Amino-acid sequence of the coeliac active gliadin peptide B 3142

Original Papers Originalarbeiten

Amino-acid Sequence of the Coeliac Active Gliadin Peptide B 3142 *

Herbert Wieser 1, Hans-Dieter Belitz ~, and Azaria Ashkenazi 2 1 Deutsche Forschungsanstalt f/Jr Lebensmittelchemie und Institut f/Jr Lebensmittelchemie der Technischen Universitfit Mfinchen,

LichtenbergstraBe 4, D-8046 Garching 2 Department of Pediatrics and Pediatric Research, Kaplan Hospital, Rehovot,

affiliated to the Nadassah and Hebrew University Medical School, Jerusalem, Israel

Aminos/iuresequenz des coeliakieaktiven Gliadinpeptids B 3142

Zusammenfassung. Das in vorangegangenen Arbeiten [1] aus einem peptisch-tryptischen Partialhydrolysat yon Gliadin fiber einen mehrstufigen Trennprozel3 ge- wonnene Peptid B 3142 wurde an einem erweiterten Patientenkreis im immunologischen Test und im Or- gankultur-Test auf Coeliakie-Aktivit/it gepriift. In beiden Tests zeigt das Peptid eine coeliakie-spezifische Wirkung. Die N-terminale Sequenzanalyse (ED- MAN-Abbau), die C-terminale Sequenzanalyse (Um- setzung mit Carboxypeptidase Y) sowie die Sequenz- analyse von Spaltpeptiden aus den mit Papain und Chymotrypsin erhaltenen Partialhydrolysaten erge- ben folgende, aus 53 Aminosfiureresten bestehende Gesamtsequenz: H-Val-Pro-Val-Pro-Gln-Leu-Gln- Pro-Gln-Asn-Pro-Ser-Gln-Gln-Gln-Pro-Gln-Glu- Gln-Val-Pro-Leu-Val-Gln-Gln-Gln-Gln-Phe-Pro- Gly-Gln-Gln-Gln-Pro-Phe-Pro-Pro-Gln-Gln-Pro- Tyr-Pro-Gln-Pro-Gln-Pro-Phe-Pro-Ser-Gln-Gln- Pro-Tyr-OH. Daraus errechnet sich eine Molmasse von 6 129 g/tool.

Summary. Peptide B 3142, which has been isolated from a peptic tryptic digest of whole gliadin by several separation steps [1], was examined for coeliac activity in an immunological test and in art organ-culture test, comparing enlarged groups of coeliac patients and control persons. In both test systems the peptide shows a coeliac specific effect.

The N-terminal sequence analysis (EDMAN degradation), the C-terminal sequence analysis (incubation with carboxypeptidase Y) and the sequence deter-

Offprint requests to." H.-D. Belitz * Supported by grants from Stiftung Voli~swagenwerk and from

Deutsche Forschungsgemeinschaft We gratefully acknowledge the excellent assistance given by Mrs. Schfitzler

ruination of peptides, obtained from B 3142 by digestion with papain and chymotrypsin, result in the following total amino-acid sequence: H-Val-Pro-Val- Pro-Gln-Leu-Gln-Pro-Gln-Asn-Pro-Ser-Gln-Gln- Gln-Pro-Gln-Glu-Gln-Val-Pro-Leu-Val-Gln-Gln- Gln-Gln-Phe-Pro-Gly-Gln-Gln-Gln-Pro-Phe-Pro- Pro-Gln-Gln-Pro-Tyr-Pro-Gln-Pro-Gln-Pro-Phe- Pro-Ser-Gln-Gln-Pro-Tyr-OH. The 53 amino-acid residues correspond to a molecular mass of 6,129 g/ mol.

Introduction

For the isolation of coeliac active peptides the peptic tryptic digest of whole gliadin was successively separated by ultrafiltration, gel filtration, cation- and anion-exchange chromatography and reversed-phase HPLC [1]. The peptide B 3142 obtained in a yield of 0.012%, based on gliadin, showed activity in an immunological test (LIF test) with all patients examined. B 3142 seems to be a pure peptide when rechromatographed by HPLC: The molar ratio of the amino-acid residues is essentially integer, and even using different buffer systems HPLC produces single symmetrical peaks. It consists of about 53 amino-acid residues with a high content of Glx and Pro. In the present report further experiments with immunological and organ- culture-tests and the determination of the total amino- acid sequence of B 3142 are described.

