a-Amylase Isozymes in GibberellicAcid-treated …ethanolfor30secandthensoakedin50%H2SO4 for15hrat0C ... glycogen complex which was prepared following the method of ... measuringtherateofstarch-I2

Plant Physiol. (1970) 46, 586-591

a-Amylase Isozymes in Gibberellic Acid-treated Barley Half-seedsReceived for publication May 12, 1970

Y. TANAKA AND T. AKAZAWASeikagaku Seigyo Kenkyu Shisetsu, Nagoya University, School of Agriculture, Chikusa, Nagoya, Japan

ABSTRACT

The presenceof multiple forms of a-amylase in gibberellicacid-treated embryoless barley half-seeds was demon-strated by separation on diethylaminoethyl-Sephadex andisoelectric focusing polyacrylamide gel disc electrophoresis.Two major a-amylase fractions (A and B), each consistingof two to three isozyme components, were purified.a-Amylase fractions A and B were distinguishable in theirreaction patterns. The optimal pH of fraction A a-amylasewas found to reside in the acidic side (pH 5.0), as was de-ternined by analyzing the reducing sugars formed as wellas the paper chromatographic detection of reaction prod-ucts. At neutral pH, 6.9, fraction A exhibited weak amylo-lytic activity in forming maltose. The a-amylase activity infraction A was markedly stimulated by heat treatment(70 C/15 minutes). Fraction B, constituting a major partof amylases in the endosperm extract, was also found to becomposed of a-amylase, as evidenced by the loss of enzymeactivity upon allowing fractions A and B to stand at pH3.3 for a prolonged period. The possible physiological func-tion of the two different types of a-amylase in the carbo-hydrate breakdown of barley seeds is discussed.

The enhanced synthesis of a-amylase induced by the gibberel-lic acid treatment of cereal seeds is an important facet of thebiological function of the hormone (3, 14, 15, 24, 25, 30-32,35). A series of studies carried out by Varner and his associates(4, 5, 7, 30-33) have conclusively demonstrated that synthesisof a-amylase in either embryoless barley half-seeds or isolatedaleurone layers occurs in response to exogenous addition ofGA. They have thus implicated that in normally germinatingseeds, GA secreted from the embryo is translocated to aleuronecells, where it promotes synthesis of the enzyme. However, thetarget of GA in promoting protein synthesis is still unknown(33). On the other hand, there are numerous reports concerningthe synthesis of a-amylase isozymes in intact barley seeds (10) aswell as in embryoless half-seeds treated by GA (12, 20, 21, 23,33). Recent experiments by Tanaka et al. (28) have demonstratedthat banding patterns of multiple forms of a-amylase detectedin extracts of embryoless (GA-treated) and embryo-attachedrice endosperm tissue on an isoelectric focused polyacrylamidegel were basically similar, if not identical. In view of the pre-dominant role of amylolytic breakdown of reserve carbohydratein germinating starchy seeds (22), the nature of such a-amylaseisozymes is of interest, and an intriguing possibility would bethat some of the isozymes may function intracellularly in a co-operative manner. In an experiment reported in this paper, wehave studied the nature of a-amylase isozymes in GA-treatedembryoless barley seeds. It has been demonstrated that there are

two types of a-amylase (fractions A and B), differing in theirenzymic properties.

MATERIALS AND METHODS

Gibberellic Acid Treatment of Barley Half-seeds. Freshlyharvested (1969) barley seeds (Hordeum vulgare var. cv. Musa-shino mugi) were used throughout this study. Seeds were firstdipped in 75% ethanol for 30 sec and then soaked in 50% H2SO4for 15 hr at 0 C (20). The seeds were dehulled, thoroughly washedin tap water, and cut into two parts with a razor blade, embryo-less and embryo-attached. The embryoless half-seeds were againsoaked in 75% ethanol for 30 sec and then in 1% NaClO for 15min. After a thorough washing with sterilized distilled H20,embryoless half-seeds were aseptically placed in Petri dishes(diameter = 9 cm), each containing 10 ml of an aqueous solutionof 2 X 10-6 M GA, and incubated at 30 C in the dark.

