Role of Proteinaceous a-Amylase Enzyme Inhibitors.pdf

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J. Agric. Food Chem. 1909, 37, 295-299Role of Proteinaceous a-Amylase Enzyme Inhibitors in PreharvestSprouting of Wheat Grain

295

Selma Abdul-Hussain and Gary M. Paulsen*

Proteins in wheat (Triticum aestiuum L.) grain inhibit a-amylase enzyme from many sources. Wecompared proteinaceous a-amylase inhibitors in wheats that differ in preharvest sprouting susceptibility;determined involvement of the inhibitors in excised embryo germination; and assessed the relationshipamong a-amylase inhibition, embryo germination, and calcium. Water-solubleproteins were fractionatedwith ethanol, purified by ion-exchange chromatography, and tested for inhibition of a-amylase fromunmalted grains. Only proteins from sprouting-resistant genotypes inhibited a-amylase in standardassays, but adding EDTA to chelate calcium induced inhibition by all genotypes. Chromatographyyieldedsix inhibitor peaks that had molecular weights of 14000-68000 by electrophoresis. None of the inhibitorsinfluenced germination of excised embryos. We concluded that proteinaceous a-amylase inhibitorsinteract with calcium but do not play primary roles in preharvest sprouting. They may have importantsecondary effects, however, by influencing a-amylase activity per unit of sprouting.

Preharvest sprouting of wheat and other cereals is amajor problem in many regions of the world. Both agro-nomic attributes, particularly tes t weight and yield, andfunctional quality for milling and baking are adverselyaltered (Belderok, 1961, 1976; Bhatt et al., 1981). a-Amylase enzyme (E.C. 3.2.1:l) activity is closely associatedwith preharvest sprouting and is often used as a measureof sprouting damage (Mathewson and Pomeranz, 1977).Proteinaceous compounds that inhibit a-amylase en-zyme from animal sources have been widely studied be-cause of their possible nutritional significance, retardationof insect infestation of grain, and protein-protein inter-action properties (Richardson, 1981). Endogenous pro-teinaceous inhibitors of cereal a-amylase enzyme have beenreported only recently (Weselake et al., 1985a). These orsimilar compounds may regulate a-amylase activity duringsprouting and other processes (Warchalewski,1977). Therelationship between the level of inhibitor and a-amylaseactivity, however, is not clear. The inhibi tor is reportedto increase during grain maturation, when a-amylase ac-tivity is decreasing, and to decrease to a low level duringgermination, when a-amylase activity is increasing (Paceet al., 1978). Other reports indicate that th e inhibitorremains active during germination (Weselake et al., 1985a).Wheat a-amylase inhibitors occur in multiple molecularforms (O'Donnell and McGeeney, 1976). They belong tothree main families having molecular weights of 12000,24OOO, and 60 OO (DePonte et al., 1976),and an a-amylaseinhibitor of 30 000 is described (Granum and Whitaker,1977). Purothionins, basic low molecuIar weight proteinsof 6000-12 000,ave alsobeen reported (Ballset al., 1942);a-amylase enzyme might be inhibited by interactions ofits Ca2+cofactor with proteinaceous inhibitors, includingpurothionins (Lang et al., 1973; Jones and Meredith, 1982).Susceptibility to preharvest sprouting and productionof a-amylase enzyme differ markedly between red andwhite wheat classes and among genotypes within each class(McCrate et al., 1981). Reasons for these differences arenot well understood, however. Objectives of th e presentinvestigations were to compare inhibitor levels in wheatclasses and genotypes tha t differ in preharvest sprouting;determine effects of the inhibitors on germination of ex-cised wheat embryos; and assess the relationship amonga-amylase inhibition, calcium, and embryo germination.

Department of Agronomy, Kansas State University,Manhattan, Kansas 66506.