Material and Methods

Material

Reagents and solvents for the Edman degradation were of sequenal grade and were obtained from Pierce (Rockford, USA). Carboxy- peptidase Y (17 U/rag) and papain (type IV, 21 U/rag) were pur- chased form Sigma (St. Louis, USA) and e-chymotrypsin (from bovine, 45 U/mg) was from Merck (Darmstadt, FRG). All other reagents and solvents were of analytical or HPLC grade and were products of Merck (Darmstadt, FRG).

Z Lebensm Unters Forsch (1984) 179:371-376 © Springer-Verlag 1984

Isolation of B 3142

Peptide B 3142 was isolated by the techniques previously described [11.

LIF-test and Organ-culture Test

The peptide fractions obtained by HPLC (B 3141 B 3146) were examined in the LIF-test [2]. Twenty-four coeliac patients on normal diet (ND), 29 coeliac patients on gluten-free diet (GFD) and 20 control persons with normal mucosa (C) were selected for this study. Ad- ditionally B 3142 was examined by the organ-culture test according to the method described previously [3]. Four coeliac patients on normal diet and two control persons were included in this test series.

Amino-acid Analysis

About 10 gg of peptide was hydrolyzed in vacuo for 24 h with 1.5 ml of HC1 (6 mol/1)/phenol (0.1 tool/l) at 110 °C. The samples were dried over KOH pellets in a vacuum desiccator and the dry residues were dissolved in 100 gl of sodium citrate buffer (0.2 mol/1, pH 2.2) and analyzed on an "LKB 4400" amino-acid analyzer. The values for Ser and Val were corrected by the factors 1.09 and 1.08, respectively, according to [4].

EDMAN-degradation

The N-terminal amino-acid sequences of peptides were determined by manual EDMAN-degradation according to the method used by Chang et al. [5] with the exception that only phenylisothiocyanate (PITC) and not dimethylaminoazobenzeneisothiocyanate (DA- BITC) was used as coupling reagent.

About 2-20 nmol ofpeptide was dissolved under N2 in 100 gl of aqueous 50% (v/v) pyridine and treated with 10 p.l of PITC. The closed tube was heated in a heating block at 56 °C for 30 rain. After 10 gl of PITC had been added the coupling reaction was repeated. The excess of reagent was removed by mixing the sample with two portions of 0.5 ml of heptane/ethyl acetate (2 + 1; v/v) and centrifuging. The upper phase was removed and discarded, the lower phase was dried over P205 in a high vacuum, dissolved under N2 in 50 ~tl of anhydrous trifluoroacetic acid and heated at 56 °C for 15 rain. The sample was dried over NaOH in a vacuum desiccator and the residue was dissolved in 50 Ixl of H20. After mixing with 150 gl butyl acetate and centrifuging, the organic phase was removed and the extraction was repeated. The aqueous phase was dried over P2Os and subjected to the next cycle. The combined organic phases were dried in a N stream, dissolved in 50 gl of aqueous 50% (v/v) trifluoroacetic acid, heated for 50 rain at 56 °C, dried over NaOH and analyzed for PTH-amino-acids.

Identification of PTH-amino-acids

The analysis of PTH-amino-aeids was performed by HPLC on ODS- Hypersil according to the method used by Lottspeich [6] under the following conditions: column: 4.6 mm x 240 ram; ODS-Hypersil (3 ~tm, Shandon) - temperature: 67.5 °C - sample: 50-500 pmol PTH-amino-acid, dissolved in 5-50 gl of the elution buffer: sodium acetate (0.01 mol/1, pH 5.2)/acetonitrile/dichloromethane (68.2+ 31.3 + 0.5; v/v/v) - flow rate: 0.7 ml/min - detection: measurement of the absorbance at 220 nm. For the separation of PTH-Gln and PTH- Ser the column temperature was lowered to 22 °C.

