Gent. Belgium

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H i n l p r i n t s e c t i o n t o c o m p l e t e » m M i o g e l d s e q u e n c e o f t h e Zn - c o t i t a I n l n o O a l n n y l O -I l l ' ^ « c U ^ v ^ b t l d o f Stfiptomyoeo J 1 > - U » G B . J o r t i , d. V a n B e e u m e n , F , C a s a g r a n d e . Cf>. G o r d . i y . J , M , F n > r c a n d J .M . G h u y s e n 11 0,1. cai CBS CB6

t'i.j. -IM, FraationatJon of peptidea TMIIO (Fig, SM) after i-e cf th-: mathioninc reeidueB ty treatment wii/i EtSH,

F a r c o n d i t i o n s , i e e F i g . 3 M .

200

fi[7. IV, separation of th« peptidD fragments prcduoed by CNBr aleavage

o,"" tH,' PCV-pr^'tiPi'n (S wncl ' . T h e p e p t i d e s s o l u b l e i n 0 . : H N H ^ I ^ C O J b u f f e r p H 8 . 6 w e r e f i l -t e r e d o n 1 2 . S ' I S O c m c o l u m n o f S e p h a d e n G - 5 0 f i n e i n t h e s a m e b i c a r b o n a t e b u f f e r . F l o w r a t e : 3 5 m l / h . V o l u m e o f t h e f r « c t i o n s : 5 , 3 m l . SO 100 ISO Fraction

Separation o / the peptide fragments pc<ydii.Jed by CSBr c Leauage

cf the SCM-protein (O.S vmoli .

A l l t h e p e p t t d e s w e r e s o l u b i l i z e d i n 50 mM a m i r o n i u m f o r m a t e b u f f e r pH 1 .S s u p p l e m e n t e d w i t h ? H g u a m d i n i v i m t h l O T i a e a n d t h e r e s u l t i n g s o l t j t n o n was f i l t e r e d on a 1 . ^ 5 • 1 5 0 cm c o l j m n o f S e p h a d e x G - 5 0 f i n e i n t h e same ammonium f o r m a t e b u f f e r t w i l t i o u t g u a r i d i r i i um c n l o r i t l e ) . F l o w r a t e : 1 5 r n l / h , 'Volum e o f t h e f r a c t i o n s : I .Z m l . 150 200 Fracl.on Fractionation of the tCijftic dirtee

chroma tog rap hy on ahrcriatohi^ada P. A f t e r t r y p t i c d i g e s t i o n , t h e m e t h i w i t h i o d o l ' " C l a c e t a m i d e . I h e sampl a c i d , was a p p l i e d t o a 0 . 8 • 2 4 . S a g a i n s t b u f f e r fl ( 5 0 mM p y r i d i r i u m was c a r r i e d o u t s u c c e s s i v e l y w i t h Of a l i n e a r g r a d i e n t o b t a i n e d w i t h 1 5 0 ml o f b u f f e r B ( 5 0 mM p y r i d i r i o f a l i n e a r g r a d i e n t o b t a i n e d w i t h 1 5 0 m l o f l i u f f e r C ( Z M p y r i d i n m r n n a l l y , b u f f e r C a l o n e . The f r a c t i o s c i n t i l l a t i o n ( g r a p h a ) a n d b y a U b ) . F l o w r a t e : 1 G . 3 m l / h ; v o l u m e the SCM-protein by onine e , d i s cm col aceta 75 m 150 m um ace 160 m a c e t a ns wer a l i n e of th residues were solved i n 30 I umn p r e e q u i l i b te pli ?,1) . El l o f b u f f e r fl; l of b u f f e r fl t a t e pH 3 . 7 5 ) : l of l i u f f e r 6 te pH 5.0) ; an e analysed hy f l u o r e s c a m i n e e f r a c t i o n s ; l a b e l l e d a c e t i c r a t e d u t i o n 3 0 0 ml and 3 0 0 ml a n d d . f i -l i q u i d ( g r a p h J . 4 ml P e ^ (-) (A)

e

o Pdo Pc^'

