Plant Physiol. (1983) 73, 1008-10120032-0889/83/73/1008/05/$00.50/0

Purification and Characteristics of an Endogenous a-AmylaseInhibitor from Barley Kernels'

Received for publication June 24, 1983 and in revised form August 12, 1983

RANDALL J. WESELAKE, ALEXANDER W. MACGREGOR, ROBERT D. HILL,AND HARRY W. DUCKWORTHGrain Research Laboratory (R. J. W., A. W M.), Canadian Grain Commission,Winnipeg, Manitoba R3C 3G8 Canada; and Departments ofPlant Science (R. J. W., R. D. H.) andChemistry (H. W. D), University ofManitoba, Winnipeg, Manitoba R3T2N2 Canada

ABSTRACT

An inhibitor of malted barley (Hordeum vulgare cv Conquest) a-amylase II was purified 125-fold from a crude extract of barley kernelsby (NH-)42SO4 fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a proteinwith an approximate molecular weight of 20,000 daltons and an isoelectricpoint of 73. The protein was homogeneous, as assessed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysisindicated the presence of about 9 half-cystine residues per mole. Theneutral isoelectric point of the inhibitor suggested that some of theapparently acidic residues (glutamic and aspartic) existed in the amideform. The first twenty N-terminal amino acids were sequenced. Somehomology appeared to exist between the a-amylase II inhibitor andtrypsin inhibitor from barley. Complex formation between a-amylase IIand the inhibitor was detected by the appearance of a new molecularweight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitorhad to be preincubated for 5 min, prior to assaying for enzyme activitybefore maximum inhibition was attained. Inhibition increased at higherpH values. At pH 5.5, an approximately 1100 molar excess of inhibitorover a-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1molar ratio of inhibitor to enzyme produced the same degree of inhibition.

Frydenberg and Nielsen (7) made an in-depth study of thepolymorphism of germinated barley a-amylase using zone elec-trophoresis on agar gel. They showed that heating of maltedbarley extracts at 70°C for 15 min resulted in disappearance ofsome a-amylase bands and enrichment of others. The heattreatment appeared to cause the conversion of a heat labile bandof a-amylase to a more stable form having a different electro-phoretic mobility. A similar phenomenon was described later byMacGregor and Ballance (14). Using isoelectric focusing, theseauthors reported the presence ofthree major polymorphic groupsof a-amylase, designated a-amylases I, II, and III, in extracts ofmalted barley. Quantitative determination ofthese groups beforeand after a 15-min heat treatment at 70°C showed almost com-

Supported by a Canadian Wheat Board Fellowship to R. J. Weselakeand by the Natural Sciences and Engineering Research Council ofCanadaGrant No. A4689.

Paper No. 522 of the Grain Research Laboratory, Canadian GrainCommission, Winnipeg, Manitoba R3C 3G8 Canada. Paper No. 648 ofthe Department of Plant Science, University of Manitoba, Winnipeg,Manitoba R3T 2N2 Canada.

plete conversion of a-amylase III to a-amylase II. More recentresults (24) have indicated that a-amylase III is a complex formedby a-amylase II and a proteinaceous factor. Furthermore, thisfactor inhibited a-amylase II of both germinated barley andwheat but did not inhibit the a-amylase I group. Because theinhibitor is heat labile, a 70XC heat treatment disrupts the a-amylase II-inhibitor complex and liberates a-amylase II thusexplaining the conversion of a-amylase III to a-amylase II duringheating.Heat stable inhibitors of cereal a-amylases have been reported

in winter wheat (22, 23) and maize (1) kernels. Purothioninshave been implicated also as inhibitors of wheat a-amylase (9).However, most research on a-amylase inhibitors has been re-stricted to a family of albumins isolated from wheat kernels (3).These albumins do not have inhibitory activity against cereal a-amylases.The current investigation describes the purification, amino

acid composition, and some physicochemical properties of thefactor that binds specifically to, and inhibits a-amylase II, con-verting it to a-amylase III. Some parameters affecting the en-zyme-inhibitor interaction will be discussed.