Analysis of the C-terminal Amino-acid Sequence

As described by Beyreuther et al. [7] and Castel et al. [8] eight samples of 60 gg of B 3142 were each dissolved in 240 p.1 of ammonium acetate (0.1 mol/1, pH 5.5) and incubated with carboxypeptidase Y (1.6 p.g enzyme protein) at 37 °C for 5, 10, 30, 60, 180, 300 and 480 min, respectively, after which they were placed in ice and 750 ~tl

of acetone were added. They were thoroughly mixed, left in ice for 1 h and filtered. The filtrate was freed of acetone in a N2 stream and freeze-dried. Finally the residues were each dissolved in 70 gl of sodium citrate buffer (0.2 mol/1, pH 2.2) and analyzed using an "LKB 4400" amino-acid analyzer.

Partial Hydrolysis orB 3142 with Papain and Chymotrypsin

A 300-gg portion of B 3142 was dissolved in 1.5 ml ofpH 8.0 buffer (0.05 tool/1 Tris/HC1, 0.005 tool/1 L-Cysteine, 0.002 mol/1 EDTA- Naz; freshly prepared under N2) and digested with 60 gg papain at 37 °C for 16 h. A 300-gg portion of B 3142 was dissolved in 3.0 ml sodium borate buffer (0.1 mol/1, pH 8.0) and digested with 60 gg of c~-chymotrypsin at 37 °C for 2 h.

The partial hydrolyzates were freeze-dried and separated by HPLC under the following conditions: column: 4.6 mm x 240 mm, ODS-Hypersil (5 gm, Shandon) - temperature: 60 °C - elution system: A) 0.01 mol/l triethylammonium formate (pH 6.0)/acetonitrile (95 + 5; v/v) and B) as for A but 60:40 (v/v) - gradient: linear, 0 to 100% B in A in 100 min (papain digest); 0 to 100% B and A in 60 rain (chymotrypsin digest) flow-rate: 2.5 ml/min - detection: measurement of the absorbance at 220 nm.

After the acetonitrile was removed in a N2 stream the collected fractions were freeze-dried and analyzed for amino-acid composition and amino-acid sequence.

Results

Isolation of B 3142from Wheat Flour

The extraction of gliadin from wheat flour of the strain "Kolibri" was performed according to [9]. For the preparation of the peptide B 3142, gliadin was digested successively with pepsin and trypsin, the partial hydrolyzate was separated by ultrafiltration, gel filtration, cation-exchange and anion-exchange chromatography and HPLC as described in [1]. B 3142 was rechromatographed by HPLC and desalted by gel filtration on Biogel P2. The yield for the pure peptide was about 0.5 mg per 100 g of flour.

Coeliac Activity

LIF Test. Preliminary experiments with seven coeliac patients have shown that among the HPLC fractions examined (B 3142, B 3143, B 3144) only B 3142 causes a LIF-response to all patients [1]. After larger amounts of peptides had been prepared, 24 coeliac patients on normal diet (ND), 29 coeliac patients on gluten-free diet (GFD) and 20 control persons (C) were chosen for further examination of the peptide fractions B 3141-B 3146. The concentrations tested were in the range of 0.1-1.0 ~tg/ml. The results are summarized in Table 1. In the group of ND patients B 3142 and B 3146 show the highest number of positive tests. If the results for the different concentrations are compared B 3142 is active for 88%, B 3146 for 80%, B 3145 for 75%, B 3141 for 67%, B 3143 and B 3144 for 50% of the patients examined. In the group of GFD patients most responses were obtained with B 3145. B 3142, B 3144, and B 3146 were less active,