e

P a ^

Fig. iM, Allo':at'i..-'i of the disul.fije tridijce in the natnii? pj-ctcir,. T h e p r o t e i n w a s d i g e s t e d b y p e p s i n a n d t h e d i g e s t s u b m i t t e d t o a n a l y t i c p a p e r e l e c t r o p h o r e s i s a t p H 6 . 5 i n d i r e c t i o n A . A f t e r p e r f o r m i c o x i d a t i o n , t h e p a p e r s t r i p w a s s u b m i t t e d t o a s e c o n d p a p e r e l e c t r o p h o r e s i s a t t h e s a m e p H i n d i r e c t i o n E T h e p e p t i d e s w e r e d e t e c t e d b y f 1 u o r e s c a m i n e . 1.0 (1) T a b l e I H . O a t a o n t r y p t i c p e p t i d e s o f C B 2 . F i g u r e s i n p a r e n t h e s e s w e r e o b t a i n e d b y s e q u e n c i n g , NU ; n o t d e t e r m i n e d ; i : I o n - e x c h a n g e c h r o t n a t o -g r a p h y ; G i O : f i l t r a t i o n o n S e p h a d e x G - 5 0 ; G ? 5 : f i l t r a t i o n o n S e p h a d e x G - E 5 - , P 6 -, f i l t r a t i o n o n B i o - G e l f 6 - . P4 ; f \ U r a t i o n a n B i o - G e l P I ; P2 : f i l t r a t i o n o n B i o - G e l P 2 ; 6 . 5 : e l e c t r o p h o r e s i s a t p H 6 , 5 ; 3 . 5 : e l e c t r o p h o r e s i s a t p H 3 . 5 ; C ; d e s c e n d i n g p a p e r c h r o m a t o g r s p h y ; 6 . 5 0 ; d i a g o n a l e l e c t r o p h o r e s i s i p H 6 . S ) a f t e r p e r f o r m i c o i i d a t i o n . Ami no a c i d Lys His Arg CmCyj Asp Thr Ser GIu Pro Gly Ala Va1 H e Leu Phe Trp Hse M o b i l i t y (pH 6 . 5 ) N - t e r m i na1 Y i e l d P o s i t i o n Number of r e s i d u e s Procedure of i s o -l a t i o n 1.0 0 , 3 1 . 5 2 . 1 3 , 9 2 . 0 4 . 1 1.2 1.0 1.3 0.7 ND [ 1

u

(2 (4 1 {1 (1 ( I [1 1 ,0 0 . 4 1.6 2 . 1 2 , 6 0 . 3 1.2 1,0 0 . 7 2 . 0 1 , 1 1,0 ND 1 1) M ^) 3) 1) 1) I ) 1) 2) 1 ) 1 .0 O.S 4 . 7 2 . 0 3 , 0 0 . 5 1 .2 1 . 1 0 . 8 2 . 3 1.3 1 ,1 NO (2 (3 ( 1 a

a

( 1 ( 2 (1 1.2 1 ,8 2 . 1 2 . 4 "1.5 4 . 4 0 . 9 1.0 1.1 0 . 5 1 . 1 ND ( ' • ) (Z) { 2 ) ( ^ ) ( 5 ) ( 1 ) ( 1 ) d l (11 1 . 6 1.0 0 . 7 1.2 0 . 5 2 , 9 4 . 2 0 . 8 0 , 9 1 , 3 NO ( 2 ' ( 1 ) (tl ( 3 ) ( ' ' : ( 1 ) (1 ( 1 - 0 . 3 7 A s p 9 . 3 t 1 2 5 ; 6 . 5 ; 3 , 5 ; C - 0 , 3 7 0 . 0 0 0 , 0 0 l i e 10.;! t 7 2 - 9 2 6 72 H e ,3 1 - 92 Val U . 9 2tj -% 17 Phe 17 . 1 53 ^ % 71 G 2 5 ; ! 3 . 5 3 . 5 ; C G 2 5 - . 6 . 5 ; 3 , 5 ; C 19 G 2 6 ; 6 . 5 3 . 5 ; C

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u:

Asp 1 hr Ser GIu Pro G1 y fl 1,1 U P Leu Tyr Phe Trp Hse M o b t l i t y (pH 6 . S ) S - t e r m i n e l r i e l d Posi t i o n Number oT res idues Procedure of i s o -l a t i o n 0.5 ( ! ) 1.0 ( n 1.0 ( l i 0.9 ( 1 ) ND • O.i 3 Val 1 0 . 7 I 1 0 2 - 1 0 8 7 C ' ' ' 1.0 1 .0 0 . 4 ?.3 o . a 1 .0 ND 0.9 * 0. flla : . i 109-7 c'" (11 ll' (1) ( 1 ) i3 I 115 6 . 5 ; O.H 1.3 1 .9 1 .1 NO • 0. Ala 5.1 48. 5 G25; O.g {[ ( 1 ) (1 (? ( I 4fl I 52 6. li, i M 1.9 (2 1.7 a 2 . 5 ( 7 ND ( 1 - 0.Z4 CinCys 6.1 i 93-10 9 0.(1 1 .1 I.i ;'.9 1.1 ND + 0 . Val 3.4 102 5 ; G 2 5 ; I H ) 'M 46 t •108 7 6 , 5 ; o c i d Lys H i s A r g CmCys Asp Thr Ser filu Pro G\y A 1.1 Val l i e Leu Tyr Phe Trp Hse M o b i l l t j / (pH 6 . 5 } K - t e r m i n a l Y i e l d Posi t i o n Number of r e s i d u e s Procedure of i s o -l a t i o n 0.9 ( 1 ) 0.9 (1 0.9 (1 0.3 2.3 ( 2 ) ND •> 0 . 4 5 Thr 7.7 I 106-110 5 G 2 5 ; 6 . 5 ; I 0 0 0 D 1 t 2 1 t .0 .1 .1 . 4 . 9 . 0 .3 .8 .0 ND I ) 1)

