Canadian Journal Journal canadien Biochemistry

Published by THE NATIONAL RESEARCH COUNCIL OF CANADA

de biochimie Publib par LE CONSEIE NATIONAL DB RECHERWES DU CANADA

The amino acid sequence of wheat P-purothioninl

A. S. M A K ~ AND B. L. JONES Department of Phnr Science, University of Manitoba, Winnij~eg, Man., Canada R3T2N2

Received February 11, 1976

Mak. A. S. & Jones, B. L. (1976) The amino acid sequence of wheat p-pursthionin. Can. J . Biochem. 54,835-842

The complete amino acid sequence sf a-purothionin, a low molecular weight, very basic, protein isolated from wheat endosperm material, has been determined. p-purothionin is toxic to some bacteria. to yeasts, and to animals when injected. The protein contains 45 amino acid residues and has a molecular weight of 4913. The 8 cysteine and 10 basic residues are distributed throughout the molecule. The primary structure of the protein shows considerable homology to those of the viscotoxins, which are toxic, small, basic proteins found in the leaves and stems of European mistletoe (Viscum albltrn k.).

Mak, A. S. & Jones, B. L. (1876) The amino acid sequence of wheat P-purothionin. Con. 3. Biochern. 54,835-842

Nous avons determine la sequence complete des acides amines de la fi-purothionine, une protkine de faible poids moleculaire, tres basique et isolke de I'endosperrne de Isle. La fi-purothionine est toxique pour plusieurs batteries, pour les levures et pour les animaux qui Ia re~oivent par injection. La protkine contient 45 acides aminCs et elle a un poids molCcuiaire de 4813. Huit rtsidus de cysteine et 10 rksidus basiques sont distribues dans la molecule. La structure primaire de la proteine montre beaucoup d'homologie avec celle des viscotoxines, petites protkines basiques, toxiques, trouvtes dans les feuilles et les tiges du gui europken (Vi.~curn album k.).

[Traduit par le journal]

Introduction

The purothionins are a group of low molecular weight proteins which are extracted from wheat flour (as lipoprotein complexes) with organic sol- vents (1-3). They are found in hexaploid, tetra-

A s a w ~ v r ~ s ~ s ~ s : PEP, pyridylethylated fi-purothionin; PECys, pyridylethylated cysteine; DFP, diisopropyl fluorophosphate; SPETC, 4-sulfophenylisothiocyanate; AN§, 2-amino- 1.5-naphthalene-disdfonic acid; PTH, phenylthiohydantoin; GLG. gas-liquid chromatography; TLC , thin-layer chromatography; BSA, N, 0-&is(tri- methylsi1yl)-acetarnidea

'Contribution No. 432, Department of Plant Science, University of Manitoba.

2Present address: Department of Biochemistry, Univer- sity of Alberta, Edmonton, Alta.

plaid, and diploid species of the genus Triticurn (4). The chemical and physical properties of these pro- teins have been studied thoroughly (5) but until re- cently there has been confusion about their molecu- lar weights. Study of the peptides released upon chymotryptic hydrolysis of a- and p-purothionins (6) indicates they have molecular weights around 5000, not 12 000, as was long believed.

The in vivo function of the purothionins is not k n ~ w n but they are toxic to animals when injected intraperitoneally (7) and to some bacteria (8, 9) and yeasts (8, 10).

The purothionins are quite different from most other flour proteins. They contain about 20% cys- tine, 10% arginine, and lysine but lack meth- ionine, tryptophan, and histidine (5, 11).

This sequence study was undertaken to obtain

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836 CAN. J. BIOCMI

information which would explain the physical and chemical properties of the purothionins and which would shed light on the genetic origins of these pro- teins and on their functions in the endosperm.

All reagent chemicals used in the Beckman model 890C sequencer were either Sequencer Grade from the Beckman Company or were Pierce Chemical Company Sequenal grade. Sequencer solvents were Distilled-in-Glass grade from Burdick and Jackson.

