endochitinase from seeds of Job's Tears (Coix lachryma-jobi)

Biochimica et Biophysica Acta, 993 (1989) 260-266 Elsevier

260

BBAPRO 33514

Purification and characterization of an insect a-amylase inhibitor/endochitinase from seeds of Job's Tears

( Coix lachryma-jobi) M a f i a B. A r y 1.2, M i c h a e l R i c h a r d s o n t a n d Peter R. S h e w r y 2 s Biology Department, Unipersity of Durham, Durham, and ~ AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpemten (U.K )

(Received 26 May 1989)

Key words: Cereal; a-Amylase inhibitor; Endochitinase; PR protein; Crop protection

A protein inhibitor of locust gut a-amylase was purified from seeds of Job's Tears (Coix lachryma-jobi) using ammonium sulphate pcecipitation, affinity chromatography on Red Sepharose and reversed-phase HPLC. It consisted of two majo¢ isomers, each a dimer el two timely similar or identical subunits of M, about 26400, and associated by inter-chain disulphide bonds. These isomers also had dosely similar amino acid compositions. The major isomer showed no inhibitory activity against amylases from other uu'ess: human saliva, pordne pancreas, Baeilhn subtilis, Aspergillm myza¢ and barley mall The manual DABITC/PITC method was used to determine about half of the amino acid seq~ of the major isofenn. T I ~ showed a high delm*e of heatele~ with previomly reported sequeaees of e ~ d o d d t i n ~ emymcs from several speeles (tobacco, porto, barley, bean), and endochitlnase activity was demomtrated by f e l l e w ~ the release of ~ i t y frmn [3Hlehitin. This novel cembination of functions may be relevant to protection of the grain from insect feerllu and f a n p l infection.

intr~'uetion A large number of protein hd'dbitors of hydrolytic enzymes (proteinases and a-amylase) have been isolated from plant tissues, notably from storage organs such as seeds, fruits and tubers. They have been classified into a number of famities on their amino acid sequences and target enzymes [1,2]. The a-amylase inhibitors are often specific for enzymes from a single source (tissue, organism or group of organisms), but only one family has been shown to inhibit endogenous plant enzymes [3,4]. In addition, several inhibitors appear to be bifunctional, inhibiting both a-amylase and proteinases [3-6]. Although many inhibitors are present in healthy plant tissues, others are synthesised in response to tissue damage or invasion by pathogens (fungi, bacteria, viruses) [7], As ouch they may form part of a wide spectrum defence mechanism, and be components of the mixture of PR ('pathogenesis-related') proteins characterized in species such as tobacco [8].

Other components of the PR protein complex are hydrolytic enzymes such as glucanases and endochitinases [9,11]. Although endochitinases are also present in some healthy tissues such as barley seeds [12] and wheat germ [13], most studies have been made on tissues where their synthesis is induced by infection with viruses (toba~o) [14], or by treatment with ethylene (bean and potato leaves) [15,16], a plant hormone that is produced in response to various stresses including damage and invasion [17]. In the present paper we report the purification and characterization of a novel protein from seeds of the tropical cereal Coix lachryma-jobi. Although initially identified as an inhibitor of insect a-amylase, the partial amino acid sequence showed strong homology with those of endochitinases from various sources. Subsequent analysis confirmed that the purified protein exhibited endochitinase activity. This is, to our knowledge, the first characterization of a plant protein with activity as an enzyme and an enzyme inhibitor. Materials ~M! Methods

Correspondence (and Present address): P.I~ Shewry, Deparunem of Agricultural Scienc¢~ University of Bristol, AFRC IACR, Lon8 A~hton Research Station, Long Ashton, Bri$toi, BSI8 9AF, U.K.

Purification of an a-amylase inhibitor. The purification was based on Kutty [18], all operations being carried out at room temperature unless stated otherwise.