11

l j 3 0 . 4 5 Gly 10.0 G c % 4 3 - 5 1 9 2 5 ; f , 5 ; 0.4 1 .1 1 .0 1.0 NO 0.4 * 0. flla 2.9 111-5 G.5; ( 1 ) (1 h ( 1 ( 1 ) 5? t US 6 . 5 ; i . O ( 1 ) 0.4 0.3 1.2 ( 1 ) 1.0 ( 1 I 1.1 U) ? . 9 ( 3 ) 0.2 MD * 0 . 5 ? Gly 1 0 . 2 I 4 3 - 4 9 7 G 2 5 ; 6 . 5 1.1 1 .3 0.3 1 .4 NO t f l . Ala Z.3 1 0 7 -4 C (1) (1) (?) 60 110 6 . 5 ; 1 . 0 ( 1 ) 1.0 ( 1 ) 0 . 1 i . O ( 1 ) NO - 0 . 5 5 A U 1 4 . 2 X 8 8 ^ 9 0 3 G25-.6 .5 T a b l e I H A m m o a c i d Lys His Arg CmCys Asp Thr Ser GIu Pro Gly Val H e Leu Tyr Phe Trp Hse M o b i l i t y (pH 6 . 5 ) N - t e r m i n a l Y i e l d Posi t i o n Number of r e s i d u e s Procedure of i s o -l a t i o n ( c o n t i n u e d b ) CB2T2fC" 0.8 ( 1 ) Z.2 ( 2 ) 0.7 ( 1 ) D.7 I D ND + 0.16 Gin 3.3 I 21-25 5 r CB2T2g 0.9 ( 1 ) 1-0 (11 2.2 ( 2 ) 1.0 ( 1 ) ND •t- 0 . 5 4 Ala 31.6 % 18-52 5 G25;6,5 CB2T3b 1.0 ( 1 ) i . l ( i ) 0.4 1,2 (1 ) 1.0 ( 1 ) O.B ( ! ) ND 0 . 0 0 Phe 7 . 4 1 5 3 - 5 7 c, G ? 5 ; 6 . S Table 2M ( c o n t i n u e d b) A m m o a c i d L y s H i s flrg CmCys A s p T h r 5 e r G ?ii P r o G l y fits Val l i e L e u Tyr-Phe T r p Hse M o b i l i t y (pH 6 . 5 ) N-te rmi na1