METHODSPlant Material. Wheat genotypes used for the inves-tigations were identified previously as having a wide range

of dormancy and afterripening requirements (McCrate etal., 1981). Newton and Clark's Cream are hard red andwhite winter wheats, respectively, that resist preharvestsprouting, and Parker 76 and KS75216 are hard red andwhite winter wheats, respectively, that sprout readily underappropriate conditions (Nielsen et al., 1984).The four genotypes and Lancota, a sprouting-resistanthard red winter wheat that provided excised embryos, weregrown with recommended agronomic practices in repli-cated plots. Grain was harvested immediately when itripened. After harvest, grain of the four former genotypeswas held at room temperature, whereas grain of Lancotawas frozen at -20 OC to arrest afterripening (Belderok,1961).Inhibitor Extraction. A modification of the O'Donnelland McGeeney (1976) procedure was used for extractingand separating a-amylase inhibitors (Figure 1). Grain ofthe four genotypes was ground through a 1-mm sieve toproduce whole wheat flours, which were suspended in threevolpmes of water and centrifuged at 4000g at 0 "C for 15min. The residue was washed with one volume of waterand centrifuged. The supernatants from the first andsecond centrifugation were filtered and lyophilized. Theresulting powder was dissolved in water heated a t 70 "Cfor 30 min to inactivate a- and 8-amylase enzymes(Warchalewski,1977), cooled, and centrifuged a t 8OOOgat0 "C for 10 min.Absolute ethanol was added to 60% (v/v) volume of theaqueous extract at 20 "C, and the precipitate was discardedafter centrifuging a t 8000g at 0 "C for 30 min. The ethanolconcentration was raised to 90% (v/v), and t he resultingprecipitate, which contained the a-amylase inhibitoryactivity (O'Donnell and McGeeney, 1976), was separatedby centrifugation as above. The precipi tate was washedwith absolute ethanol three times and dried a t 30OC ov-ernight. The precipitate was dissolved in 0.01 M Tris-HC1buffer (pH 9.2) containing 1 mM NaCl and dialyzedagainst the same buffer at 4 "C overnight. Any cloudinessthat developed was removed by centrifugation.The extract was applied to a 1.5 X 40 cm DEAE-Se-phacel column th at was equilibrated previously with th ebuffer. Four column volumes of buffer were eluted, andthen the extract was eluted with a continuous linear gra-dient of 0-0.5 M NaC1. The flow rate was 0.33 mL min-',

0021-8561 18911437-0295$01.50/0 0 1989 American Chemical Society


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298 J. Agric. Food Chem.,Vol. 37, o. 2, 1989 AbduCHussain and Paulsenwater (3:1:6), stained in methanol-acetic acid-CoomassieBrilliant Blue R (8:2:0.05), and destained in methanol-acetic acid-water (5:7.5:87.5). Molecular weights of a-amylase inhibitors were calculated from their electropho-retic mobilities relative to those of the standard proteins.Embryo Bioassay of a-Amylase Inhibitors. FrozenLancota seeds were warmed to room temperature, sur-face-sterilized with 70% (v/v) ethanol for 10 min and 0.1mM sodium hypochlorite for 10 min, and rinsed thoroughlywith sterile distilled water. Embryos were excised bysectioning seeds with a sharp blade; one side of the em-bryos contained a small amount of pericarp and seed coat,and the other, a small amount of endosperm.For bioassays, 25 embryos were placed endosperm sidedown on double layers of sterile filter disks in 15 X 100mm Petr i dishes. The filter disks were moistened with 4mL of sterilized water or 1mL of Newton chromatographyeffluent plus 3 mL of sterilized water. Germination re-sponse of embryos was recorded daily, and a promptnessindex (PI) (George, 1967) was calculated from the formula4D1 30, +20, +D4, whereD,=the number of embryosgerminating tha t day. All tests were replicated four times.Calcium Content and Depletion. Water extracts ofthe four genotypes were tested for a-amylase inhibitionand calcium content before and after adding 0.2 mMEDTA. Treated extracts were stirred a t room temperaturefor 30 min and dialyzed against distilled water at 4 "Covernight. a-Amylase inhibition was measured as de-scribed above.Calcium content was determined by digesting extractsin nitric acid-perchloric acid solutions, diluting them withlanthanum chloride, and reading them by atomic absorp-tion spectrophotometry at 422.7 nm.Other s tudies determined the effect of dialysis ofKS75216 genotype extracts on a-amylase inhibition.Chromatographic fractions of the sprouting-susceptiblegenotype were collected as described above, dialyzedagainst 0.01 mM Tris-HC1at 4 "C overnight, and assayedfor a-amylase inhibition and protein content as describedpreviously.RESULTS