372

Table 1. LIF activity of peptide fractions obtained by HPLC

Fraction ND" GF D C

lag A b B A B A B

B3141 0.10 0/2 2/3 0/1 0/1 0.25 0/2 (67%) 0/1 (0%) 0.50 2/3 0/1

B 3142 0.10 10/21 3/29 0.25 11/22 21/24 6/24 13/29 0.50 10/24 (88%) 5/29 (45%) 0.75 2/5 0/1 1.00 2/6 2/6

B 3143 0.10 0/2 1/2 0/4 0/4 0.50 1/2 (50%) 0/4 (0%) 1.oo 1/2 0/4

B 3144 0.I0 0/2 1/2 0/3 1/4 0,50 1/2 (50%) 1/4 (25%) 1.00 0/2 0/4

B3145 0.10 1/4 3/4 8/14 10/14 0.25 1/4 (75%) 4/14 (71%) 0.50 2/4 2/14

B 3146 0.10 3/5 4/5 1/7 3/7 0.25 3/5 (80%) 2/7 (43%) 0.50 2/5 0/7

0/20 1/20 2/20 1/20 (10%)

0/2 1/8 0/2 (13%) 1/8

0/2 0/8 0/2 (0%) 0/8

a ND =coetiac patients on normal diet; GFD=coel iac patients on gluten-free diet; C=control persons

b A=number of positive tests/number of performed tests B=number of persons reacting with at least one concentration/ number of all persons tested

Table 2. Effect of B 3142 in the organ-culture test a

B 3142 lag

10 20 50 100

Coeliac patients 1 42 13 74 2 100 74 67 3 62 44 87 4 183 273 -

Control persons 1 66 101 - 2 94 179 126

91

" Alkaline phosphatase activity (%) according to the activity without peptide ( = 100%)

B 3141 and B 3143 were inactive. Altogether B 3142, B 3145, and B 3146 seem to be the most antigenic fractions for both groups of patients. The activities are obviously coeliac-specific, because control persons responded only in a few cases.

Table 3. Incubation of B 3142 with carboxypeptidase Y. - (liberated amino-acids ~)

Incubation time rain

5 10 30 60 120 180 300 480

Tyr 0.03 0.07 0.12 0.23 0.44 0.55 0.78 0.92 Gln+ Ser - - - 0.15 0.43 1.22 2.24 2.50 Pro - - 0.10 0.34 0.83 1.83 1.95 Phe - - - 0.05 0.15 0.30 0.48 0.54

mol/mol peptide

Organ-culture Test. Additionally the coeliac activity of B 3142 was examined in organ cultures with mucosa samples from four ND-patients and two control persons (Table 2). The reduction of alkaline phosphatase activity in the cultures incubated for 24 h was taken as a measure of toxicity. Cultures incubated with B 3142 were compared with cultures without peptide (= 100% activity). The amounts of B 3142 examined were in the range of 10-100 gg. It induced a strong reduction of phosphatase activity with patients 1 and 3, whilst the effects with patient 2 were weaker. The op- timum concentration of B 3142 was about 20-50 gg/ ml. With patient 4 no toxic effect could be observed. With the exception of one sample, cultures from the control persons did not show any significant effect.

N-terminal Amino-acid Sequence. The N-terminal sequence of B 3142 was determined by manual ED- MAN degradation. The PTH-amino-acids were identified by means of HPLC on an ODS-Hypersil. 14 degradation cycles lead to the following sequence: Val- Pro-Val-Pro-Gln-Leu-Gln-Pro-Gln-Asn-Pro-Ser- Gin-Gin-. B 3142 proved to be a pure peptide, because only one PTH-amino-acid per cycle was liberated.

C-terminal Amino-acid Sequence. For the C-terminal sequence analysis B 3142 was treated with carboxypeptidase Y. Table 3 shows the amounts of free amino-acids liberated according to the incubation time. Because Gln and Set were not separated under the standard conditions used for amino-acid analysis, their amounts were determined as a total. From the asymmetric peak in the chromatograms of the higher digestion times (180, 300, 480 rain) it can be concluded that besides Gln also Ser was liberated. The data obtained suggest that Tyr is the C-terminal amino-acid and further that Gln, Pro (two residues each), Phe and Ser (1 residue each) also occur in the C-terminal section.