The method of Wedman and Fisher (12), as modified by Mak and Jones (6), was used to prepare a crude mixture of a- and &purothionins. These were separated by ion- exchange chromatography on carboxymethyl cellulose (Whatman CM 52) (6). The p-purothionin thus obtained was free of protein contamination as assayed by starch gel elec trophoresis in aluminum lactate buffer at pH 3.1 (13).

The ppurothionin was hydrolyzed to peptides suitable for sequencing with chymotrypsin (Worthington Biochem- ical Corp.) by a method previously described (6). When large peptides were needed, the native protein was hy- drolyzed for 15 f i n and the resulting peptide mixture was reduced (14) and alkylated with freshly distilled 4vinyl pyridine (15) before separation of the peptides on carbox- ymethyl cellulose (6). When smaller peptides were desired, the protein was reduced and alkylated before chymotryptic hydrolysis. The PEP was then hydrolyzed for 30 min and the resulting pyridylethylated peptides were separated as before.

Amino acid compositions of the separated peptides were determined on a Beckman 121 amino acid analyzer after hydrolysis, under nitrogen, in 6 N HCI at 110 "C for 24 h. The pH of the sodium citrate buffer used to elute the short column was altered to pH 5.5 to allow determination of the PECys residues (15, 16).

C-terminal residues of the protein and various peptides were d termined with carboxypeptidase B or C (17)- For pepti+ digestion, reduced and pyridylethylated peptide (0.5 pmol) was dissolved in 200 pl 0.02 M sodium phos- phate buffer (pH 8.0). Thirty microlitres of carboxypep- tidase B (EC 3.4.2.2, Worthington, DFP treated, 6 mg/ml) was added and the solution was incubated at 37 "C. Ali- quots (one-third of the reaction mixture) were removed after 15,60, and 120 min. Each aliquot was acidified with 50 pl of acetic acid. A slightly different method was used with PEP. The pyridylethylated protein (1.6 pmol) was dissol- ved in 8.5 ml of 100 mM NaCl and 25 mM Tris buffer. pH 7.65. Carboxypeptidase B (Sigma, DFB treated, 2 mglml) (38 pi) was added and the solution was incubated at 37 "C. Aliquots (1.0 ml) were removed at appropriate intervals, acidified with 20 pl of acetic acid, and freeze-dried.

A carboxypeptidase C purified from germinated wheat (18) was used to determine the C-terminal sequence of the native protein. Protein (1.9 ymol) was dissolved in 9 ml of 25 mM sodium acetate buffer, pH 4.0. Three millilitres of carboxypeptidase C enzyme with an activity of 3.0 pmol glycylglycine per millilitre (19) was dialyzed against the sodium acetate buffer to remove low molecular weight contaminants. The substrate and enzyme solutions were filtered through millipore bacterial filters, prewarrned to 35 "C, and mixed. Two-millilitre aliquots were removed at intervals and immediately freeze-dried. All carboxypep-

tidase assay aliquots were dissolved in (or adjusted to) 1.0 ml with sodium citrate buffer (0.2 N, pH 2.2) and analyzed for free amino acids on an amino acid analyzer.

Two methods were used to alter the charge properties of nonpolar small peptides so they could be sequenced au- tomatically. The method of Braunitzer (20) as performed by lnman (21) was used to couple SPITC to lysine- containing peptides. Alternatively, the method of Foster eb al. (22) was used to attach ANS to the C-terminal residues of some peptides.