MATERIALS AND METHODS

Purification of Inhibitor. The inhibitor was purified frombarley (Hordeum distichum cv Klages) kernels, essentially asdescribed by Weselake et al. (24). Flour prepared from dehuskedbarley kernels (85 g) was extracted for 1 h (4C) with 420 ml of20 mm sodium acetate buffer (1 mm CaC12, pH 5.5). Subsequentpurification procedures were performed at 4°C. After centrifu-gation, the material precipitating from the supernatant between40% and 70% (NH4)2SO4 was resuspended and dialyzed against5 mM Tris-HCl buffer (1 mm CaC12, pH 8.0). After dialysis, thematerial (15 ml) was applied to a DEAE-Sephacel (Pharmacia)ion-exchange column (2 x 44 cm) equilibrated with the samebuffer. After sample application, the column was washed withapproximately one bed volume of equilibration buffer at a flowrate of 40 ml/h. A linear gradient consisting of 300 ml equilibra-tion buffer and 300 ml of buffer containing 150 mm NaCl thenwas applied. The same flow rate was maintained and 5.3-mlfractions were collected. Conductivity measurements were madeat room temperature on every tenth fraction using a YSI modelConductivity Bridge (Yellow Springs Instrument Co.) equippedwith a type CDC 314 conductivity probe (Radiometer). Inhibitoractivity and A at 280 nm were determined. The inhibitor solu-tion, eluting in a volume from 405 to 450 ml, was pooled andconcentrated by pressure ultrafiltration (Amicon UM2). Theconcentrate (10 ml) was applied to the bottom of a Bio-Gel P 60(100-200 mesh) gel filtration column (2.6 x 77 cm), equilibrated

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BARLEY a-AMYLASE INHIBITOR

with 5 mm Tris-HCl buffer (1 mM CaC12, pH 8.0). The columnwas eluted using an upward flow rate of 15 ml/h and 5.6-mlfractions were collected. Active fractions, eluting in the range of220 to 270 ml were pooled, concentrated (Amicon, PM 10), andfrozen at - I5C for subsequent analysis.

Preparation of a-Amylase II. One kg of green malt fromConquest barley was extracted with 2 L of 200 mm acetate buffer(1 mM CaCl2, pH 5.5). After centrifugation (10,000g, 10 min),the extract was heated at 70°C for 15 min to remove ,B-amylase,was cooled, centrifuged, and dialyzed extensively against 20 mMacetate buffer (1 mM CaC12, pH 4.75). The extract was added toa column (4.3 x 36 cm) of carboxymethyl cellulose equilibratedwith the same buffer and, after thorough washing with startingbuffer, the column was eluted sequentially with 80 mm acetatebuffer (pH 4.75) to remove a-amylase I (13) and 200 mm acetatebuffer (pH 4.75) to remove a-amylase II. This enzyme wasdialyzed against 100 mm acetate buffer (1 mm CaCl2, pH 4.75)and purified further on a column (2 x 90 cm) of carboxymethylcellulose using a linear gradient of 1 L of 100 mm acetate bufferand 1 L of200 mm acetate buffer (both at pH 4.75 and containing1 mM CaCl2). Affinity chromatography (21) was used as a finalpurification step to yield an enzyme of high purity as assessed byisoelectric focusing followed by protein and enzyme activitystaining.

Protein Determination. The protein content of inhibitor anda-amylase solutions was determined by the method of Lowry etal. (12). Absorbance measurements at 280 nm were used todetect calibration proteins after gel filtration on Bio-Gel P 100.Assay of a-Amylase and Inhibitor Activity. All enzyme and

inhibition assays were performed at 35°C. The Briggs (2) assayfor a-amylase was adapted to measure inhibitor activity at pH8.0 (40 mm Tris-HCI, I mm CaC12) during purification steps.One ml of inhibitor solution was preincubated for 15 min withI ml of appropriately diluted barley a-amylase II, containing 100Ag of BSA. Control digests without inhibitor were prepared. Thereaction was started by adding 2 ml of ,B-limit dextrin (0.65 mg/ml) and, after 15 min, stopped by adding 10 ml of acidified I2-KI (0.05 N HCl, 0.5 mg KI/ml, 0.05 mg I2/ml). Loss of iodine-binding-capacity was determined at 540 nm.