Preparation and Analysis of Fragment Peptides. For the further sequence analysis, fragment peptides were

373

A97~Iul 2

0.08-

0,04 -

6 7 g

I

~°/, CH3CN i

1

~20

E I

~ ~Io

i D I

10 mio 30 Fig. 1. Hydrolysis of B 3142 with papain: HPLC of fragment peptides on ODS-Hypersil

A22o

o.o

1 ,.~*/o CH3CN

2 ~ J l

f2o I I I I I I

10 min 3'0 Fig. 2. Hydrolysis ofB 3142 with c~-chymotrypsin: HPLC of fragment peptides on ODS-Hypersil

Table 4. Peptides from B 3142

Peptide Amino-acid Amino-acid sequence composition

p A . a

1 BP2SZ4 b Not determined 2 PYZ QPY 3 PYZz Blocked 5 LPZ 2 LQPQ 6 P2 YZ 1 - z QPYP(Q) 7 FGPZ 2 FPGQQ 8 FP2SZ 2 QPFPSQ 9 P 2 V 2 z VPVPQ

10 F2P6SZ 5 QP~P~?SQ 11 FP3SZ 3 QPQPFPSQ 12 LPV 2 VPLV 13 LPV2Z 2 QVPLVQ

CT -a 1 BL2P6SV3Zlo 2 FaGPgVYzZ14

VPVPQLQPQNPSQQQPQEQVP. VQQQQF P GQQQPFPPQQPYPQPQPFPSQQP.

" PA = papain; CT = chymotrypsin b One-letter-code for amino-acids: B =Asx; E =Glu ; F = P h e ; G = G l y ; L = L e u ; N = A s n ; P = P r o ;

Q =Gln ; S = Ser; V = Val; Y = Tyr; Z = Gix

prepared from B 3142 by digestion with papain and e- chymotrypsin. The partial hydrolyzates were separated by HPLC on ODS-Hypersil. The 16-h-incubation with papain yielded a relatively high number of peptides (Fig. 1). The dominating fragments (PA-1 to PA- l4) were collected. From the 2-h-incubation with chymotrypsin two main fragments (CT-1, CT-2) were obtained (Fig. 2).

The collected fragments were analyzed for amino- acid compositions and partially for amino-acid sequences (Table 4). The hydrolyzate from the papain digest consisted of tri- to octa-peptides. PA- 1 was not analyzed for sequence; from the amino-acid composition it can be concluded that the known N-terminal section of B 3142 contains this peptide. PA-3 is proba- bly blocked by pyroglutamic acid. PA-6 consists of two peptides with the sequences Gln-Pro-Tyr-Pro and

Gln-Pro-Tyr-Pro-Gln, similarly PA- 10, which is over- lapped by PA-11. Thus the sequence Gln-Pro-Phe- Pro-Pro-Gin can be deduced for PA-10. PA-8 shows a partial sequence of PA-11 and PA-12 a partial sequence of PA- 13. Fractions PA-4 and PA- 14 were also observed in the blank experiment and have atypical amino-acid compositions.

For the fragment peptides from the chymotryptic digest sequences consisting of 21 and 30 amino-acid residues, respectively, were determined. Obviously CT-1 is derived from the N-terminal section and CT-2 from the C-terminal section of B 3142.

Total Amino-acid Sequence of B 3142. The analyses of N-terminal and C-terminal sequences, the sequences of the two fragment peptides CT-1 and CT-2, obtained by chymotryptic digestion and the sequences of

374

V P V P Q L Q P Q N

P s Q O O P O E Q V

P L V Q Q Q Q F P G . . . . . . . 2 . . . . . . . . . L . . . . . .

Q Q Q P F P P Q Q P . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Y P Q P QP F P S Q

Q P Y

Fig. 3. Amino-acid sequence of B 3142. - - ~ E D M A N degradation,

degradat ion with carboxypeptidase Y, - - peptides f rom papain digest, - - - peptides f rom chymotrypsin digest. One-letter-code for amino-acids: see Table 4

fragment peptides from the papam digest, especially the sequences of PA-13 and PA-2, establish the total primary structure of the coeliac active peptide B 3142, which is shown in Fig. 3. B 3142 consists of 53 amino- acid residues with the composition Gln 21, Pro a 6, Val4, Phe3, Leuz, Ser 2, Tyr 2, Asn 1, Glyl, Glul. This corresponds to a molecular mass of 6,129 g/tool.