All sequencing was done on the automated sequencer. Peptide or protein sample (0.5 to 2 pmol) was dissolved in 650 pl of water or 163% acetic acid and applied to the reaction cup. All samples were dried using the Beckman sample application routine No. 02772. The Beckman DMAA-peptide program No. 802974 was used for all de- gradations. The first cycle was usually started with the cleavage step so that the protein was washed with chlorobutane to remove contaminating substances before proceeding to the actual degradation. Ethanethiol was added to the chlorobutane (0.1%, vlv) to stabilize the thiazolinone derivatives (23). To avoid loss of the volatile ethanethiol, chlorobutane extracts were not automatically dried in the fraction collector. When serine was suspected to be the degradation product, conversion was performed as soon as the cleaved thiazolinone was delivered.

Conversion of the dried thiazolinone derivative to the PTH derivative was performed manually by heating the thiazslinone at $0 "C for 18 min in 1 .OAT HCI containing 1% v/v ethanethid. The PTH amino acids were extracted with ethyl acetate (twice, with 63.7-ml aliquots) and dried with a stream of N2. The dried sample was dissolved in 50 pl of ethyl acetate for analysis. The polar PTH-amino acids, PTH-Arg and PTH-PECys, remain in the aqueous phase during the ethyl acetate extraction. The aqueous phase was dried with N, and redissolved in 25 pl of methanol.

A combination of GLC, TLC, and spot tests was used to identify the PTH-amino acids. All GLC analyses were performed on a Hewlett-Packard 5711A gas chromato- graph equipped with a flame-ionization detector and an au- tomatic sample injector. The procedures of Pisano eb 661.

(24) and Hermodson ef al. (23) were adapted. A silanized glass column (4 in. x 4 M) was packed with deactivated, acid-washed chromosorb W coated with 10% SP-400 (Supelco, Hnc.) and conditioned at 300 "C for 16 h. Nitrogen was used as carrier gas at a flow rate of 65 crn3/min. Sam- ples were analyzed directly as PTH-amino acids and (or) after "'on column" trimethylsilanization with BSA (In Se- quence, 1972, pp. 62-5, Beckman Company). The GLC temperature program started with an isothern~al elution (170 "C) for 8 min, followed by a linear temperature gra- dient (8 "Chin) from 170 to 290 "C and ended with an isothermal elution (298 "C) for 2 min. The PTH-amino acids in the ethyl acetate extract were separated with this temp- erature program. PTH-PECys was identified in the methanol solution by GLC using an isothermal 220 "C program.

Thin-layer chromatography, either one- or two-di- mensional, was used to identify or confirm labile PTH- amino acids. To distinguish PTH-Asn from PTH-Asp, and PTH-Gln from PTH-Glu, the solvent system (chloro- form-methanol, 98:10, v/v) of Bsenner ef c t l . (25) was used with silica gel plates (5 x 5 cm, silica gel 1B2F, J . T. Baker Chemical Company). The 2D systems of Sum-

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TABLE 1 . Amino acid compositions of /3-purothionin and of the chymotryptic peptides sequenced during analysis of fi-purothionin. Results are expressed as mole ratios with

respect to the amino acid marked * in each column

Amino acid PEP chi31 chp3 chp14 chi31 5

EY s Arg 4-PECysd ASP Th r Ser Glu Pro G ~ Y Ala Val Leu TYH Phe

6 . 3 (6)" 4.3 (4) 8 .4 (8) 4 .4 (4) 2:o (2) 4 .0 (4) 1.0 (1) 2 .2 (2) 3.4 (3) 3.0 (3) 1 .0 (1) 5 .4 (5) 1 . 0* (I) 1.0 (1)

0 .3 (0) 1.0 (I ) 1 .o (1) 0.1 (0)

2.2 (2) 1.8 (2) 1.0 (1)

1 .0 (1) 2.8 (3) 1.3 (1) 1 .0 ( I )

0 .9 (1) 1.0 (1) 1.8 (2)

"Pyridylethylated /3-purothionis. *Peptide numbering system is that of reference 6. 'Number in parenthesis has been adjusted to the nearest integer. dPyridylethylated cysteine.