In other experiments, with purified inhibitor, a reducing sugarassay (20) was used to determine a-amylase and inhibitor activ-ity. One hundred g1l of inhibitor solution was preincubated with100 ,ul of a-amylase II, containing 1 mg/ml of BSA. The reactionwas started by adding 200 ,d of 1% soluble starch (Baker) andterminated by adding alkaline copper reagent. One unit of a-

amylase activity liberated 1 gmol glucose equivalent/min.Under the conditions of the assay:

Inhibitor Activity = (Amylase Activity Without Inhibitor) -(Amylase Activity With Inhibitor)

One unit of inhibitor activity nullified 1 unit of a-amylaseactivity.

Preincubation time required for maximum inhibition to occurwas studied at pH 5.5 (200 mm sodium acetate, 1 mM CaCi2)and at pH 8.0 (40 mm Tris-HCI, I mm CaCI2). Solutions ofenzyme and inhibitor were preincubated for different time pe-riods prior to addition of soluble starch. Enzyme solution wasused to initiate the reaction where the effect ofzero preincubationtime was studied. Enzyme reactions in these time dependentstudies were allowed to proceed for 7 min.The effect of different concentrations of inhibitor on a fixed

amount of enzyme (0.10 ,ug) was studied at pH 5.5 (200 mmsodium acetate, I mm CaCl2) and 8.0 (40 mM Tris-HCI, 1 mMCaCl2). Enzyme and inhibitor were preincubated for 15 minprior to the addition of soluble starch. Enzyme reactions wereallowed to proceed for 8 min at pH 5.5 and 22 min at pH 8.0.Mol Wt Estimation. The mol wt ofthe inhibitor was estimated

by SDS-PAGE using the Laemmli (I 1) system. Marker proteinswere from Pharmacia and 10 Mg of purified inhibitor was used.Mol wt ofinhibitor, a-amylase, and enzyme-inhibitor complex

were estimated by gel filtration on a column (1.6 X 54 cm) ofBio-Gel P 100 (100-200 mesh) at pH 8.0 (40 mM Tris-HCI, 1

mM CaCI2, 4C). Samples were applied to the bottom of thecolumn in 0.50 ml of equilibration buffer. The column waseluted with an upward flow rate of 6 ml/h and 1.93-ml fractionswere collected. Calibration proteins were from the Sigma Chem-ical Co.Twenty Mg of inhibitor were applied to the column and frac-

tions were assayed for inhibitor activity at pH 8.0. In a separaterun a-amylase II (9 ,g) was applied to the column and fractionswere assayed for a-amylase at pH 5.5 (200 mm sodium acetatebuffer, I mM CaCl2). A mixture of inhibitor (76 ug) and a-

amylase II (9 ug) was preincubated for at least 15 min prior tocolumn applications. Elution of a-amylase II-inhibitor complexwas detected by assaying fractions for a-amylase activity at pH5.5 and elution of excess inhibitor was detected by assayingfractions at pH 8.0 for inhibitor activity.

Isoelectric Point Determination. Analytical polyacrylamide gelisoelectric focusing (1C) of the inhibitor and subsequent visual-ization was done as described previously (24). The gradient ofpH in the gel was determined using a surface electrode.Amino Acid Analysis and Sequencing. The method for amino

acid determination has been described by Duckworth and Bell(5). Tryptophan was determined as described by Edelhoch (6)and half-cystine was determined after oxidation to cysteic acidby the method of Moore (17). Amino-terminal sequencing wasperformed using a Beckman 890C Sequencer, with 2 mg Poly-brene in the spinning cup. A 0.1 M Quadrol program was used,Beckman catalogue no. 030176. Phenylthiohydantoins ofamino

Table I. Purification ofa-Amylase Inhibitor

Total Total SpecificFraction Volume Total Inhibitor Activity Purification RecoveryPoen Activitya

ml mg anti units anti -fold %mg ~~~~units/mgCrude extract 353 1272 561,100 440 1 10040-70%(NH4)2SO4(after dialysis) 15 240 235,500 980 2 42

DEAE-Sephacel(after concn.) 10 7.8 167,000 21,410 49 30

Bio-Gel P 60(after concn.) 5 1.9 105,000 55,260 125 19' Based on Briggs (1961) assay.