Isolation and Purification of a-Amylase. After 3 days of in-cubation, seeds (300 g fresh wt) were subjected to the isolationof a-amylase according to the method of Loyter and Schramm(17), with slight modification. The entire contents of a Petridish were macerated in a mortar and squeezed through threelayers of cheesecloth. Residual matter was washed again with200 ml of 4 m, NaCl and 3 mm CaCI2 solution (NaCl-CaCI2solution). The filtrates were combined and centrifuged at 15,000rpm (20,000g) for 20 min, and the supernatant liquid was broughtto 50% saturation with (NH4)2S04, the pH being adjusted to5.8 with 1 N NH40H. After 30 min the suspension was centri-fuged at 8,000 rpm (7,000g) for 60 min, and the precipitate wasdissolved in the NaCl-CaCI2 solution. The preparation was thenpassed through a column (3.8 x 41 cm) of Sephadex G-25,which had been equilibrated with 0.01 M phosphate buffer (pH6.9) containing the NaCl-CaCl2 solution. Then, the enzyme-glycogen complex which was prepared following the method ofLoyter and Schramm (17) was washed twice with 40% ethanolcontaining 0.01 M phosphate buffer (pH 8.0). After dissolving theenzyme-glycogen complex in 0.02 M phosphate buffer (pH 7.0)containing 7 mm NaCl and 3 mm CaCI2, the preparation waskept at 25 C for 1 hr to digest glycogen. A small amount of pre-cipitate that had formed was removed by centrifugation, andthe supernatant fraction was passed through a column (22 X2.4 cm) of Sephadex G-25, which had been equilibrated with 0.01M tris-HCl buffer (pH 8.5) containing 0.1 M NaCl and 3 miCaCl2. The enzymically active fractions were applied to a column(2.4 X 18.5 cm) of DEAE-Sephadex A-50, which had beenequilibrated with 0.01 M tris-HCI buffer (pH 8.5) containing0.1 M NaCl and 3 mm CaC12. Employing a linear gradient ofNaCl (0.1-0.7 M), the enzyme was fractionated. All the prepara-tive steps were carried out at 0 to 4 C.

Isoelectric Focused Polyacrylamide Gel Disc Electrophoresisand a-Amylase Zymogram. The multiple forms of a-amylaseby isoelectric focusing in combination with polyacrylamidegel disc electrophoresis were detected by the method of Momo-tani (6, 28, 34). The pH gradient used was in the range of either4 to 6 or 5 to 8 using LKB Ampholine (Uppsala, Sweden). A

586

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a-AMYLASE ISOZYMES IN BARLEY SEEDS

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10 20 30 40 50FRACTION NUMBER

FIG. 1. Purification of a-amylase by DEAE-Sephadex A-SO colurmnchromatography. Purified preparation of a-amylase obtained by themethod of enzyme-glycogen complex formation, as explained in thetext,was equilibrated with 0.01 m tris-HCI buffer (pH 8.5) containing 0.1 mNaCi and 3 imM CaCd2. Ten-mililiter fractions of eluates were used formeasurements of ultraviolet absorbance at 280 nm and enzyme ac-tivities. Aliquots of fractions A and B were used for enzyme assay withthe standard system described in the text (1 yl for starch hydrolysis innon-buffered system and 5 Al for maltose formation [pH 6.91). Amount(mg) of starch hydrolyzed or maltose formed under the assay condition(30 C for 5 min) was calculated.

glass plate (8 X 9 cm) with a thin film of 1% egg albumin as anadhesive was heated at 120 C for 1 hr. Then, a thin layer of asolution containing 30%O cyanogum-41, 1.0 M acetate buffer(pH 5.3), 0.004%C riboflavin, and 1.5%l, soluble starch (2.5:1:1:4,v/v) and a few drops of N,N,N',N'-tetramethyl ethylenediaminewas added to each plate and photopolymerized. The gels to betested for a-amylase were placed between two such starch-acrylamide plates in sandwich form and incubated for 30 minat 30 C. Then the plates were stained with the 12-HCI solution.Electrofocused zones containing amylase activities hydrolyzedthe starch film and appeared as decolorized bands on a bluebackground. The focused gels were also used for staining proteinbands with Amido black lOB after the elimination of Ampholineand for carbohydrate constituents in protein using the fuchsin-sulfite reagent (1, 11).