DEGRADATION CYCLE

FIG. I . Quantitative yields of PTH-Leu, PTH-GIy. PTH-Ala, and PTH-Val obtained from a typical sequencer run with PEP. Six milligrams (1040 nnmsl) of PEP was subjected to 33 degradation cycles. The amounts of the various PTH-amino acids formed during each cycle were calculated and corrected for Boss by comparison with an internal standard (PTH-Met). The slope and intercept were calculated by regression analysis. A , BTH-Leu; 0 , PTH-Gly ; x , PTH-Ala; e, PTH-Val.

mers et a[. (26) on double-faced polyamide sheets 65 x 5 cm, Cheng-Chin polyamide or Schleicher and Schuell mi- cropolyamide sheets) were used to identify PTH-ser and PTH-Lys. PTH-Arg was detected by the spot test of Yamada and Itano (27) as modified by Hermodson e f ai. (23). The ammonia vapor spot test (28) was used to confirm the identity of residues believed to contain PTH-61y.

All proteins and peptides were sequenced at Beast three different times. Figure 1 shows the yields d selected PTW-amino acids obtained from a typical sequencing ex- periment, showing a repetitive yield of about 90% and an initid yield of 60%. No evidence of contamination by non- purothionin protein was found.

Results and Discussisn

The electrophoretically pure ,8=purothionin used in this study did not contain any His, Met, Hle, or Trp and its amino acid composition (Table 1) was in good agreement with that reported by other laboratories (5,6, 12). We have previously shown (6) that when native P-purothionin was hydrolyzed for IS min, it was cleaved into two peptides (chPI4 and chfiE5, using the naimbering system of reference 6). Since one of the resulting peptides (chpl4, Table 1B con- sisted of 13 amino acid residues and contained a C-terminal Tyr residue (61, while native 6-puro- thionin had Lys at its C-terminus, it was apparent that residue 13 from the N-terminus of the protein was Tyr. Hydrolysis at this residue occurred read- ily, but n o cleavage at the single $he residue could be detected.

A sample of reduced and pyridylethylated P-pu- rothionin was therefore subjected to 18 cycles of automated sequence analysis, the results of which are shown in Fig. 2A. The first 13 residues from the N-terminus of the protein accounted for the corn-

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CAN. J. BIBCHEM. VOL. 54, 1976

(33) (38) 20 25

Lys-Leu-Thr- - -Leu- - -Pro-. . . . . . . ..* -Lys-COOH a . ~ ~ ~ ~ I ~ ~ ~ L

(34)(35)(36)(37) 1 2 3 4

C chpl W2N-Thr-Ser-Gly-Leu-COON Modified with ANS a>--

(38) (4) (45) 1 3 8

D chP3 (i) H2N- -Cys-Pro- -Asp-Phe-Pro- -COOH Modified with SPBTC -2-A-2-A

(ii) H2N-Ser-Cys-P~cP-Lys- -Phe-Pro- -CQOH Modified with ANS ->-\-ass-

(iii) H,N-Ser-Cys-Pro-Lys-Asp-Phe-Pro-Lys-COOH Complete sequence

FIG. 2. Partial amino acid sequence of PEP and complete or partial sequences of chymstryptic pegtides used for determination of the complete sequence of fi-purothionin. (A) Sequence of the first 18 residues from the N-terminus of PEP. (B) Partial sequence of cfBPI5. (C) Complete sequence of chpl. (D) Complete sequence of chp3: (i) sequence obtained for the SPITC modified peptide; (ii) sequence obtained for the ANS modified peptide; (iii) complete sequence of the peptide based on information from i and i i . The C-terminal Lys was determined by subtraction. (E) Sequence of PEP from the (3-terminus (using carboxypeptidase hydrolysis method). Number in parenthesis represents position of residue in the parent protein. Number below each parenthesis represents position of residue in the peptide concerned. Arrow indicates direction of degradation. All Cys residues were identified as PECys.

plete amino acid content of the peptide chpl4 (Table I ) and the N-terminal amino acid was Lys, as had been reported by other investigators (1 1). ChS15, the other peptide formed by limited hydrolysis sf the p-purothionin, was then sequenced (Fig. 2B). Re- sidue 27 of this peptide was identified but residues 22.23.25, and 26 could not be determined. Residues 1 to 5 of chB15 overlapped with residues 14 to 18 of whole /3-purothionin, confirming chymotryptic cleavage at Tyr residue 13. Hydrolysis of chp15 with carboxypeptidase B revealed that the @-terminal residue was Lys, as expected.