Levels of a-amylase inhibition in extracts obtained fromthe four genotypes by procedures outlined in Figure 1areshown in Table I. Inhibition was observed only in extractsfrom the two genotypes that resist preharvest sprouting,Newton and Clark's Cream, and not in the sprouting-susceptible genotypes, Parker 76 and KS75216. Th e lowinhibitory activity in crude water extracts was not in-creased by heating. Ethanol precipitation, however, greatlyincreased the concentration of inhibitor in the two geno-types in which it occurred. Ion-exchange chromatographyfurther concentrated the inhibitor into six peaks (Figure2) that varied in activity (Table I). Chromatography ofthe same extracts from the two sprouting-susceptiblegenotypes, on the other hand, yielded only one majorprotein peak, which was not inhibitory.A n electrophoretogramof the chromatography peaks ofone of the sprouting-resistant genotypes, Newton, is il-lustrated in Figure 3. Electrophoresis separated two tofive bands in each chromatography peak; estimated mo-lecular weights of the proteins in the different bandsranged from 14000 to 68000 (Table 11).The relationship among a-amylase inhibitor activity inchromatography fractions of Newton wheat, calcium con-tent of the fractions, and germination of excised embryosin the presence of the fractions is shown in Table 111.Inhibition ranged from 0% by fractions that contained 0.93hg 8-l calcium to 36.6% by fractions tha t contained 0.18

WHOLE WHEAT FLOUR(1 SAMPLE: 3 WATER)tSTIRtCENTRIFUGE

WASH F I L ~ E Rt tCENTPIFUGE LYOPYYLlZEC&BINE D I S S ~ LV ESUPERNATANTS tHEAT

tCENTRIFUGEn 1DISCARD ETOH PRECIPITATIONtCENTt?IFUGE

WASH D I S ~ A R DttDISSOLVECENTRIFUGEIISCARD CHROMATOGRAPHY

ELECTROPHORESlSIFigure 1. Separation and purification procedure for endogenouswheat a-am ylase enzyme inhibitors.and 6-mL fractions were collected. Protein in the effluentwas monitored continuously at 280 nm.All extracts and fractions were tested for inhibition ofa-amylase (see below). Chromatography effluents thatwere inhibitory were combined within genotypes, dialyzedagainst 0.03 M NH4HC03, yophilized to dryness, and heldfor electrophoresis. In a separate study, fractions obtainedfrom chromatography of Newton inhibitor were assayedindividually for a-amylase inhibition, calcium content, andeffect on embryo germination.a-Amylase Inhibition. a-Amylase enzyme inhibitoractivity in crude and purified extracts was measured bya modification of the methods of Barnes and Blakeney(1974) and Pace et al. (1978). All assays used enzymeobtained by NaCl extraction of KS75216 wheat seed ger-minated inInhibition was measured by diluting 0.5 mL of the testsolution (adjusted to pH 5.5) with 3.5 mL of NaCl ex-traction solution and adding 5 mL of a-amylase extract.The mixture was held at room temperature for 20 min andequilibrated at 50 "C for 10 min, and a Cibacron Bluelabeled amylose tablet was added to each tube. Themixture was agitated vigorously every 2.5 min during in-cubation at 50 "C for 30 min. The reaction was stoppedby adding 1mL of 0.5 N NaOH, and the tube contentswere filtered through Whatman No. 4 paper. Absorbanceof the filtrate was measured at 620 nm against a blankfrom which inhibitor was excluded. Percentage a-amylaseinhibition was calculated by the formula (AB - As)/AB X100, where AB =absorbance of the blank and As =ab-sorbance of the sample. Inhibition was expressed eitheras percentage as described or as percentage per unit pro-tein as measured by the Bradford (1976) procedure.PAGE-SDS Electrophoresis. Molecular weights ofthe a-amylase inhibitors were determined by the electro-phoretic method of Weber and Osborn (1969). Chroma-tographic fractions that were inhibitory and standardproteins (bovine albumin, trypsinogen, @-lactoglobulin, ggalbumin, lysozyme) were run on a continuous system. The10% (w/v) gel and buffer contained 1% (w/v) SDS, 1%(v/v) 2-mercaptoethanol, 0.01% (v/v) bromophenol blue,and 0.2 M phosphate (pH 7.0). Samples (10-80 pL) werelayered on the gel and run a t 2 mA for 30 min and at 8mA for 5-6 h. Gels were fixed in methanol-acetic acid-