Y i e l d Posi t i o n Number o f r e s i d j e s Procedure of i s o -l a t i o n c B 2 : ? f 0.4 1,0 0 . 1 0,2 1.0 1.0 1.0 1 . 1 0.9 ND - 0 fll 16. 37 G25 (1) (1) {1-) ( 1 ) ( 1 ) (11 .3B a 0 % -4 2 6 ; 6 . 5 CBZC21 1.2 1.1 0 . 9 0 . 8 ND + 0 As 9 . 7 0 G25 ( 1 ) ( 1 ) ( 1 ) ( 1 ) . 3 7 n 6 % -73 4 ; 6 , S CB2 0.5 0.9 1.2 1.0 ND - 0 . flsp 2 9 . 5 1-4 4 G 2 5 ; C3a (11 (1) ( 1 ) (1) 90 Z 6.5 CB2C3b 0.6 0.4 2.0 0.6 0.9 0.5 1.1 1.3 ND - 0 . flls 2.8 (1) (1) ( 3 ) ( I ) (1) ( I J ( 1 ) (1) 64 1 88-97 10 G25;6 . 5 CB2C3d 0 . 3 0 . 9 1 .Z ] . 2 0 . 7 MD - 0 Gl 6. 54 G25 ( 1 ) (1) ( 1 ) (1) .53 n 8 £ -57 4 ; 6 , 5 CBZC3e I.O 0.3 0 . 1 1 , 1 0,9 ] . D 0.9 0.4 0.3 0.9 ND - 0 . flla 20.B 3 7 -6 G25; (1) (1) (1) (1) (1) ( 1 ) 37 t 12 6 . S T a b l e 2 M , flmi no a c i d Lys His Arg CmCys flsp Thr Ser GIu Pro Gly Ala V.J ! l i e Leu Tyr Phe Trp Hse M o b i l i t y (pH 6 . 5 ) N - C e r m i n a l Y i e l d P o s i t i o n Number of r e s i d u e s ' r D c e d u r e of i s o -Oat Tab e I M .on chvmotryptic B2Clb .3 .7 .0 .9 . 1 . J .? .0 ND 0. Gin 3D.B 7 1 -n G26; ( 1 ) ( 1 ) 1 ^ ) ( 1 ) ID (1) (1) 74 t 65 6.5 CB2Clf^ 0.9 0.7 0. 9 3.0 3 .0 0.9 0.8 0.8 ND - 0 . Gly 14.6 1) I ) i) 3) 3) I ) 1) 1) 8 I 5S-I59 12 G25; 3.5 . 5 ; e p t i Q e s • C B 2 C l f e 0.9 2.8 2 . 9 2 . 1 1 . 1 1 . 0 1 . 0 ND - 0 . Ser 7.6 12-12 G 2 5 ; 3 . 5 (1) (3) (3) ( ^ ) (1) ID ( 1 ) 28 t i?2 6 . 5 ; . t 0 C B 2 : i h 1.0 0.3 0.7 2,4 3.0 2.2 1 , 1 0.7 1 ,3 ND - 0 . Ser 2 5 . 5 7-2 16 G 2 5 ; (1) [ I ) I ^ ) ( 3 ) ( 3 ) ( 1 ) (1) ( 2 ) 17 1 2 6.5 d 1 1 ue i.d C B 2 C l j a -0 . 7 ( 1 ) 0 . 5 ( I ) 1 . 3 ( 2 ) 2 . 3 ( 2 ) 2 . 1 ( 2 ) 0 . 7 ( 1 ) ND D.OO Ser 2 . 1 X 9 8 - 1 0 6 9 G 2 5 ; 6 . S ; C 1.5 1 0 I 0 1 1 3 1 1 1 1 1 ^ 1 s ee CB2C1 .0 .3 .0 .3 .8 .2 .1 .6 .1 .0 .0 .0 ND 0. Gin 1 6 . 9 23-14 G 2 5 ; 1 i 2 1 1 1 1 1 ^ 6 5. T a b l e 2M fl[iiino a c i d Ly5 H i s A r g CmCys Asp Thr Ser G1 u Pro Gly flla y a i H e Leu Tyr Phe Trp Hse M o b i l i t y (pH 6.S) N-termina1 Y i e l d Posi t i o n Number of r e s i d u e s Procedure of i s o -l a t i o n ( c o n t i n u e d c CB2C3f 0.9 n 0.4 1 ?.9 (3 1.2 { ! 0.5 ND ( I - 0.29 Asn 4.4 1 gi-gi" 7 G 2 5 ; 6 . 3 . 5 CB2C3JC--1 . 0 ( CB2C3JC--1 ) 1.3 ( l ) 1.1 (1) 0.6 (1) NO D.OO Gin 7.4 1 54-57 4 5; r . 2 5 i 6 . S ; C CB2C3n 1.0 ( 1 ) 1.0 ( 1 ) ND + 0.7 2 flrg 23.5 1 52-53 2 G25;6.5 Ce2C4e 1.1 ( 1 ) 1.0 ( 1 ) ND 0 .00 Thr 8 . 0 X 86-87 Z G25-,6.5 CB2C5f 1.0 (1) NO ( 1 ) 0.00 Thr 9.4 X 5-6 2 G25;6.5