0167-4838/89/$03.50 ~ 1989 Elsevia Science Publishers B.V. (Biomedical Division)

261 400 g milled seed was defatted with acetone and then stirred for 5 h with 2 1 of 0.1 M HCI containing 0.I5 M NaCi. After centrifugation ( 1 0 0 0 0 x g , 30 rain, 40C) the pH of the supernatant was adjusted to 7.0 by the dropwise addition of 1 M NaOH and the precipitated proteins removed by a further centrifugation. Ammonium sulphate was added to the supematant to 60% saturation and, after standing overnight at 4°C, the precipitate removed by centrifugation and redissolved in 0.05 M Tris-HCl buffer (pH 7.0), containing 0.1 M NaCI. After dialysis against the same buffer for 24 h, undissolved protein was removed by centrifugation. The dissolved protein was applied to a column of Red Sepharose CL-6B (8.5 x 3.0 cm) equilibrated with 0.05 M Tris-HC! (pH 7.0), containing 0.1 M NaCI. The column was washed with equilibration buffer until all unbound protein was removed, and then eluted with the same buffer but containing 3 M NaCI. Active fractions were dialysed against distilled water, lyophilized, dissolved in 0.1 M trifluoroacetic acid containing 6 M guanidinium chloride and injected onto a Vydac widepore preparative reverse-phase C~s HPLC column. Elution with a bradient of acetonitrile in 0.1% trifluoroacetic acid gave two major active components. Preparation of a-amylase from Locasta migratoria migratorioides (African migratory locust). Whole guts were prepared from mature larvae (nymphs) by dissection in distilled water at 40C and then homogenised with a motorised teflon pestle. After centrifugation at 10000 rpm for 30 rnin at 4 ° C , the supernatant was stored at - 2 0 ° C. Extract from four guts was us'~ally made up to a final volume of 1_.0ml. a-Amylase assays, a-Amylases from Bacillus subtilis, porcine pancreas, human saliva, Aspergillus oryzae and barley malt (a mixture of a-amylase and /t-amylase), were purchased from Sigma Chemical Co. The activity of a-amylase was determined using the method of Bernfeld [19] using 0.05 M sodium acetate buffer (pH

7.0). Electrophoresis. SDS-PAGE was carried out using a modified Laemmli system [20], with 4 M urea in the stacking and separating gels [211. lsoelectric focusing. Protein was dissolved in 10 mM glycine (pH adjusted to 8.0 with "Iris), containing 6 M urea and 2% (v/v) 2-mercaptoethanol. Samples were separated using an LKB Multiphor system, the gels containing 6 M urea and 2~ (w/v) ampholyte (pH range 3.5-10) [221. Gels were washed with 10~ (w/v) trichloroacetic acid to remove ampholyte and stained as described by Blakeslee and Boezi [23]. To determine the pH gradient, gel strips were macerated with 1 ml distilled deionised water and the pH determined after standing for 1 h at 20 * C. Amino acid w~alysis. Duplicate samples of protein (--100 pg) were hydrolysed under N 2 with 1 ml of constant boiling (5.9 M) HCI, for 21 h at 110"C. The

hydrolysates were reduced to dryness and the amino acid compositions determined using an LKB Alpha Plus amino acid analyser, Amino acid sequencing. Samples of the reduced and S-carboxymethylated protein were digested separately with trypsin and chymotrypsin. The mixtures of peptides were separated by reverse-phase HPLC on a Vydac analytical C~s column, using variable gradients of 0-50% acetonitrile in 0.1% trifluoroacetic acid. The peptides were then sequenced using the DABITC/PITC double coupling method as in Ref. 24. Endochitinase assays. The protein was incubated with [3H]colloidal chitin (prepared as in Ref. 12) in 0.1 M citrate buffer (pH 5.0) for 15 min at 37°C. The total reaction volume was 200 ~tl and the reaction was stopped by the addition of 300 ~1 of 10% (w/v) trichloroacetic acid and 100 t*! of 1% (w/v) bovine serum albumin. The precipitated protein and undigested chitin were removed by centrifugation (10000 × g for 5 rain) and the released [3H]diacetyl chitobiose present in the supernatant determined by liquid scintillation counting. The enzyme activity was calculated from the specific activity of the [3H]ehitin substrate. Results and Discussion