mM GAB (gibberellic acid) for 5 h.


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&Amylase Inhibitors in Wheat J. Agric. Food Chem., Vol. 37 , No. 2, 1989 207

0 .5

0.1q

T a b l e I. Percentagea-Amylase Inhib i t ion per Mil l ig ra m o f P ro t e in a nd P ro t e in Con te n t o f F ra c t ions Ex t ra c t e d f rom Fou rWhe a t G e no type sgenotype

-0 - 1 s10

6I , ,

Newton Par ker 76 Clarks Cream KS75216inhibn, protein, inhibn, protein, inhibn, protein, inhibn, protein,fraction % m g- *S E m g I S E % mg-l mg % mg-*SE mg*SE ?& mg-l mg

water extractheated extractethanol pptbefore dialysisafter dialysischromatographypeak 1peak 2peak 3peak 4peak 5peak 6

002 * 04 f O

75 *7109 f 943 *424 f48 f632 *83

5558 f 331850 9719 f3 f O0.1 00.2 *00.1 *00.2 00.1 *00.1 *0

0000000000

T a b l e 11. Est ima te d M ole c u la r We igh t s of P ro t e in sSe pa ra t e d by S D S G e l E le c t ropho re s i s of Chroma tog ra phyP e a k s f r o m N e w t on W h e a t R e l a t iv e t o S t a n d a r d P r o t e i n schromatogr electrophoresis band

peak 1 2 3 4 51 68000 65000 630002 65000 630003 59000 55000 530004 60000 58000 55000 17000 140005 60000 58000 55000 140006 65000 63000 58000 57000

N e w t o ng 0. 5 162 0.2

0.18

32 64 96 128 16 0 1 9 2 2 2 4Eluflon Vo l u mo (MI

1 2 0 ia r k o r 76

6 5 -c0. 15 2 6 4 96 12 6 16 0 192 224

85 2 6 4 96 12 6 16 0 192 224

0.40. 5

N 0. 240. 1

Elutlon Vo Iu mo (mll

5 2 64 96 12 6 160 192 2 2 4Elutlon Vo l u mo (ml )C l a r k s C r e a m0.4E-

EIuflon Volumo l m l )Figure2. DE AE Sep hace l chromatograms of th e alcohol fractionof four wheat genotypes after elution with an NaCl gradient.pg g-l calcium. The r value between a-amylase inhibitionand calcium content was negative and significant. Ger-mination PI of embryos treated with the chromatographyfractions ranged widely but correlated neither with a-amylase inhibition nor with calcium content.

2879 0 2300 f 125 0 3417535 0 546 f 43 0 85841 0.8 *0.1 26 f 2 0 4939 0.8 k 0.1 15 f 1 0 39