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of C F o r n i l

a c i d CB2AH2b CB2AM2d CB2AH2f CB2AM3

H i s Ai'9 CiilCys A s p T h r Ser G l u P r o G l y A l a V s l M e Leu T y r Phc T r p Hse H o b i l i t y (pH 6.5) N-terflina 1 Y i e l d Posi t i o n dumber of residues 'rocedure of i so-la t i o n 0 . 8 0 . 9 1 .4 1 .2 ! , 2 1 .3 2 . 9 0 . 7 I.a 1 . 0 1 ,0 0 . 7 1.7 1 .!-0 . 9 NO - 0 1 I 2 7 z 2 J 1 2 1 1 1 2 2 ( 1 ( 1 , 5 5 L y s 7! 3 V 1 0 0 3 0 G50 ; 6 . 0 . 6 1 .« D.S 4 . ! 5.2 4 . 8 6 . 0 1.4 6 . 1 5 . 6 2,2 1.7 3 . 0 1 .6 1.2 ND - 0 . 2 1 I 4 4 6 2 9 5 I 2 1 I ' 2 A s n 0 , 1-16 46 G 5 0 ; s. 0 . 7 0 . 7 1 . 9 1 .0 0 . 9 1 .8 0 . 7 2 . 5 6 . 3 1 . 0 O.a 0 . 9 0 . 7 1 . 5 NO + 0 1 ( 1 2 1 2 1 3 6 1 1 ( I ( 1 .17 L y s I 4 3 . 17 • 2 G50 6 1 70 4 ; 6 . 1 . 3 0 . 9 1 . 1 . ' . 1 1 .0 0 . 2 4 . 6 2 . 1 0 . 9 ND 0 . 3 + C ( 1 ) ( 1 ) (1 ) ( ; ' 1 ! I) I &l 1?) 11} ( 1 ) 45 L y s 1 .1 1 0 1 -V 115 15 G50 6 . 5 I .3 1 .3 1 ,D I . b 0 . 9 0 . 3 5 . 0 1 . 9 1 . 0 ND 0 . 7 * 0 ( 1 ( 1 ( 1

u

1 ( 5 ( ? ( 1 (• . 6 5 L y s 0 . 1 0 1 G50 4 i D.8 1 ,C I .a 0 . 9 0 . 9 t .1) 0 . 7 2 . 6 6 .3 1 .7 0 .9 \ .0 0 . 8 1 . 8 NO t 0 . 1 1 ) 2 [ 2 3 6 1 I [ 1 2 7 L y s l b . 6 I -115 4 7 - 7 0 I S -,6. 21 GSO Tab I D 6M . D o l j on t r y p t i c p e p t i d e s o f CB3. F o r a l l d e t a i l 5 , see Amlna a c i d L y s H i s A r g CmCys Asp t h r SBr G l u P r o G l y A U Va1 H e Leu T y r Phe T r p Hse M o b i l i t y (pH 6 . 5 ) N - t e r m i n a 1 Y i e l d P o s i t i o n Number o f r e s i d u e s P r o c e d u r e o f i s o -l a t i o n C 8 3 T l d CB311h C B 3 T n C B l T l j CB3T2fl CB3T2b CB3T2i> - 0 . 2 5 - 0 . 0 6 6 ( 1 ) 0 . 0 0 + 0 . 2 9 - 0 . 5 1 - 0 . 1 9 + 0 . 5 3

Tyr Tyr ftsn flsn Phe Phe Tyr

6 . 2 I 2 1 . 2 % 1 1 . 4 % 2 1 . 8 X 1 . 6 i 1 2 . 9 i 2 . 4 I 1 5 5 - 1 7 0 1 5 5 - 1 7 6 1 7 7 - ^ 2 0 3 1 7 7 - ^ 2 0 3 2 0 4 - 2 1 2 2 0 4 - 2 1 2 1 5 5 - 1 5 7 16 22 27 27 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 G 2 5 ; 6 . 5 T a b l e 7H. D a t a ort t h e r m o l y t i c p e p t i d e s Of CB3. F o r a l l d e t a i l s , see T a b l e 4M. D a t a on Staph^U-'i^oi^cu