Purification of an inhibitor of locust gut a-amylase The crude enzyme preparations from locust gut eataiysed the hydrolysis of starch, with a pH optimum of about 7 (Fig. 1A). The reaction was linear with 2.5 to 20 pl of enzyme preparation per assay (Fig. 1B), but became non-linear at higher volumes. A volume of 15 pl was therefore used for all assays. The amylolytic activity of the locust enzyme preparation was strongly inhibited by the neutralised protein extract from Coix seeds (Fig. 2), and by the two fractions precipitated between 0 --, 30% and 30 --* 60% ammonium sulphate (Fig. 2). No inhibition occ'arred with the Coix proteins precipitated between 60 and 90% ammonium sulphate (data not shown). The proteins precipitated between 0 and 60% ammonium sulphate were, therefore, bulked and applied to a column of Red Sepharose CL-6B. This is an affinity absorbant with a procion red HE-3B ligand. Although marketed as an absorbant for purifying NADP-delx~ndent enzymes and carboxypeptidase G, it has previously been shown to bind inhibitors of a-amylase [18]. The molecular basis for this binding is not known. Inhibitory activity was present in the unbound fractions and in the fraction eluted with 3.0 M NaCI (Fig. 3). However, when the unbound fraction was re-applied to a second column all the inhibitory activity bound, and could then be eluted as before (results not shown). The bound protein from the two columns was then bulked and separated by HPLC, using a preparative Vydac Cin column (Fig. 4). Two major peaks were eluted, at acetonitrile concentra-

262

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10 20 30 40 50 Locust enzyme preparation (pI)

pH

Fig. 1. (A) Effect of p H on hydrolysis of soluble starch at 30 e C by gut homogcnates of larvae of locusts (Locusta migratoria migrotori:)ides). The following buffers were used at 50 mM concentrations: acetate (pH 5.0-5.5), phosphate (pH 6.0-7.0) and Tris-HCI (pH 7.5-10). Relative activities were determined from duplicate analyses. (B) Starch hydrolyzin$ activity ~f the crude enzyme preparation from locust Set The amyla~ activity was measured as described in the text using 0.05 M sodium acetate buffer (pH 7.0).

tions of 46 and 47~ (called peaks 1 and 2, respectively, in Fig. 4). Both peaks inhibited the amylolytic activity of the locust gut preparations, the total yield being about 1.0 mg per 400 g flour ( = 0.007~). The first peak (at 46~ ac~tonitrile) accounted for about 75~$ of the

100

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total.

m

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Volume of Inhibitor Preparation

(ml)

Fig. 2. Inhibition of the locust gut amylase by the neutralised protein extract from Coix seeds (o) and by two fractions precipitated between 0-30~ (4) and 30-60~$ ([3) ammonium sulphate. Increasing volumes of each sample were mixed with the same volume of the locust [gut preparation. The residual amylase activity was measured as described in the text.

i

2.0 1.8

Characterization o/ the a-amylase inhibitor. SDSPAGE analysis of the two major peaks from the HPLC separation gave identical patterns. In each case a single band of Mr about 52 500 was observed under non-reducing conditions, which was replaced by a lower Mr band in the presence of 2-mercaptoethanol (Fig. 5). The M r of this latter band was determined using reduced and carboxymethylated material from the first (46q[ acetonitrile) peak as 26400. Isoelectric focusing of the two fractions under denaturing and reducing conditions showed similar patterns, with several diffuse bands with pl values of between about 8.5 and 9.0 (results not

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Fig. 3. Separation of the 60~ ammonium sulphate precipitate on a column (8.5 x 3.0 cm) of Red Sepharose CL-6B. The protein was applied to the colunm in 0.05 M Tris-HCi buffet containing 0.1 M NaCI. After removal of unbound protein with the same buffer the bound material was eluted by the addition of 3 M NaC1. Fractions (10 ml) containing unbound and bound material were tested for inhibition of locust amylase.

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60 70 8 9 0 1 0 110 120 130 (rain) Fig. 4. Reverse-phase HPLC of the bound fraction from the Red Sepharose CL-6B colmtm on a preparative Cta column (25 cm×22 nun, Vydac 218TP1022), equilibrated in 0.1% trifluoroacetic acid and eluted with a gradient of ace~onitrile in 0.1% trifluoroacetic acirJ at a flow rate of 10 ml-mi. -1. The two major peaks eluted at 46% (peak 1) and 47% (peak 2) (v/v) acetomtrile inhibited the amylolytic activity of the locust gut preparation. Time

s h o w n ) . T h e i r a m i n o a c i d c o m p o s i t i o n s ( T a b l e I) w e r e also shnilar, with high contents of aspartate/asparagine, glutamate/glutamine, glycine and alanine.