53 f 0.1 0 020 *1 0.3 0 09 * 1 0.5 f 0 017 f 1 0.2 f 0 091 *7 0.1 f 0 022 f 1 0.1 f 0 0T a b l e 111. Pe rc e n ta ge a -A myla se Inh ib i t i on , Ca lc iumCon te nt , a nd Ef fe c t on Exc i sed Em bryo G e rmina t ionP r o m p t n e s s I n d e x ( P I ) o f C h r o m a t o g r a p h y F r a c ti o n s f r o mN e w ton Whe a t

chromatogr a-amylase Ca embryofraction, inhibn, content, germination,no. % f SE pgg- PI & S E22 0 0.9 f 0.1 7 * 123 0 0.9 0.1 12 *124 11f 1 0.4 f .1 4 f O25 20 f 2 0.6 f .1 4 f O26 16 f 1 0.6 f 0 .1 4 * 027 8 f 1 0.7 *0 .1 4 * 028 23 & 2 0.3 & 0.0 10 f 129 9 * 1 0.4 f 0.1 4 * 030 37 f 2 0.2 0.0 6 * 131 22 f 2 0.5 f 0 .1 6 * 132 13 f 1 0.9 *0.2 5* 133 6 f 1 0.3 f 0.0 2 * 0r , a-amylase inhibn -0 .6 P -0.09Ca content 0.29a Significant a t P =0.05 level.

Ta b le IV . Pe rc e n ta ge a -A myla se Inh ib i t i on a nd Ca lc iumCon te n t of W a te r Ex t ra c t s f rom Fou r Whe a t G e no type sbe fo re a nd a f t e r A dd ing ED TAbefore adding EDTAinhibn, Ca content, inhibn, Ca content,aenotvpe % S E fi g g-l SE % *S E pa a-l f SE

after adding EDTA

Newton 23 f 2 26 2 37.3 6.1O f 0 3 2 * 2 3 * 0 6 * 1arker 76ClarksCream 6 f 1 2 6 f 2 1 3 * 1 8 * 1KS75216 o * o 3 3 f 2 7 f 1 6 * lTab le V. P ercentag e a -Amylase Inhib i t ion per Mil l ig ra m o fP r o t e i n a n d P r o t e i n C o n t e n t o f KS75216 Whe a tChroma tog ra ph y Pe a ks a f t e r D ia lys i s a ga in s t Tr i s -H C1B u f f e r

chromatogr a-amylase inhibn, protein content,peak no. % mg protein-1 *SE mg f S E1 37 * 2 0.1 f 02 61 4 0.1 03 90 f 5 0.1 f 04 80*4 0.1 05 193 *8 0.1 *0

Adding EDTA to crude water extracts increased thea-amylase inhibition in wheat genotypes in which it waspreviously noted (Table IV). It also prompted inhibitionby extracts from Parker 76 and KS75216, genotypes thatwere not inhibitory before adding EDTA. Calcium contentof crude extracts without EDTA was slightly higher in thetwo sprouting-susceptible genotypes than in the two re-sistant genotypes.


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29 8 J. Agric. Food Chem., Vol. 37,No. 2, 1989 AbduCHussain and Paulsen

Peak 1Peak 2Peak 3mPeak 6 11 I \\ IIII I

Albumln, BovlneTrypr lnogen

6-Lactoglobulin ]Lysozyme

Albumin, Egg

Figure 3. SDS-PAGE electrophoretogramsof a-amylase nhibitorchromatography fractions of Newton wheat. The lower linerepresents the marker, and other lines represent movement ofthe separated bands relative to the marker.Dialysis of the chromatography effluent of KS75216extract against Tris-HC1 also caused substantial inhibitionof a-amylase (Table V) compared with undialyzed effluent(Table I) . Activity separated into five distinct peaks thatwere similar in magnitude to activity of extracts fromsprouting-resistant genotypes shown in Table I.