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x

I

Eri?aZ0I.

c0

N ..

Gouswic<00'

0.51.

0.41.

< 0.3ELUTION VOLUME (ml)

FIG. 1. Ion-exchange chromatography on DEAE-Sephacel ofthe 40%to 70% (NH4)2SO4 fraction of the barley kernel extract. The inhibitorwas eluted using a gradient of 300 ml of 5 mM Tris-HCI (1 mM CaCl2,pH 8.0) and 300 ml of the same buffer containing 150 mM NaCl.

0.2.

0.1

0.08

0.06 Ec0co

0.04 u

O0

0.02 <

50 150 250 350ELUTION VOLUME (ml)

FIG. 2. Gel filtration on Bio-Gel P 60 (100-200 mesh) ofthe inhibitorpeak eluted from the DEAE-Sephacel column. The gel filtration columnwas eluted with 5 mm Tris-HCI (I mM CaC12, pH 8.0).

ONA

I--

a0

wL

wj

0.8F

0.61_

0.41_

n o4.2 4.4 4.6

LOG MOL. WT.4.8 5.0

FIG. 3. Mol wt of a-amylase inhibitor estimated by SDS-PAGE.

4.0 4.2 4.4 4.6LOG MOL. WT.

4.8 5.0

FIG. 4. Mol wt of inhibitor, a-amylase, and the a-amylase-inhibitorcomplex estimated by gel filtration on Bio-Gel P100 (100-200 mesh).The column was eluted with 50 mm Tris-HCI (I mm CaC2, pH 8.0).

Table II. Amino Acid Composition ofa-Amylase InhibitorAmino Acid Residues/20,000 gAsp 19.0Thr 10.0Ser 8.4Glu 14.7Pro 13.4Gly 17.5Ala 20.8Val 14.0Met nilIle 8.2Leu 9.9Tyr 6.0Phe 5.3His 4.6Lys 7.0Arg 13.1Trp 2.6Half-cystine 8.8

acids were identified by HPLC on a Perkin-Elmer HS-3 reversedphase column, using a Perkin-Elmer System 4 Liquid Chromato-graph and an elution system recommended by the manufacturer.

RESULTS AND DISCUSSION

The inhibitor was purified 125-fold from the crude extractwith an overall recovery of approximately 20% (Table I). Almost60% of the total inhibitor activity present in the crude extractwas lost after (NH4)2S04 fractionation and dialysis. It is possiblethat part ofthe a-amylase inhibitory activity present in the crudeextract was due to components other than the protein inhibitor.For example, polyphenolics have been implicated as cereal a-

a

x

Ec.a

0I-

* RIBONUCLEASE A

-INHIBITOR (20,000)\*-a-CHYMO TRYPSINOGEN A

-a-AMYLASE U (32,000)P-LACTOGLOBULIN-'

COMPLEX (41,000)--

OVALBUMIN-@ r=-0.987

BOVINE SERUM ALBUMIN-I I I I X

0

x

I._

"'I-_ 4

zo

z

-SOYBEAN TRYPSIN INHIBITOR-a-AMYLASE rl INHIBITOR (21,000)

-CARBONIC ANHYDRASE

OVALBUMIN-'r=-0.996

BOVINE SERUM ALBUMIN-.

PHOSPHORYLASE A--I I I

bff-- 'IL

v.A 0

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BARLEY a-AMYLASE INHIBITOR

Table III. N-Terminal Sequence ofa-Amylase Inhibitor Compared toBarlev Trypsin Inhibitor

A single sequenator run on 0.46 mg of a-amylase inhibitor (23 nmolassuming 20,000 D) gave 112% coupling (26 nmol alanine in cycle 1)and a repetitive yield of 91.4%.

a-Amylase Inhibitor

1 AlabAspProPro

5 ProValHisAspThr

10 AspGlyHisGluLeu

15 ArgAlaAspAlaAsn

20 Tyr

aFrom Odani et al. (18).b N-terminal amino acid.