D i s c u s s i o n

Coeliac Activity

In the immunological test (LIF-test) B 3142 was active for 88% of the patients on normal diet, the optimal concentration was in the range of 0.1-0.5 ~tg/ml. As expected from previous studies [10] patients on gluten- free diet responded less frequently (45%). The results for the control persons (58 out of 60 tests are negative) indicate that the effect of B 3142 is coeliac-specific. Since larger amounts of the peptide are necessary for the organ-culture test, only a few tests with patients on normal diet and with control persons could be performed. The experiments confirm tile results of the LIF- tests, as well as the results of both test-systems were generally in good agreement in previous investigations [1, 9].

Analytical Methods

The amino-acid compositions of peptides were determined by standard methods. To prevent loss of Tyr during the hydrolysis, phenol was added to the HC1 [11]. The N-terminal sequence was determined by a manual EDMAN degradation according to the tech- nique described by Chang et al. [5]. Phenylisothiocya- nate (PITC) and not dimethylaminoazobenzeneisothiocyanate (DABITC) was used as coupling reagent, because only PTH-amino-acids, but not DABTH-amino-acids can be separated by the HPLC method using an isocratic elution system [6, 12].

With B 3142 16 degradation cycles were performed. PTH-amino-acids of cycles 15 and 16 could not be identified because of flow yields. The increase of cy-

cle numbers, which would have been possible with a higher amount of peptide (200 gg), was not conside- red, because the sequence analysis by means of fragment peptides seemed to be more promising.

For the C-terminal sequence analysis carboxypeptidase Y, which is able to cleave bonds involving Pro, was used. The results obtained are an indication for a possible sequence, which have to be confirmed by sequence analysis of fragment peptides.

For the preparation of fragment peptides from B 3142 two endopeptidases, one with a high (chymotrypsin) and one with a low specifity (papain), were used. According to that a relatively high number of peptides was obtained by the digestion with papain and only two fragment peptides were produced by the chymotryptic treatment.

Primary Structure of B 3142

The purity of B 3142 is assured, because - the molar ratio of the amino-acid residues is essen-

tially integer [1], - rechromatography on ODS-Hypersil using diffe-

rent buffer systems produces single symmetrical peaks [1], only one PTH-amino-acid per cycle is liberated by EDMAN-degradation. The total amino-acid sequence of B 3142 (Fig. 3) is

joined together by N-terminal and C-terminal sequences and by sequences of four fragment peptides: CT-1 and CT-2 from the chymotrypsin digest and PA- 2 and PA- 13 from the papain digest. PA-2 is the C-terminal tripeptide and PA-13 is the overlapping fragment for the peptides CT-1 and CT-2. The sequences of the other fragment peptides from the papain digest are inserted into the total sequence without contradic- tions.

According to the known specifity of the endopeptidases used papain mainly cleaves the peptide bond GIn-X, especially Gln-Gln. c~-Chymotrypsin, which is known to hydrolyze preferentially bonds like Phe-X, Tyr-X, but also bonds like Leu-X and Ile-X (X + Pro) [13], cleaves only one bond of B3142, namely Leu- Val, because all aromatic amino-acids are attached to Pro.

The amino-acid composition of B 3142, deduced from the total sequence is different from that determined by amino-acid analysis [1] in four amino-acid residues (Tyr: + 1; Pro: + 1; Gin: -2 ) , the total of 53 residues is unchanged. Similar to whole gliadin and to single gliadin fractions [9] B 3142 has a high amide content (95.7%). The molecular mass (6,129 g/mol), calculated from the total sequence, is somewhat lower than the molecular mass range determined for fraction B by gel filtration [1].