Sequence analysis of the peptides released by limited chymotryptic hydrolysis of the protein thus disclosed the positions of 36 of the 45 amino acid residues. We have previously shown (6) that hy- drolysis of denatured (reduced and pyridylethy-

lated) purothionins by chymotrypsin fur 30 min caused scission of the protein at bonds adjasent to Leu as well as Tyr residues, producing a total of 15 relatively small peptides, which were separated by chrsrnatography. Amino acid anaHyses of the smal- ler peptides indicated that peptide chbl, a tetrapep- tide, contained one residue each of TInr and Leu (Table 1). Since this peptide was known to possess a C-terminal Leu residue (6), it seemed probable that It occupied the position from residue 34 through 37.

When peptide chpl was sequenced, only one re%- idue (Thr) could be determined because of its small size and nonpolar nature. The peptide was there- fore reacted with ANS (22) and resequenced. All four of the residues were then determined (Fig. 2@), including the C-terminal Leu. The tkiazolinone de- rivative of this Leu (modified with ANS) was appar-

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MAK AND JONES 839

FIG. 3. Release of amino acids from the C-terminus of native p-purothionin by a wheat carboxypeptidase C. hrothionin (1.9 pmoi) was hydrolyzed with a carboxypep tidase C enzyme isolated from germinated wheat (18). Ali- quots were removed from the reaction mixture at intervals and free amino acids were determined on an amino acid analyzer.

ently partially (-70%) hydrolyzed to PTM-Leu dur- ing the conversion steps, since PTH-Leu was found in the ethyl acetate extract of cycle 4.

Peptide chp3 contained all eight of the residues not then positioned, including the two Pro residues of P-purothionin, one of which was known (Fig. 2B) to occupy position'40. This peptide was modified with SPITC (21), to increase its polarity, and se- quenced (Fig. 2D. a). Since SPITC reacts with all free amino groups, the W-terminal residue and all Lys residues were not determined. To position these

residues, the peptide was derivatized with AN% and sequenced (Fig. 2B, ii). That no residue was found at position 42 after derivitization with AWS (which specifically reacts with free carboxyl groups) confirms the fact (as determined from TLC) that the Asx at that position was Asp and not Asn. That Lys occupies position 45 was determined by subtraction and confirmed by the fact that this residue reacted with SPITC (Fig. 2D, i),

It then remained to determine whether the Lys residue at position 45 (as determined from chp3) was the C-terminal Lys of /3-purothionin. Preliminary experiments with a- and p-purothionins , using a mixture sf carboxypeptidases A and 8 , had indi- cated that the penultimate residue of both proteins was probably Arg (29). When PEP was subjected to hydrolysis with a DFP-treated carboxypeptidase B completely free of chymotryptic activity, however, only a single Lys residue was released and no Arg was detected in the hydrolysate, indicating the penultimate residue was not Arg. To confirm the C-terminal sequence, the native protein was hy- drolyzed with carboxypeptidase C isolated from germinated wheat (18). The results (Fig. 3, the aver- age of two experiments giving almost identical re- sults) indicate that the sequence of amino acids at the C-terminus is -Asp-Phe-Pro-Lys-C88H (Fig 2E). This sequence is identical with that of the last four residues of chp3. Since this portion contains the only Phe residue of P-purothionin. chp3 must be the C-terminal peptide, and the Eys at position 45 is the C-terminal residue. The original incorrect results apparently arose from the fact that the carboxypep- tidase A was contaminated with chymotrypsin, which we have found (unpublished observation) will readily hydrolyze -Arg-PECys- bonds, leaving the Arg susceptible to attack by carboxypeptidase B.