Preparative Isoelectric Focusing. The isoelectric focusing elec-trophoresis was carried out following a similar method, as re-ported by Matsumoto et al. (16, 19). A linear sucrose gradient(0-50%X-) containing 1 % of LKB Ampholine (pH 4-6) was pre-pared with the aid of a mixing cylinder. Three milliliters of theenzyme preparation obtained by DEAE-Sephadexl A-50 ionexchange column chromatography (fraction B in Fig. 1) in 25%Csucrose was applied to a column. Electrophoresis was performedat a constant voltage of 400 v for 48 hr, in a cold room (4 C).At the end of the run, 25 drops (about 1.5 ml) of each fractionwere collected, and both pH and a-amylase activities were deter-mined.Enzyme Assay. Activities of a-amylase were determined by

measuring the rate of starch-I2 decoloration following the methodof Shuster and Gifford (26). (a) Nonbuffered system. One-milliliter of appropriately diluted enzyme preparation in theNaCl-CaCl2 solution was incubated with 1 ml of 0.067%o soluble

I Abbreviation: DEAE: diethylaminoethyl.

starch dissolved in 0.06 M NaH2PO4 solution. After 5 to 10 minof incubation at 30 C, the reaction was stopped by adding 1 mlof I2-HCl solution, and the absorbance at 620 nm was determinedafter adding 4.0 ml of H20. As can be seen in Figures 5, 9, and10, enzyme activities as measured represent the initial reactionvelocity. (b) Constant pH system. Soluble starch (0.1 or 1.0%)dissolved in either 0.05 M acetate buffer (pH 5.0) or 0.02 M phos-phate buffer (pH 6.9) was used as substrate.

In a separate analysis, an increase of reducing power of thereactant (maltose formation) was measured by the method ofBernfeld (2). An appropriately diluted enzyme preparation (0.5ml) dissolved in the NaCl-CaCl2 solution was incubated at 30 Cfor 5 to 10 min with 0.5 ml of 1.0% soluble starch dissolved in

(A)

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LC-IpH 4-6

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Experimental details for gel electrophoresis and staining of gels are de-scribed in the text. A: a-Amylase zymograms of enzyme preparationsat various purification steps; B: Amido black lOB and fuchsin-sulfitestaining of fraction B.

Plant Physiol. Vol. 46, 1970 587



TANAKA AND AKAZAWA Plant Physiol. Vol. 46, 1970

60,000 RPM I16.6mg/ml)

FIG. 3. Analytical ultracentrifugation of a-amylase (fraction B). Purified preparation of a-amylase (fraction B) obtained from DEAE-SephadexA-SO (cf. Fig. 1) was applied to analytical ultracentrifugation with a Hitachi model UCA-1 -A analytical ultracentrifuge (60,000 rpm at 20C; barangle of 70°).

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10 20 30 40 50 60 70 80FRACTION NUMBER

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FIG. 4. Preparative type isoelectric focusing of a-amylase (fractionB). a-Amylase preparation (fraction B) was subjected to a preparativetype isoelectric focusing, as explained in the text. Collected fractions(25 drops) were subjected to measurements of ultraviolet absorbance at280 nm, and aliquots were used for measurements of enzyme activitiesin the presence or absence of Ca2+ (5 IAI for starch hydrolysis in non-buffered system and 20,l for maltose formation [pH 6.9]). Reactionwas at 30 C for 10 min. Other conditions of enzyme assay were the sameas those explained in Figure 1.

either 0.05 M acetate buffer (pH 5.0) or 0.02 M phosphate buffer(pH 6.9) containing 7 mM NaCl. The reaction was stopped byadding 1.0 ml of 1% alkaline 3,5-dinitrosalicyclic acid reagent,and the absorbance at 540 nm was determined after adding 5 mlof H20.The heat treatment (70 C) of the enzyme preparation was per-

formed in 0.05 M tris-HCl buffer (pH 8.5) containing 5 mMCaC12 (28). An aliquot, 0.1 ml for starch hydrolysis and 0.05 mlfor maltose formation, was then used for determining the residualenzyme activities by employing the method described above.The method of low pH treatment was described previously (28).