For a l l d e t a i l s , see p r o t e a s e d i g e s t o f CB2AM2a,

r Ami no" a c i d CB2AM2aSAd L y s His Arg CinCys Asp Thr Ser GIu Pro Gly A l a W»1 H e Leu T y r Phe T r p Hse M o b i l i t y (pH 6 . 5 ) N - t e r m i n a i Y i e l d P o s i t i o n Number o f r e s i d u e s Procedure of i s o -l a t i o n 1.0 ( 1 ) 0.5 { l j 3 . 9 ( 4 1 1.9 ( 2 ) 3 . 1 ( 3 1 1.0 ( 1 ) 0 . 4 1.0 ( 1 ) 0.6 (1) 1.0 ( 1 ) 1.4 ( 1 ) 2 . 0 (2) 0,8 ( 1 ) ND - 0.35 L y s 31 .9 % 71-89 1 Q L 3 6 . 5 1 . 0 ( 1 ) 0 . 6 ( 1 ) 2.3 ( 3 ) 1.3 ( 2 ) 1 . 9 1 2 ) 0 . 9 ( 1 ) ND ( 1 1 - 0 . 1 8 Leu 4 0 . 8 -t 9 0 - 1 0 0 ^ ]^ 6 . 5 Amino a c i d L y s H i s flrg CmCys flsp Thr Ser G1u P r o Gly AU Val l i e Leu T y r Phe T r p H o b i I i t y (pH 6.5) N-termira 1 Vield P o s i t i o n Number of residues Procedure of i so-la t i o n T a b l e IH I : B 3 T H 1 2.8 2 . 2 1.7 D. 9 1 , 9 3 . 3 0 . 8 O.S 0 . 9 D.6 ND + 0 . Phe 1 . 6 1 3 0 -17 G25; 13 (2 ( 2 ( 1 ( 2 ( 3 ( I ( 1 { 1 ( 1 13 % CBJTHle 3 . 3 2 . 0 1 . 2 2 , 1 3 . 3 0 . 9 0 . 8 1 . 1 ND + 0 . He 1 5 . 7 196 1 8 3 -6 . 1 1 G25; ( 3 ) ( ^ ) ( 1 ) ( 2 ) ( 3 ) ( U ( 1 ) f l ) 38 I 1 9 6 6 . 5 CB3TH2e D . 9 1 ,0 0 . 9 1 .2 4 . 1 3 . 0 1 .3 1 , 0 NG ( 1 ) ( 1 ) ' 1) ' 1-) ; 4 ) ( 1 ) ( 1 ) O.DO T y r 1 . 9 1 5 5 -13 G25; % 167 CB3TH21 0 . 9 0 . 9 1 . 0 1 . 0 1 . 6 ND <• 0 , A l a 7.7 171-6 G25 ( 1 ) ( 1 ) ( 1 ) ( 1 ) il) 8 1 1 1 7 9 6 . 5 :B3TH3ad" 0 . 5 1 .4 D . 9 1 . 5 I .2 0 . 9 ND - 0 . Ser 2 . 2 206-7 G25" C ( 1 ) ( 2 ) ( 1 ) ( 1 ) ( 1 ) ( 1 ) 37 It 212 6 . 5 ; T a b l e 7H [ c o n t i n u e d a )

Table 5H. Time c o u r s e o f d e g r a d a t i o n o f 10 nmol o f CB2AH2aSAa ( 7 1 - 8 9 ) by c a r b o n y p e p t i d g s e f

flmount o f a m i n o a t i d r e s i d u e r e l e a s e d : ' ^ ' " ' * A s n Phe T h r T y r A l a G l u

m i n r•mo^ n n o ) n n o l nmol nmol nmo1 0 10 20 4D 60 0.00 0.00 0.00 0.00 D . O O 0.00 O.OQ 0 . 2 5 0 . 8 1 0 . 9 4 0 . 0 0 Q . 4 9 0.81 1.03 1 .02 O.OD Q.53 0.84 0.95 0.94 0.00 0.66 0.74 1.03 1 .00 0.00 Q.9S 1 .00 1 .07 1 .09 Ami no a c i d L y s H i s flrg CiiiCys flsp T h r S e r G l u P r o G l y A U V a l He Leu Tyr Phe Trp M o b i l i t y (pH 6 . 5 ) N-terrni na 1 Y i e l d P o s i t i o n Number o f res i dues Procedure o f i s o -l a t i o n CB3TH3cf CB3TH4ef-' CB3TH5b 0 . 7 ( 1 ) 0 . 3 0 . 8 0 . 9 3 . 0 2 .0 0.7 0 . 8 NO - 0 . Leu 2 . 1 1 6 1 -10 G25; C ( 1 ) ( 1 ) ( 1 ) ( 3 ) ( 2) ( 1 ) ( 1 ) 29 I 170 6 . 5 ; 0 . 9 1 .0 3 . 0 1.2 0 . 9 ND ( I ) ( 1 ) ( 3 ) ( 1 ) 0 . 0 0 Val 9.0 1 9 7 -7 G25; 3 . S S 203 6 . 5 ; 1.0 1.3 1 .7 0 . 7 ND ( 1 ) ( 1 ) ( 1 ) 0 . 0 0 Tyr 1 5 5 -6 G25; C 160 6 . 5 ; 1-1 ( 1 ) 1 .0 ( 1 ) 1.0 ( 1 ) ND 0 . 0 0 Leu 1 0 . 7 I 171*173 3 G 2 5 ; 6 . 5 ; C 1 .0 1.1 0 . 9 NO - 0 . Phe B . I 1 8 0 -3 G25; (^) ( 1 ) ( 1 ) 52 % 182 6.5