The complete amino acid sequence of P-puro- thionin is shown in Fig. 4. The molecular weight of the protein is 4913 (assuming four disulfide bonds, since the protein has no free -SH groups). This value is quite close to the value proposed by Wimmo eb a/ . ( 5 ) but is less than half that accepted up to the

PIG. 4. Complete amino acid sequence of fl-purothionin. All Cys residues were identified as pyridylethylated Cys (PECys).

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840 CAN. J . BIOCHEM. VOL. 54, 1976

1 5 10 15

Viscotoxin A, H2N- -Pro-ASHI- -HBe- Tyr-Asw- Ala- Cys-Arg Leu-Th-

8-purothionin H2N- -Lys-Ser- -eys- n Tyr-Asn- Leu- n cys-Arg -Lila-kg-

Fro. 5. Comparison of amino acid sequences of viscotoxin A3 and p-pursthionin. Numbering of the residues is based on the sequence of viscotoxin A3. Chemically identical or similar residues are enclosed by boxes. Chemically similar residues are defined as Arg = Lys, Leu =Val = Ile, Thr = Ser. Tyr = Phe. Insertions at residues 25 and 45 and a deletion at residue 42 are made to maximize homsle~gies between the two proteins.

time this study was instituted. The cystine residues are scattered throughout the molecule except that they are missing from the extreme C-terminal reg- ion. The basic Lys and Arg residues are likewise distributed throughout the molecule. The very basic nature of the protein is enhanced by the h c t that of five Asx and Glx residues only one (residue 42) has an acidic side chain.

Since the purothionins are toxic to animals, bac- teria, and yeasts, the primary structure of /3-puro- thionin was compared with those of toxins from other sources. None of the toxins from animals have structures like P-purothionin, but there is a group of small proteins, the visestoxins, which have been isolated from the European mistletoe (Wscunz album L .), are toxic to animals in small doses (301, and have primary structures (31. 32) very similar to P-purotkionin (Fig. 5). It is particularly striking that the first four amino acids of p-palrothionin and of the viscotoxins are exactly the same. In addition. if one assumes that a residue is deleted from P-purothionin at position 25 of viscstoxin A3 (Fig. 5) all of the Cys residues of the viscotoxin are aligned with Cys res- idues of the purotk.lisaain. p-hrothionin does, how- ever, contain two more Cys residues than do the viscotoxins.

The disulfide bridging between Cys residues of viscotoxin A3 has been determined (33) and these bridges form the molecule into two small loops and a large loop (Fig. 6A), If one assumes that the homologous Cys residues of P-purothionin form disulaide bonds identical with those of viscotoxin (Fig. BB), then the two extra Cys residues of

purothionirm (residues 12 and 29) are aligned so they can form a disulfide bond and still maintain the basic three-loop structure of the viscotoxins. Bf these loop structures are indeed found to have been highly con- served during the evolutionary process they are probably quite important in determining the function of the proteins.

The %act that very similar proteins have been found in plants as different as wheat (a mono- cotyledonous plant) and mistletoe (a dicotyle- donous plant) indicates that the proteins must per- form some important role in 11ivo. Since they are toxic to some bacteria and yeasts they may play some part in protecting the plant from attack by pathogenic microbes.