I |

2 4

PREINCUBATION T

FIG. 5. Effect of preincubation time onamylase activity at pH 8.0 (A) and pH 5.5was 0.30 gg a-amylase and 0.20 ug inhibitor0.15 ug a-amylase and 1.90 Mg inhibitor. Enzto proceed for 7 min.

amylase inhibitors (4). The developmethe inhibitor would resolve this questioiDuring DEAE-Sephacel chromatogra

activity emerged in one peak at a condi

z2

zu-wCL

Barley Trypsin InhibitorI PhebGlyAspSer

5 CysAlaProGlyAsp

10 AlaLeuProHisAsp

15 ProLeuArgAlaCys

20 Arc,

100°

801-A

601-

40

20

20 60 100

TOTAL INHIBITOR (Mg)FIG. 6. Effect of inhibitor concentration on a-amylase activity at pH

5.5 (A) and pH 8.0 (B). A fixed amount of a-amylase (0.10 Mg) was used.Enzyme and inhibitor were preincubated for 15 min prior to the additionof starch solution. Enzyme reactions were allowed to proceed for 8 minat pH 5.5 and 22 min at pH 8.0.

Thr 500 uMho in the salt gradient. The peak fraction had a saltTyr concentration of approximately 70 mm NaCl and the peak of

inhibitor activity coincided with a peak of A at 280 nm. Somestarch hydrolyzing activity, as detected by the reducing sugarassay, was eluted later in the salt gradient and was due probablyto f-amylase. Overall, ion-exchange chromatography was themost effective purification step, giving a purification of about25-fold (Table I).The inhibitor peak from DEAE-Sephacel chromatography was

separated into two major protein peaks by gel filtration on acolumn of Bio-Gel P 60 (Fig. 2), but only one of these hadinhibitory activity against a-amylase II. Total inhibitor activityfrom both chromatographic steps was determined after concen-tration of the active fractions. In each series of chromatographyand concentration, a 10% loss was incurred (Table I). Some ofthis loss may have been due to adsorption of the inhibitor to theultrafiltration membrane.The homogeneity of the isolated inhibitor was demonstrated

by the appearance of only one protein band after SDS-gel elec-trophoresis. High purity of the preparation also was supportedby the sequencing of the first 20 N-terminal amino acids whichreadily identified a unique amino acid with each cycle of thesequenator. Previous analysis by polyacrylamide gel isoelectric

B focusing concomitant with silver staining for protein similarlyrevealed the high purity of the isolated inhibitor (24).

Electrophoresis of the inhibitor on SDS gels indicated a moll______________lwtof 21,000 D (Fig. 3). Gel filtration on Bio-Gel P 100 (Fig. 4)6810 indicated a mol wt of approximately 20,000 D for the inhibitor6 8 I 0 which is in good agreement with the electrophoretic data and

IME (min) suggests that the inhibitor is not a dimer. The mol wt of a-amylase II was estimated to be 32,000 D (Fig. 4) which is

per cent inhibition of a- anomalously low compared to the generally accepted value of(B). Protein used at pH 8.0 about 45,000 D (8). Similar low mol wt have been reported for. Protein used at pH 5.5 was wheat a-amylases using Bio-Gel as a sieving matrix (15).zyme reactions were allowed Gel filtration of inhibitor plus a-amylase II at pH 8.0 yielded

a new peak having a mol wt of 41,000 D (Fig. 4). This peak,presumably, was due to formation of an enzyme-inhibitor com-

nt of a specific assay for plex and would correspond to a-amylase III. Weselake et al. (24)n. have demonstrated the formation of an enzyme-inhibitor com-phy (Fig. 1), the inhibitor plex by isoelectric focusing of a solution of a-amylase anductivity between 400 and inhibitor. The complex appeared to correspond to a-amylase III.