These r e s u l t s i n d i c a t e t h a t t h e p r o t e i n s i n t h e t w o m a j o r H P L C p e a k s a r e closely r e l a t e d . I n e a c h c a s e t h e native protein appears to be a dimer, consisting of two

TABLE I Comparison of the amino acid compositions (expressed as tool%) of the Coix peaks eluted at 46 and 47% (v/v) acetonitrile with those reported for endochitinmer from other species n.d., not determined. Amino acid

Coix 46%

47%

Barley

Wheat

Bean

Tobacco (basic)

Potato

12.9 5.3 6.0 9.5 2.6 15.5 11.7 2.7

12.7 5.6 6.3 10.1 2.5 13.4 11.2 2.7

9.1 5.4 6.9 7.q 6.6 12.0 14.5 2.8

9.4 7.4 8.1 6.7 5.1 17.5 9.1 40

9.7 7.3 8.7 7.3 6.7 12.3 8.7 5.3

11.3 4.9 8.3 6.3 7.3 14.9 7.0 5.7

13.2 4.0 7.6 6.0 7.3 14.6 7.0 5.6

V~

4.7

Met I~

1.0 3.~

5.1

4.9

4.7

3,3

2,7

3.0

1.1 3.5

0.6 4.2

1.0 3.0

0.7 3.7

1.3 5.0

1.0 5~

~u ~e His

3,3 5.6 5.4 1.8

3.7 5.8 5.3 1.9

4.4 4.6 4.9 1.9

4.4 4.7 4.7 1.3

5.7 5.0 4.3 1.0

4.7 3.7 4.7 1.3

4.6 3.6 5,0 1.3

~s ~g T~

5.1 3.6 n.d.

5.1 4.1 md.

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2.3 6.0 2.7

2.6 6.0 2.6

-

[121

[13]

115]

[14i

[161

Asx a Thr ~r G~ b Pro Gly Ala Cys

Tyr

Ref.

" Asp/Asn. Glu/GIn.

264

ab Fig. 5. SDS-PAGEof the peak eluted from the revereed-pkaseHPLC colunm at 46~ aeetonitrile in the native state (track a) and after reduction and c.admxymcthylation (track b). The humbert indicate

the position of marker proteins of known molecularweight. These are: 1, bovine serum albumin (66000); 2, egg albumin 145000); 3.

glyceraldehyde-3-phosphate dehydmsenase (36000); 4~ carbonic anhydrase(29000); 5, trypsinogan(7.40G0);6, soybeantrypsininhibitor (20000); 7, a-laetalbumin(I,t200).

identical or closely similar subunits. The effects of reducing agents on the SDS-PAGE patterns also indicate that the dimers are stabilized by disulphide bonds. Subsequent characterization was carried out only on the first (46% acetonitrile) HPLC peak. Inhibitory properties. Fractions from the purification stages (the nentralised acidic extract, ammonium sulphate fractions and Red Sepharose fractions) were tested for inhibition of a-amylases from Bacillus subtills, porcine pancreas and human saliva. No inhibition was observed even when the inhibitor fractions were pre-incuhated with the enzyme for 3 h at 37°C in 0.1 M acetate buffer (pH 7.0) containing 20 mM NaCI and 100 mM CaLl 2. The addition of bovine serum albumin (0.4 nag mi) also had no effect. The purified inhibitor was re-tested against these a-amylases and also against enzymes from Aspergillus orjzae and barley malt. The enzymes and purified inhibitor were mixed at molar ratios of 1 : 1 and allowed to stand for 25 rain at 370C before assay. No inhibitory activity was observed.