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• c t d CBlSAla COSSftlbs" C03SA2»b» CD3SA?c H U Arg CuCys Asp Tftp S i r &)u Pro Gly Al* Va 1 l i e Ltu Tyr Phe Trp Hse M o b i l i t y (pH 6 . 5 ) N-terra i na1 Y i e l d P o s i t i o n Number of r e s i due s Procedure of Iso-l a t i o n ; I 0 I 1 3 4 6 1 0 2 : .0 . ? .& , ! ,3 , 2 .9 , 2 .8 ,8 , 3 ND i 1 \ 3 2 I 2 0,00 Tyr 1,9 IS5-P 28 4 ; ff I 3 .1 I .0 0 ,b 0 . 9 1 .k 0 . 2 i . O 7 . 1 0^7 1.7 0 , 8 0 , 3 O.b NO . 0 , l i e i 82 1 8 3 -30 P4 ; 6 C J I \ 1 1 2 I 1 1 I ; i 5 0 , 6 0,H 0 5 1 \ I O!H O.G ND - 0 , Ala 3 . 9 2 0 7 -6 P4 ; 6 C 1 1 1 , 1 8 I 0 ,4 0 , 1 1 ,3 i 9 i l l 1 ,0 0 , 9 ND - 0 . Aid 2 . S 12 1 6 3 -10 P 4 ; 6 (1 (1 j l (^ (I (1 28 t 0 , 9 0 , 7 1 , . ' 11. 1 1 ,2 3 , 9 4 ,0 I ,6 0 , 6 1 ,! ND - 0 . Tyr 2 , ? 1 7 2 1 5 5 -.5 17 P4 ;6 1

li

1) l ) '') •'1 2 1 1 5 i 71 5 a c i d Lys His Arg CmCys A5p I h r Ser G1u Pro Gly AlB Val H E Leu l y r Phe T r p Hse Mobil I t y (pH 6 , 5 ) N- t e r m i n a l y i e l d Posi t i o n Number o f r e s i d u e s P r o c e d u r e of i s o -l a t i o n CDICb 0 . 9 ( ) ) 2.2 (2) 1.0 ( I ) 0 . 5 0.9 (1) 2 , 9 l i ) 1,1 ( 1 ) 1 .1 ( 1 ) 0,7 ( 1 ) 0 . 0 0 G l y 4 0 , 5 X 126-136 6 , 5 CS4C4 0 . 9 1.6 0 , 3 3,0 0 . 8 3 ,3 Z.B 1.1 2.0 0 , 7 + 0 , A r g 36,0 1 2 1 3 I 4 z 1 2 1 ) 4 I 137-154 18 6 . T a b l e 8M Ammo a c i d L y s H i s A r g CmCys Asp Thr Ser SIu Pro Gly A l a l i e Leu Tyr Phe T r p Hse M o b i l U y (pH 6 , 5 ) N - t e r m * n a l Y i e l d Posi t i o n Number o f r e s i d u e s Procedure of i s o -l a t i o n ( c o n t i n u e d C83SA 0 . 8 0 . 9 0 , 5 1 , 3 0 , 9 0 , 8 2 , 6 0 , 1 2 , 3 3 . 7 0 . 9 1 .7 NO - 0 , S e r 6 . 5 2e ( 1 1 1 [ 1 ( 1 ( 1 ( 3 ( ? ( 1 ( 1 {^ 48 I 165-13 18 r V , 5 •• C83SA2et." 0 , 6 (11 1.1 (1) 2 . a ( 3 ) ?,8 ( 3 ) 1,0 ( 1 ) 0 , 5 ( 1 ) NO 0.00 T y r 22,4 I 155-164 10 P 4 " 6 5 ' C CB3SA3i 1,1 ( 1 ) 1.2 ( 1 ) 2,6 ( 3 ) 2.7 ( 3 ) 1,1 ( 1 ) 0 , 9 ( 1 ) NO D,00 T y r 14,0 I 155-164 10 P 4 ; 6 , 5