DISCUSSIONPreharvest sprouting resistance of wheat has been at-tributed to endogenous catechin-tannin compounds, car-yopsis structure, and embryo responsiveness to inhibitors(Belderok, 1976; McCrate et al., 1982; Stoy and Olsen,1980). Present results suggest that proteinaceous a-amy-lase inhibitors may influence preharvest sprouting butprobably are not primary determinants of genotypic var-iation in sprouting susceptibility. Proteinaceous inhibitorsmay exert an important secondary effect, however, byregulating a-amylase enzyme activity per unit of sprouting.Genotypes such as Clarks Cream that produce little a-amylase activity per unit of sprouting and also resistsprouting are doubly useful for alleviating adverse effectsof inclement conditions on wheat functional properties(Nielsen et al., 1984; Upadhyay e t al., 1984).Endogenous inhibitors of preharvest sprouting slowgermination and production of a-amylase enzyme con-currently (McCrate et al., 1982). The lack of any dis-cernable effect of proteinaceous a-amylase inhibitors onexcised embryos indicates that these compounds are nota major factor in preharvest sprouting. Sprouting teststhat are based on a-amylase activity in whole flour extractsmay lead to contrary conclusions, however (Mathewsonand Pomeranz, 1977) . They do not distinguish betweengermination per se and activity of enzymes that are in-duced by germination.An association of proteinaceous a-amylase inhibitorswith calcium is indicated by the negative correlation be-tween the two parameters and effects of treatments tochelate or remove calcium. Calcium is required for sta-bilizing a-amylase enzyme, and chelation of the metal byEDTA has been shown to inactivate malt a-amylase(Fisher and Haselback, 1951). The effect of proteinaceousinhibitors appears to be similar to that of MHP (myoi-nositol hexaphosphate), which suppresses a-amylase ac-tivity in sprouted wheat meal by chelating the calcium thatis needed for enzyme activity or by interacting directly with

the enzyme protein moiety (Jacobsen and Slotfeldt-El-lingsen, 1983; Sharma et al., 1978). Calcium may alsomediate the interaction between a-amylase enzyme andproteinaceous inhibitors by electrostatic effectsas reportedfor NaCl in barley by Weselake et al. (1985b). Removingcalcium by EDTA or dialysis may enhance inhibition ofa-amylase in the four wheat genotypes tha t we studied byfacilitating interactions between the proteinaceous inhib-itors and the enzyme.It is apparent t ha t several molecular species of protei-naceous compounds inhibit a-amylase in wheat. Most ofthem are in the range of molecular weight 60000 f 5000,the largest reported to date (DePonte et al., 1976). Noneof them resemble the intermediate species of molecularweight 30000 (Granum and Whitaker, 1977), and only afew approach the low molecular ,weight of purothionins(Balls et al., 1942; Jones and Meredith, 1982). The severalspecies of inhibitors that occur may contribute to theheterogeneity of different wheat genotypes and even dif-ferent chromatography fractions. It seems likely, however,tha t t he most quantitatively important proteinaceous in-hibitors in wheat lie in the upper range of molecularweights.Genotypic differences in the amounts and distributionof proteinaceous a-amylase inhibitors suggest that they canbe manipulated genetically. Altering inhibitor levels couldreduce the level of a-amylase per percentage of sprouting,if not the susceptibilityto preharvest sprouting. This couldimprove the functional quality of wheat flour and perhapseven the nutritional properties of leavened products.LITERATURE CITEDBalls, A. K.; Hale, W. S.; Harris, T. H. A Crystalline ProteinObtained from a Lipoproteinof Wheat Flour. Cereal Chem .Barnes, W. C.; Blakeney,A. B. Determinationof a-Amylase Usinga Commercially Available Dye-labelled Substrate. Die StarkeBelderok, B. Studieson Dormancy in Wheat. R o c . Znt. Seed TestBelderok, B. Physiological-biochemical Aspectof Dormancy inWheat. Cereal Res. Commu n. 1976, 4, 135-137.Bhatt, G. M.; Paulsen, G. M.; Kulp, K.; Heyne,E. G. PreharvestSproutingin Hard Winter Wheat: Assessment of Methods toDetect Genotypic and Nitrogen Effects and Interactions. CerealChem. 1981,58 , 300-302.Bradford, M. M. A Rapid and Sensitive Method fo r the @an-titation of Microgram Quantities of Protein Utilizing thePrinciple of Protein Dye Binding. Anal. Biochem. 1976, 72 ,DePonte,R.; Partamenti, R.; Petrucci, T.; Silano,V.; Tomasi,M.Albumin a-Amylase Inhibitor Families from Wheat Flour.Cereal Chem . 1976,53 , 805-820.Fisher, E. H.; Haselback, C. H. Contribution a 1Etude de 1Al-pha-amylase de Malt-Sur les Enzyme Amylolytiques. Helu.Chim. Acta 1951, 34, 325-334.George, D. W. High Temperature Seed Dormancy in Wheat. CropGranum,P. E. ;Whitaker,J. R. Purification and Characterizationof a-Amylase Inhibitors in Wheat. J.Food Biochem. 1977,1 ,Jacobsen, T.; Slotfeldt-Ellingsen, D. Phytic Acid and MetalAvailability: A Study of Ca and Cu Binding. Cereal Chem .Jones, B. L. ; Meredith, P. Inactivationof a-Amylase Activity byPurothionins. Cereal Chem. 1982,59 , 321.Lang, J. A.; Talley,D. J.; Saunden,R. M. Protein Characterizationand Kinetic Studies of a-Amylase Inhibitors Isolated fromWheat . Fed. Proc. Abstr . 1973, 32 , 554.Mathewson, P. R.; Pomeranz,Y .Detectionof sproutedwheat bya rapid colorimetric determinationof a-Amylase. JAOAC 1977,