375

The N-terminal sequence of B 3142 (positions 1- 25) corresponds to the positions 3-27 of the N-terminal sequences, described for some gliadin components (e2-, e8-, eg-, elo-, e11-, e12-, fis-, 71-gliadins) [14]. From this it follows that B 3142 derives from the N- terminal sections (positions 3-55) of the gliadins men- tioned; the N-terminal dipeptide Val-Arg is split offby trypsin. This is a confirmation of previous results by which coeliac activity was ascribed to e-, fl- and 7-gila- dins [9].

Thus the sequence of B 3142 give first insights into a larger part of the N-terminal sections of gliadins. Partial sequences, especially typical for gliadin [15], seem to begin at some distance from the N-terminus. Gin occurs both isolated and accumulated (Gln2, Gin3, Gln4), Pro mainly occurs isolated. The most fre- quent dipeptide sequences are Gln-Pro (7 x ) and Pro- Gln (6 x ) and dominating tripeptide sequences are Gin-Gin-Pro (4 x ), Gin-Gin-Gin (4 x ), and Gin-Pro- Gin (3 x ).

The average hydrophobicity [16, 17] of B 3142 is 1,234 cal/mol, a value much higher than that for whole gliadin (980 cal/mol). The hydrophobicity changes along the peptide chain: A rather hydrophobic section (1,675 cal/mol) at the N-terminus (positions 1 8) is followed by a long hydrophilic section (position 9-19, 471 cal/mol), a short hydrophobic section (positions 20-23, 2,100 cal/mol) and a long hydrophilic section (24-33, 455 cal/mol). The C-terminal section (positions 34-53) is relatively hydrophobic (1,694 cal/mol).

Using the rules of Chou and Fasman [18] for pre- dicting conformations of proteins and peptides, e-he- lix- and /~-bend-structures seem to be unlikely in

B 3142. In contrast, formation of fi-sheet-structures especially within the positions 19-28, seems possible.

References

1. Wieser H, Belitz H-D, Ashkenazi A, Idar D (1983) Z Lebensm Unters Forsch 176:85

2. Ashkenazi A, Levin S, Idar D, Rosenberg J, Handzel TZ (1978) Lancet I:627

3. Ashkenazi A, Idar D, Maimon M, I.iegesh E, Frank E, Hahn T, Wolman Y, Levin S (1984) J Pediatr Gastroenterol Nutr (in press)

4. Tkachuk R, Irvine GN (1969) Cereal Chem 46:206 5. Chang JY, Brauer D, Wittmann-Liebold B (1978) FEBs Lett

93:205 6. Lottspeich F (1980) In: Kaiser UJ, Franzen KH (eds) K6nigstei-

her Chromatographie-Tage 1980. Waters GmbH, K6nigstein, p 274

7. Beyreuther K, B6hmer H, Dimroth P (1978) Eur J Biochem 87:101

8. Castel A, DeGraaf JM, Kraal B (1979) J Chromatogr I74:474 9. Wieser H, Springer G, Belitz H-D, Ashkenazi A, Idar D (1982)

Z Lebensm Unters Forsch 175:321 10. Ashkenazi A, Levin S, Idar D, Or A, Barzilai N, Handzel ZT

(1981) Lancet I:914 11. Blackburn S (1968) Amino Acid Determination. Marcel Dekker,

New York, p 13 12. Wilson KJ, Rodger K, Hughes GH (1979) FEBS Lett 108:87 13. Croft LR (1980) Introduction to Protein Sequence Analysis.

John Wiley and Sons, Chichester New York Brisbane Toronto, p 14

14. Bietz JA, Huebner FR, Sanderson JE, Wall JS (1977) Cereal Chem 54:1070

15. Wieser H, Seilmeier W, Belitz H-D (1984) Z Lebensm Unters Forsch (in press)

16. Tanford C (1962) J Am Chem Soc 84:4240 17. Ney KH (1972) Z Lebensm Unters Forsch 149:321 18. Walton AG (1981) Polypeptides and Protein Structure. Elsevier,

New York Oxford, p 54

Received June 29, 1984

376

Documents

Amino-acid sequence of the coeliac active gliadin peptide B 3142