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A mol wt of about 52,000 D would be expected for the complexbut the lower value obtained suggests that the complex may notbehave like a globular protein during gel filtration.Amino acid analysis (Table II) showed that the inhibitor

contained about 9 half-cystine residues/mol but no detectablemethionine. At this stage, the degree of disulfide bonding hasnot been determined. The presence of a relatively high proportionof aromatic amino acids accounts for the absorbance of theinhibitor at 280 nm. The ratio of aspartic and glutamic acidresidues to basic residues was 1.4. This would suggest that someof the apparently acidic residues exist as amides since the isoe-lectric point of the inhibitor was found to be 7.3. The aminoacid composition indicates clearly that the inhibitor is not apurothionin analog (19).The relatively high content of half-cystine is a characteristic

shared by barley trypsin inhibitor (16) and a-amylase inhibitoralbumins from wheat kernels (3). However, a-amylase II inhibi-tor has a greater proportion of basic amino acid residues thandoes inhibitor albumins from wheat.

Results obtained from twenty cycles of the sequenator areshown in Table III. Despite the high half-cystine content of theinhibitor (Table II), no half-cystine was found in the first 20 N-terminal residues. Some homology appears to exist between thea-amylase inhibitor and trypsin inhibitor from barley (Table III).The amino acid residues were numbered sequentially from theN-terminal amino acid. Residues 14 to 16 (Leu-Arg-Ala) of theinhibitor coincided with residues 16 to 18 of the barley trypsininhibitor. Once this degree of homology was established, proline5 and tyrosine 20 of the inhibitor coincided then with proline 7and tyrosine 22 of the barley trypsin inhibitor. However, therewas no strong homology in the N-terminal region between inhib-itor albumins from wheat (10, 18) and a-amylase II inhibitor.

Inhibition of enzyme activity did not occur instantaneouslyupon addition of inhibitor. Maximum inhibition was attainedafter 5 min of preincubation of enzyme and inhibitor (Fig. 5).The preincubation time required for maximum inhibition wassimilar at both pH 5.5 and 8.0. No further changes were observedafter extended preincubations of up to 60 min. Similar preincu-bation requirements have been reported for the inhibition ofanimal a-amylase by inhibitor albumin from the wheat kernel(3).

Inhibition studies carried out between pH 5.5 and 8.0 showedthat the inhibitor became increasingly more powerful as pH wasincreased. A detailed investigation of the effect of inhibitorconcentration on a-amylase activity was made at pH values of5.5 and 8.0 (Fig. 6). Under the experimental conditions used, anapproximately 1 100 M excess of inhibitor over a-amylase II wasrequired to attain 40% inhibition at pH 5.5 (Fig. 6a), assumingmol wt of 20,000 and 45,000 for inhibitor and a-amylase,respectively. At pH 8.0, however, much higher levels of inhibitionwere achieved at significantly lower inhibitor concentrations (Fig.6b). Only I mol of inhibitor/mol of enzyme was required toachieve a 40% inhibition of a-amylase II and 90% inhibitionwas achieved with a 23 M excess of inhibitor. These results offeran explanation for the a-amylase activity exhibited by a-amylaseIII (a-amylase II-inhibitor complex) after isoelectric focusing (24)or after gel filtration experiments carried out at pH 8.0 in thisstudy. Although a strong complex between a-amylase II andinhibitor can be formed at pH 8.0 (forming a-amylase III) whenthe pH is lowered to 5.5 for a-amylase activity analysis, thecomplex is weakened. Thus, some a-amylase II is liberated andits activity then may be detected.At present, the effect of pH on inhibitor activity is not clearly

understood and more detailed studies must be made to clarify

this effect as well as to determine the precise mechanism ofinhibition. In the germinating seed, compartmented pH effectsand location of the inhibitor could have an impact on physiolog-ical regulation of a-amylase. Immunochemical methods arebeing developed for the localization and specific determinationof inhibitor in cereal kernels.Note Added in Proof. Since this manuscript was sub-

mitted, Mundy et al. (1983 [CarlsxbergRes. Commun. 48: 81-90]and Hejgaard et al. (1983 [Carlsberg Res Commun. 48: 91-94]have published a similar study.Mundy J, IB Svendsen, J Hejgaard 1983. Barley a-amylase/

subtilisin inhibitor. I. Isolation and characterization. CarlsbergRes. Commun. 48: 81-90.