Registry No. Ca, 7440-70-2;a-amylase, 9000-90-2.

1942, 19, 279-288.

1974, 26 , 193-197.A SSOC .961, 26 , 697-760.

248-254.

S C ~ .967, 7, 249-253.

385-401.

1983, 60, 392-395.

60, 16-20.


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J. Agric. Food Chem. 1989, 37, 299-303 299McCrate, A. J.; Nielsen, M. T.; Paulsen, G. M.; Heyne, E. G.Preharvest Sprouting and a-Amylase Activityin Hard Red andHard White Winter Wheat Cultivars. Cereal Chem. 1981,58,McCrate,A. J.;et al. Relationship Between Sprouting in Wheatand Embryo Response to Endogenous Inhibition. EuphyticaNielsen,M. T.; McCrate,A. J.;Heyne, E. G.; Paulsen,G. M. Effectof Weather Variables during Maturation on PreharvestSprouting of Hard White Winter Wheat. Crop Sci. 1984,24,ODonnell, M. D.; McGeeney,K. I. Purification and Propertiesof an a-Amylase Inhibitor from Wheat. Biochem. Biophys.Acta 1976, 22, 159-169.Pace,W.; Parlamenti, R.; Urrab, A.; Silano,V.; Vittozzi, L. Proteina-Amylase Inhibitor from Wheat Flour. Cereal Chem.1978,Richardson, M. Protein Inhibitors of Enzymes. Food Chem.1981,Sharma,C. B.; Goel, M.; Irshad, M. Myoinositol Hexaphosphate

as a Potential Inhibitor of a-Amylase.Phytochemistry 1978,

424-428.

1982,31, 93-200.

779-182.

55, 244-254.6, 235-253.

17 , 201-204.

Stoy,V.; Olsen,0. Inheritanceof a Factor Affecting the Responseto Germination Inhibitors in Excised Wheat Embryo. CerealRes. Commun. 1980,8, 03-208.Upadhyay, M. P.; Paulsen, G. M.; Heyne, E. G.; Sears, R. G.;Hoseney, R. C. Development of Hard White Winter Wheatsfo r a Hard Red Winter Wheat Region. Euphytica 1984, 3,Warchalewski, J. R. Isolation and Purificationof Native a-AmylaseInhibitors from Winter Wheat. Bull. Acad. Pol. Sci. 1977,Weber, K.; sborn, M. The Reliability of Molecular Weight

Determination by Dodecyl Sulfate-Polyacrylamide Gel Elec-trophoresis. J. Biol. Chem. 1969, 44 , 4406-4412.Weselake,R. J.; MacGregor,A. W.; Hill, R. D. Endogenousa-Amylase Inhibitor in Various Cereals. Cereal Chem. 1985a,62, 120-123.Weselake, R. J.; e t al. Effect of Endogenous Barley a-AmylaseInhibitor on Hydrolysis of Starch under Various Conditions.J . Cereal Sci. 1985b, ,249-259.

865-874.