20 30 40 5 1 0 15INCUBATION TIME IN MINUTES

FIG. 5. a-Amylase activities of fractions A and B at pH 5.0 and 6.9.The time-dependent enzyme activities (starch hydrolysis) of each frac-tion (A and B) were determined at pH 5.0 and 6.9, following the methodas explained in the text. The same quantities of each fraction (20 ,l for1.0% soluble starch and 5 ,ul for 0.1% soluble starch) were diluted to 1.0ml so as to compare the enzyme activities quantitatively.

After treatment of the enzyme preparation (fractions A and B inFig. 1) by allowing it to stand at pH 3.3 overnight at 0 C, aliquotswere used for measurements of enzyme activities by employingthe methods described above, except that 5 mm EDTA was addedto the reaction mixture. In these experiments, same quantitiesof each fraction diluted from the stock solution of same concen-tration (0.12 mg/ml) were compared for their activities (Figs.5-10).Paper Chromatography. Aliquots of the enzyme preparation

fractions A and B in Fig. 1) (40 and 80 Al) were diluted to 0.5 ml,with the NaCl-CaCI2 solution. Then it was incubated with 0.5ml of 1% soluble starch dissolved in either 0.05 M acetate buffer(pH 5.0) or 0.02 M phosphate buffer (pH 6.9) at 30 C for 40 min.At the end of reaction, the reactant was immer ed in a boilingwater bath for 2 min, an aliquot (50 ul) was applied to WhatmanNo. 1 paper, and the paper was subjected to descending paperchromatography following the method reported previously((22). The solvent used was 1-butanol-pyridine-H20 (6:4:3' v/v)and sugars separated were located by spraying the chromato-grams with the reagent of Trevelyan et al. (29).

Analytical Ultracentrifugation. Purified enzyme preparation,fraction B separated by DEAE-Sephadex A-50 (see Fig. 1) wassubjected to analytical ultracentrifugation with a Hitachi modelUCA-1-A analytical ultracentrifuge. A protein sample (16.6mg/ml) dissolved in 10 mm tris-HCI buffer (pH 8.0) containnigthe NaCl-CaCl2 solution was centrifuged (20 C) at the rotor

588



~P1nthysolVo.4,170a-AMYLASEISOZYMES IN BARLEYSEEDS58

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FIG. 6. Paper chromatogram of reaction patterns of fractions A and B. Experimental details are described in the text.

speed of 60,000 rpm, and the sedimenting bands were detected

by the schlieren optics.

RESULTS

The a-amylase preparation obtained after enzyme-glycogen

complex formation was reported to be very pure, approachingthe crystalline enzyme in its specific activities (17). However, as

shown in Figure 1, two maj'or amylase fractions (A and B) were

separated by further applying the preparation to DEAE-Sepha-dex A-5O ion exchange column chromatography. Homogeneity

of each fraction was examined by an a-amylase zymogram em-

plcy1dng isoelectric focusing in conjunction with polyacrylamide

gel disc electrophoresis (Fig. 2). Multiple isozymes detected in

,each fraction represent the enzyme constituents present in the

original crude extract as well as in the enzyme preparation after

,enzyme-glycogen complex formation.