It is concluded from these assays that the inhibitor is only active against insect a-amylase, although we have not tested enzymes from species apart from locust. Amino acid sequencing. The amino acid sequences of a number of tryptic and chymotryptic peptides were determined using the manual D A B I T C / P I T C method (see Fig. 6). The total number of residues sequenced in this way was about 130, equivalent to almost half of the total number expected from the Mr of the protein subunit. No heterogeneity in the sequence was observed. Comparison of the peptide sequences with the pubfished amino acid sequences of other proteins showed unexpected homologies with partial and complete amino acid sequences of endochitinases from a number of sources including barley seeds, and leaves of tobacco, potato and beans. In fact, almost all of the tryptic and chymotryptic !~eptides could be aligned with these sequence, including a continuous region of about 70 residues (Fig. 6). None of the peptides corresponded to the N-termini of the other proteins, and attempts to determine the N-terminal sequence of the intact Coix protein using automated solid-phase and gas-phase sequencers showed that it was blocked. It is of interest that the bean and potato endochitinases extend for a number of residues (46 in the bean enzyme, possibly more in the potato) beyond the N-terminus of the barley enzyme. This part of the bean and potato enzymes is strongly homologous with the N-terminus of wheat germ lectin [25], and has therefore been proposed to have a role in the recognition and binding of chitin rather than in catalysis. A closely related N-terminal sequence has also been reported for herein, a protein of unknown function from rubber latex [26]. Our identification of a peptide with the characteristic Cys-Cys-Ser sequence indicates that at least part of this "lectin-tike" sequence is present in the Coix protein. The two purified ~eaks from Coix also had similar amino acid compos, dons to endochitinases from other sources (Table I), most notably high contents of glycine (--12-17 mol%) and fairly high contents of aspartate/asparagine (9-13 mol%) and alanine (7-14 mol%). Endochitinase activity. Incubation of the Coix protein with tritiated chitin resulted in the release of TCA-soluhie radioactivity, demonstrating endochitinase activity. 1.0 rag of protein released about 5 nmol of N[3H]acetylglucosamine during the 15 min a ~ a y period. This activity is lower than t.L,o~ i6ported tor enzymes from wheat [13] and barley [12], which may be due to partial denaturation during the lengthy purification procedure. General d i j o n Recent studies indicate that endochitinases are widespread in higher plants. They are produced con-

265

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C C C C

S S S S

Q F KF K F K F

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GWC GWC G WC G YC

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S N N L

S0 T D ~ C - kGC qS QC-GGP S PAP T N D¥C G S G N CQ S Q C P G GG P G PG P G N D Y C G P G N C q S q C P G G- P T P PG G DA¥

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~rley ~ ~tato ~eeo(bs81c) ~

101 150 A D A R K* * ~ ~ TATRKREIAAFLGQTSH ETTGGW&TAP~GP~A~GYC-FY~ NPSTYC~ N ARMR g I AA F FAGTS H L TTG G~A S A P DG P ¥ A~G ~CF L RBRG N PG DYC T TARKR E I AA F F A QT S H E TT G GUA TAP DG P Y A WG Y C~ L- R g q G S P G DYC T FAHVTH ~TG HHAYCD i

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151 S AT Pq~ PP8 $ q~ PS-GqW

P CA PGQQ ~ Y GP G P I q I S H ~ IN YG QC GRA PCA PGKKY F GP G P I q I S H ~ YN ¥G P C GRA PCA PGRE T F GRGP I q I $ H H ~N YG PC GKA

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201 e* MTAQP PKP S V I S F K S A L W F WM T A Q S P K P S V X S FKTALW--MT PQ 5 PKP S V X S- KS ALW F MMT PQ $ PK P$

S S C C

HAV HD V RD V H DV

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AG T S I G I O

qWS K WT KWN RMQ

PDGA DgAAGRV PG PS S A DVAARR L PG P S 5 ADRAAMRL PG P $:~ A D P . A A N R L PG

14 I

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2.50 T N I T lq l TII X Tll X

C31

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Cjj 251 I N I N I N I N I N

FG Y Z G T P GV P O V

l

tstl+ e

Edtrley Bean Potato Tobacco(b~tc) Tobacco(acidic) Colx

%,

Tj, I

Barley Bean Potato Tobacco(baste)

I GV D L L~ g P DLVAT DS I GV DLLH N PDLVATD P [ GV DL L B N PDL VATD P * * * V A T D P I OH DGLG N PDR VAQ DA

C1~ 300

* * G G O G O O GG

* L L i. L

* g E E g

C C C C

G G O G

R G R O R G RG

q T T T

D S KVQ D M gVq B S R VQ DS ~VQ

D R D R D K DR

I I I I V

G G G G S

F ' F K R ]~ F YR R ¥ F ][ K R ¥ F YRRY 3[ Y K q y )i

C C C C C

T: t

D S S S q

L I I I Q

L ~ b L L

; v G ~ V T ; V S ; VS ~,V D

¥ P P P P

G G G G G

N D D D P

N N N N N

L b L L I.