Hejgaard J, IB Svendsen, J Mundy 1983. Barley a-amylase/subtilisin inhibitor. II. N-terminal amino acid sequence andhomology with inhibitors of the soybean trypsin inhibitor (Kun-itz) family. Carlsberg Res. Commun. 48: 91-94.

Acknonledgments-We thank Joan Morgan and Helen MacDougall for excel-lent technical assistance.

LITERATURE CITED

1. BLANco-LABRA A, FA ITURBE-CHINAS 1981 Purification and characterizationof an a-amylase inhibitor from maize (Zea mavs). J Food Biochem 5: 1-17

2. BRIGGS DE 1961 A modification of the Sandstedt, Kneen and Blish assay ofa-amylase. J Inst Brew 67: 427-431

3. BUONOCORE V, T PETRUCCI, V SILANO. 1977 Wheat protein inhibitors of a-amylase. Phytochemistry 16: 811-820

4. DAIBER KH 1975 Enzyme inhibition by polyphenols of sorghum grain andmalt. J Sci Food Agric 26: 1399-1411

5. DUCKWORTH HW, AW BELL 1982 Large-scale production of citrate synthasefrom a cloned gene. Can J Biochem 60: 1143-1147

6. EDELHOCH H 1967 Spectroscopic determination of tryptophan and tyrosine inproteins. Biochemistry 6: 1948-1954

7. FRYDENBERG 0, G NIELSEN 1965 Amylase isozymes in germinating barleyseeds. Hereditas 54: 123-139

8. GREENWOOD CT, EA MILNE 1968 Starch degrading and synthesizing enzymes:A discussion of their properties and action patterns. Adv Carbohydr Chem23: 281-366

9. JONES BL, P MEREDITH 1982 Inactivation of alpha-amylase activity by puro-thionins. Cereal Chem 59: 321

10. KASHLAN N, M RICHARDSON 1981 The complete amino acid sequence of amajor wheat protein inhibitor of a-amylase. Phytochemistry 20: 1781-1784

11. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

12. LOWRY OH, HJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-ment with the Folin phenol reagent. J Biol Chem 193: 265-275

13. MACGREGOR AW 1977 Isolation, purification and electrophoretic propertiesof an a-amylase from malted barley. J Inst Brew 83: 100-103

14. MACGREGOR AW, BALLANCE D 1980 Quantitative determination of a-amylaseenzymes in germinated barley after separation by isoelectricfocusing. J InstBrew 86: 131-133

15. MARCHYLO B, JE KRUGER,GN IRVINE 1976 a-Amylase from immature hardred spring wheat. 1. Purification and some chemical and physical properties.Cereal Chem 53: 157-173

16. MIKOLA J, E-M SUOLINNA 1969 Purification and properties of a trypsininhibitor from barley. Eur J Biochem 9: 555-560

17. MOORES 1963 The determination of cystine as cysteic acid. J Biol Chem 238:235-237

18. ODANI S, T KOIDE, T ONO 1982 Sequence homology between barley trypsininhibitor and wheat a-amylase inhibitors. FEBS Lett 141: 279-282

19. OZAKI Y, K WADA, T HASE, H MATSUBARA, T NAKANISHI, H YOSHIZUMI1980 Amino acid sequence of a purothionin homolog from barley flour. JBiochem 87: 549-555

20. RoBYTJF, WJ WHELAN 1968 The a-amylases. In JA Radley, ed, Starch andits Derivatives. Chapman and Hall Ltd., London, pp 430-476

21. SILVANOVICH MP, RD HILL 1976 Affinity chromatography of cereal a-amylase.Anal Biochem 73: 430-433

22. WARCHALEWSKI JR 1977 Isolation and purification of native alpha-amylaseinhibitors from winter wheat. Bull Acad Pol Sci Ser Sci Biol 25: 725-729

23. WARCHALEWSKI JR 1977 Isolation and purification of native alpha-amylaseinhibitors from malted winter wheat. Bull Acad Pol Sci Ser Sci Biol 25: 731-735

24. WESELAKE RJ, AW MACGREGOR, RD HILL 1983 An endogenous a-amylaseinhibitor in barley kernels. Plant Physiol 72: 809-812

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