Purothionin-Hike proteins have been extracted from endosperm material of wheat and barley ( 6 2 ) and from diploid species of Trittcurn and Aegilops (41, but were not found in rye flour ( I I ) , although rye is more closely related to wheat than is barley (34). The finding that homologous proteins occur in mis- tletoe leaves and stems as well as in wheat seeds indicates that they are probably more widely distri- buted than has been thought. It may well be that they have not been found in rye and some other seeds (1 1) because the extraction process used with wheat flour extracts only that plarothionin which forms a complex with lipid material. The lipoprotein is prob- ably an artifact and if this artifact does not form, either because no lipid is present or for some other reason, any purothionin-like material would be over- looktd. The viseotoxin purification method extracts the protein without need of the lipoprotein complex

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"2 Cys - Tyr - 4

=@ * A r p - c ~ ~ - ' ' ' - ~ s @ . <@

29 9, @0 s 2 5

* z 4a, C 9 I \

FIG. 6. Cc~rnparison of the positions of disulfide bonds in viscotoxin A3 and those (predicted) in ,&purothionin. (A) Arrangement of the disulfide bonds in viscotoxin A3 determined by Sarnuelsson and Pettersson (33). The exact positions of the disulfide linkages between residues 3 and 4 and residues 32 and 40 are not certain. (B) Predicted arrangement of the disaalfide bonds in /3-purothionin.

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and, if applied to rye flour, might yield a puro- 14. Redman, D. G. 8 Elton, G. A. H. (1969) 9. Sci. Food thionin-like protein. Likewise, application sf the Agric. 28,546549 method to parts of the wheat plant other than the 15. Friedman, M . , Krull, L. PI. & Cavins, J . F. (1970) 9.

endosperm might yield purothionin. Such an inares- BioE. Chern. 245,3868-38'91

tigation is, however, impeded by the lack of a suit- 16. Cnvins, J. F. & Friedman, M. (1968) J . Bird. Chern.

able assay method for the desired proteins. 243, 3357-3 360

17. Ambler, R. P. (1972) Methods Enzpmob. XXk', 143-154

Acknowledgments 18. Preston, K. 8. & Kmger, J . (1976) Plant Physiol. in press We thank John Merritt and 19- Preston, K. R. (1975) Cereul Chcm. 52,451458

for their technical assistance, and the NRCC for 20. Braunitzer, G., Schrank, B. & Ruhfus, A. (1970) supporting the research; we are especially indebted Hoppe-Seyler's Z . Physiol. @hem. 391, 1589-1590 to Dr. K. Preston for s u p ~ l ~ i n g the carbox~pep- 21* Inman. J. K., Hannon. J. E. & Amella, E. (1972) tidase C enzyme and helping with the C-terminal Biochem. BiopFzys. Res. Cornfizun. 46: 2075-2081 determinations using this enzyme. 22. Foster, J. A., Bruenger, E., Hu, C. E., Albertson, J.

& FranzMaw, C. (1973) Biochem. Biophps. Res. 1. Balls, A. K.. Hale, W. S. $ Harris, T. H. (1942) Cornmun. 5.3,7&74

Cereal fiean. 19,279-288 23. Hermodson, M. A.. Ericsson, L. H., Titani. K.. 2. Nimmo, C. C., B'Sullivan, M. T. & Bernardin, J. E. Neurath, H. & Walsh, K. A. (1972) Biochemistry 11,

(1968) Cereal Chem. 4P,28-36 44934502 3. Fisher, N., Redman, D. 6. && Elton, g;. A. H. (1968) 24. Pisano, J . J. , Bronzert, T. J. 2% Brewer H. B., ~ r .

Cereal Cliern. 45,48-57 (19'92) Anal. Biochern. 45,43-59 4. Carbonero, p. Garcia-Blmedo, F. (1569) 25. Brenner, M., Niederwieser, A. & Pataki, G. (1961)

Experientin 25, 1110 Experientiu 17, 145-153

5. Nirnrno, C. C., Kasarda, D. D. $t Lew, J . E. (1974)~. 26. Summers, I'd. Ha., Smytkers, G. W. B ~ r o s z l a n , S. Sci. Food Agric. 25,607-6 17 ( 1973) Anal. Biocbtem. 33,424-628

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