D D D D

C Y C V C G CG

S N N N

q T P q R g Q R S QRS

F F F F

G G G G

NS L NA L N G L M GL

L L L L

( C,s

301 Bean Potato Tobecco(b881c) Tobacco(acidic)

L $ D L V T S Q *****

V D T b ***** V D T H ***** V D T H *****

* P r o t e i n B - t e r L t n u s (deduced by homology f o r p o t a t o p r o t e i n ) ~* N - t e r a l ~ s o f cyanogen bromide f r o n t *** N - t e r ~ t n a l r e s i d u e encoded by p a r t i a l cDNA c l o n e **** C - t e n t t n s l r e s i d u e of p a r t l e l d l r e c t l y d e t e r m l n e d sequence ~ * * * * C - t e n ~ n a l r e s i d u e of p r o t e i n Fi& 6. A l i g n m e n t o f the a m i n o acid s e q u e n c e s o f the t r y p t i c a n d c h y m o t r y p t i c peptides o f the C o i x a . a m y l a s c i n h i b i t o r / e n d o c h i t i n a s c with thosc r c p o r t c d for basic cndochitina.scs f r o m b a r l e y [12], b e a n [15] a n d p o t a t o [16] a n d f o r b a s i c [14] a n d acidic |10] e n d o c h i t i n a s ~ f r o m tobacco. T h e s e q u e n c e s arc aligncd to m a x i r a i s c h o m o l o g y , resulting in the i n t r o d u c t i o n o f s o m e g a p s . S t a n d a r d sinKle letter a b b r e v i a t i o n s a r c used. I n d i v i d u a l C o i x p c p t i d c s a r e s h o w n by a r r o w s a n d n u m b e r e d .

266 stimtivcly in some tissues such as oat leaves [27], potato tubers [16] and seeds of barley [12] and wheat [13], but also produced in response to damasc or invasion b y

pathogens (in leaves of tobacco, bean, potato and maize) [10,11,15,16]. They have Mr values by SDS-PAGE of between 2.5000 and 30000, and can be classified into subsroups with low and high pl values (called 'acidic" and 'basic' endochitinases, respectively). Although all of the complete amino acid sequences shown in Fig. 6 correspond to basic endocbitinases, Jamet and Friti8 [28] have shown that the acidic endochitinases of tobacco leaves are sefolosicalIy related to the basic type, while Hooft van Huijsduljnen et al, [I0] have shown that partial amino acid sequences of the two types are about 70~ homologous to each other and to the bean endochitinase (see Fill. 6). The enzyme from Coix is clearly of the basic type (pl &5-9.0), and has extensivc homology with all the published amino acid sequences. However, whereas the other enzymes appear to be mononters, the Coix enzyme is a 4imet. The observation of only a single band on SDS-PAGE and the absence of heterogeneity in the amino acid sequence indicates that these two subunits are closely similar, if not identical.

Although some hetemSeneity is demonstrated by the separation of two peaks of holoprotein by HPLC and by I E F of their reduced and denatured subunits, thi~

m y have resulted, at least in part, from modification during extraction and purification (most notably during the low pH extraction). The ¢onvermon of the dimer to monomer in the presence of a reducing agent indicates

that the holoprotein is stabilized by disulphide bonds. None of the other characterized endochitinases appear to have been tested for inhibition of a-myiases, and the identification of a protein with this mmbinatiou of functions is the most unexpected and interestin$ result of the present study. W e used the locust m a m y l a ~

for our ass.ys as this was the most readily available insect sonr¢¢. It would, however, clearly be of interest to determine its activity against et~ymes from a wider

range of insects, including natural pests from its area of orisin (South East Asia) and its current areas of cultivation (South East Asia, Japan and Southern Brazil). Our identification of a bifun~Jonal protein with activity as an enzyme and as an enzyme inhibitor raises the possibility that other characterized proteins may also have two or more functions. The increxsin8 number of amino acid sequences should facilitate their identification.

A

~

We are grateful to Dr. Joern Mikkclsen (De D~nskc Sukkerfabrikker) for discussions and for assisting with the endochitina~e assays, and to Dr. J. Mundy (Durham)

for supplying the locust larvae, MBA is supported by a 8rant from CAPES (Brazil).

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