X X V , 125-729.

Received for review July 20,1988. Accepted September 2,1988.

DNA Breakage by Flavan-3-01sand Procyanidins in the Presence ofCupric Ion

Sanetaka Shirahata,* Hiroki Murakami, Kazuo Nishiyama, Koji Yamada, Gen-ichiro Nonaka,Itsuo Nishioka, and Hirohisa Omura

DNA breakage by flavan-3-01s and procyanidins in th e presence of cupric ion was investigated by gelelectrophoretic and computer analysis. Flavan-3-01s and procyanidins cleaved A-DNA in a concen-tration-dependent manner. Th e DNA-breaking activity was remarkably enhanced approximately inconnection with the increase of the number of dihydroxy or trihydroxy groups in a molecule. Althoughthe presence of cupric ions was indispensable for DNA cleavage by (-)-epicatechin (EPI),excess amountsof cupric ions inhibited the activity. A number of single-strand breaks occurred prior to double-strandbreaks. A maximum rate of DNA cleavage by EPI occurred at pH 7.0. A correlation was not observedbetween the oxidation rate of EPI and the cleavage activity. DNA breakage by EPI was strongly inhibitedby the addition of catalase or various kinds of radical scavengers. From the results obtained, a breakingmechanism was proposed that DNA chains were broken by oxygen radicals generated by a local oxidationof EPI in th e vicinity of DNA via DNA-Cu2+-EPI complexes.

Polyphenols including hydrolyzable, condensed, andother tannins are widely distributed in the plant kingdomand consumed daily by humans in milligram to gramquantities (Brown, 1980). It has been reported that tannicacid has antimutagenic effects (Gichner et al., 1987), andhydrolyzable tannins have antitumor activities (Miyamotoet al., 1987). Contrary to these reports, tannic acid isknown to be a naturally occurring hepatoxin (Handler andBaker, 1944) and to induce nucleolar changes in hepacytes(Rao et al., 1987) and liver cancer (Korpdssy, 1959).Such in vitro genotoxic assays as he DNA breakage testwill be helpful to assess the potential genetic hazard byfood components to humans. In the previous paper(Shirahata e t al., 1985), we reported that hydrolyzableDepartment of Food and Nutrition, Shokei JuniorCollege, Kuhonji, Kumamoto 862, Japan (S.S.), Depart-ment of Food Science and Technology, Faculty of Agri-culture, Kyushu University, Hakozaki, Fukuoka 862, Japan(H.M., K.Y., H.O.), Department of Agricultural Chemistry,Faculty of Agriculture, Miyazaki University, Kumano,Miyazaki 889-21, Japan (K.N.), and Faculty of Pharma-ceutical Sciences, Kyushu University, Maidashi, Fukuoka812, Japan (G.N., I.N.).

00 2 1-856 118911437-0299$0 1.50lO

tannins can cleave DNA chains in the presence of Cu2+.In order to clarify the relationship between the structuresof hydrolyzable and condensed tannins and DNA breakingactivity, we developed a rapid and exact method forquantitative determination of nucleic acid breaking activitywith a combination of gel electrophoresis and microcom-puter analysis.Flavan-3-ols are constituent units of condensed tannins,and procyanidins belong to condensed tannins. This paperdescribes the DNA-breaking activity of flavan-3-01s andprocyanidins in the presence of Cu2+analyzed by this newmethod.MATERIALS AND METHODSDNA and Reagents. Double-stranded DNA from A -phage was purchased from Biotech Co. Plasmid pBR322DNA was prepared as described before (Shirahata et al.,1985). Catalase was obtained from Sigma Chemical Co.Superoxide dismutase was purchased from Toyobo Co.Various radical scavengers were all commercial grade. Allflavan-3-01sand procyanidins were isolated from variouskinds of plants, and their chemical structures were de-termined as reported previously. Flavan-3-01s: (+)-catechin (CAT) (Nonaka and Nishioka, 1982), (-)-epi-catechin (EPI) (Nonaka et al., 1983), (+)-gallocatechin

0 1989 American Chemical Society

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