Table l l M , Oata on t r y p t ic I i n i ted J F t a i l s r s e e la&le l H , of the S P E - p r o t e i n , For a l l A m i n o a c i d T L 2 g T L 3 g c Lys His Arg PeCys Asp Thr Ser G1u Pro Gly Ala Val l i e Leu Tyr Phe Trp Met Mobil i t y (pH 6 , 5 ) N - t e r n i i n a l Y i e l d Pos i t i o n Number of r e s i d u e s P r o c e d u r e of i s o -l a t i o n 0 G 0 1 2 1 5 5 1 0 1 1 \ ,8 -9 NO ,9 . 8 .9 ,7 . 1 . 5 , 9 ,9 ,1 .0 .0 HO Val 7 6 , 5 2 6 -27 pg U (1 (I {2 (3 (2 (6 | l 1 (1 (1 I £2 0 . 8 I . O 0 , 8 NO 2 , 1 0 . 9 0,7 0 . 5 2,7 l . l 1,0 0,7 0 , 6 ND 0 . 3 * 0 , Hfs 9 . 9 1 2 3 -15 P 6 ; 6 C 1 1 (1 (2 (1 (1 [3 {1 (2 (1 (1 (1 10 i u ,5 2 . 6 1,2 ND 3 . 9 1,9 I . O I . I 1,9 6 . 2 1,3 1,1 0 , 9 1.1 0 . 9 1.0 ND + 0 , Asn 2 5 . S 1 7 7 -27 P 6 ; 6 (4 (1 ( * (^ {1 {2 (1 (1 {1 (1 {1 (1 19 I 10 .5 3.8 1 ,0 ND 3.6 1,8 1.2 1.9 6 . 3 1.0 0 . 8 O.e 1.1 0 . 9 0 . 9 ND + 0 , Asn 3 0 , 6 1 7 7 -27 P6-6 '4 ' 1 '4 [ I (1 (2 (7 (1 (1 (1 [1 (1 (1 24 % 20 .5 1.0 NO 1.3 l . a 2 . 2 3 . 4 1,2 0 . 9 1.0 0 , 8 ND - 0, Asn 8 . 6 ( 1 ) (1) ( 1 ) {2) C i ( ^ ) ( 1 ) ( 1 ) ( 1 ) {!) ( 1 ) 32 % I-ZO 20 P 6 ; 6 ,5 1.0 ND 2 . 0 O.a 1.2 ND <• 0 , Gin 2 5 . 6 2 1 -5 P6;6 3 .5 (1) \2] (1) ( I ) 59 % 25 • 5 ; T a b T e 9 K , O a t a o n c f y m o C r y p t J c p e p t i d e s o f C B 3 T I J . F o r a l l d e t a i l s , see" Table TH, Am i n 0 a c i d CE3TijCd C;B3TljCf CB3TljCh L y s H i s A r g CmCys Asp Thr Ser G1u Pro 61 j -Ala Val H e Leu Jyr Phe Trp Hse H o b i 1 i t y (pH 6 . 5 ) N-termina1 y i e l d P o s i t i o n Numher of res Idues Procedure of i s o -l a t i o n 0,8 ( 1 ) 0 , 9 ( 1 ) 3 . 2 ( 3 ) 0 . 9 ( 1 1 0 , 6 ( I ) HD 0,00 V a l 6 . 9 I 197-203 7 6 , 5 3 , 0 1 , 9 L . S o.a 1.9 2 . 8 0 , 7 0 . 8 0 . 7 ND t 0 , T h r 2 6 . 9 l e w 16 6 , ( 3 ( 2 {2 { 1 (2 ( 3 ( 1 { I | 1 13 i 1.1 ( 1 ) 0.8 ( 1 ) 1.2 [ 1 ) 0 - 9 . ( 1 ) ND + 0.35 A s n 13 ,4 % 196 177-180 b 4 6 , 5 T a b l e I I M ( c o n t i n u e d Arnino a c i d H i s A r g PeCys A s p T h r S e r G l u P r o G l y Ala Val H e Leu T y r Phe T r p M e t M o b i l i t y (pH 6 . 5 ) ^ - 1 e rm 1 n a 1 Y i e l d P o s i t t o n Number of r e s i d u e s ' r o c e d u r e of i s o -l a t i o n TL3gd 1,5 [I] NO 1.3 ( 1 ) O.e ( 1 ) NO 0,5 ( I ) + 0 . 5 9 H i s 3,5 1 153-157 5 P 6 ; 6 . 5 ; 3 . 5 TL4ac ND 1.1 (1) 1.2 (1) 1,0 (1) 0,8 j l ) 0,9 ( I ) ND O . O D Phe 2 . 9 % 53-57 5 P6;6,5-. 3 . 5 TL4b ND ( 1 1.5 (2 1 , 0 0 . 9 0 , 9 1 ] 0 . 9 ( 1 0 , 9 (1 ND ( 1 * 0.13 Phe 3 9 . 0 I 2 0 4 - 2 1 9 P 6 ; S . 5 TL5 ND 0 , 3 1.0 j l ) 0 . 9 ( 1 ) 0 . 3 l ! 2 ( 1 ) 0 . 9 ( 1 ) 1,1 [lj ND 0 , 0 0 Phe 24,3 S 53-57 S P6-6

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