Since the quantity of fraction B was greater than fraction A,its purification was undertaken. An analytical ultracentrifuga-tion picture showed an apparent homogeneity of fraction B

(Fig. 3), but staining with Amido black lOB and fuchsin-sulflte

reagent of electrofocused gels clearly showed that there were at

least three isozymes (B1, B2, B3 of Fig. 2). Since the staining

intensities of B1 band tended to increase during the later stages of

purification, this component may be an artifact (see "Discus-

sion"). By applying fraction B to a preparative type isoelectric

focusing, two protein fractions were separated as detected by

their ultraviolet absorbances at 280 nm (B and B', Fig. 4). In

the presence of Ca2+ in the reaction mixture, both fractions

showed activities of starch hydrolysis (12-starch reaction) as

well as maltose formation. However, in the absence of Ca2+,

fraction B' had negligible enzyme activity. Fraction B' is con-

sidered to be a modified form of B, as its banding patterns on a

polyacrylamide gel disc electrophoresis with Ca2+-containingmedium were indistinguishable from those of fraction B as pre-

sented in Figure 2.

As can be seen in Figure 1, the amylolytic activities of fraction

A are sharply contrasted to those of fraction B, little maltose

being formed in the reaction. Thus enzyme properties of the two

fractions were further compared with the same quantities of each

fraction for assay. We first compared the optimum pH of the

two fractions, with various buffer systems. It was found that

fraction A has the normal bell-shaped pH-activity curve, the

optimum at 5.3. On the other hand, B has a broad curve and

activity at pH 5.0 is nearly the same as at pH 6.9. Results pre-

sented in Figure 5 clearly show that the enzyme activities of frac-

tion A measured at pH 5.0 were nearly the same or even higher

compared with fraction B. This behavior can be further envisaged

from the results of paper chromatography, showing the different

40.,

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589Plant Physiol. Vol. 46, 1970

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TANAKA AND AKAZAWA Plant Physiol. Vol. 46, 1970

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40

FIG. 7. Effect of heat treatment on a-amylase activities of fraction Aand B (I). After heat treatment (70 C) of fractions A and B for differentperiods, an aliquot (0.1 ml) was used for measurements of enzyme ac-tivities as explained in the text. Incubation was 4.5 min for fraction Aand 6 min for fraction B, respectively, in starch hydrolysis and 10 minin maltose formation. The same quantities of each fraction were di-luted so as to compare the enzyme activities quantitatively.

1.2

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pH 50 (Acetoce buffer) PH 6.9 (Phosphate buffer)0.6-

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10 20 30 40 10 20 30 40

ENZYME SOLUTION ADDED PI')

FIG. 8. Effect of heat treatment on a-amylase activities of fractions Aand B at pH 5.0 and 6.9 (II). After heat treatment (70 C for 15 min), thesame quantities of fractions A and B prepared by the manifold dilutionof each stock solution were incubated with 1.0% soluble starch dis-solved in either 0.05 M acetate buffer (pH 5.0) or 0.02 M phosphatebuffer (pH 6.9). Reaction was at 30 C for 5 min. Straight lines: Noheat treatment controls.

action patterns of two fractions (Fig. 6). It must be noted thatthere is a shift in the reaction products of fraction B when thepH goes from pH 5.0 to 6.9. At pH 5.0 the reaction product ispredominantly glucose, and at pH 6.9 equal amounts of glucoseand maltose are formed.Another uniqueness of the enzyme activities of fraction A lies

in its heat activation. As can be seen in results of Figure 7, frac-tion B lost approximately 30% activity as a result of the heattreatment. When heated at 70 C for 30 min (pH 6.9), enzyme ac-

tivity of fraction A (maltose formation) was enhanced approxi-mately 2-fold, although it was impossible to prove that all theisozymes were equally activated. As presented in Figure 8, theenhancement of enzyme activity caused by heat treatment atpH 5.0 was not as marked as that by heating at pH 6.9.A question will be raised as to the nature of enzyme activity

elicited by fraction A, since its optimal pH was found to resideon the acidic side, typical of f3-amylase. However, in addition tothe heat activation, the loss of enzyme activity upon allowing thepreparation to stand at pH 3.3 overnight makes this possibilityunlikely (Figs. 9 and 10). The enzyme inactivation caused by the

B

InE-1.0~ A 1.0-0 -'0 /1m~~~~~~~~~~~~~~~~~~0, 082/

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TItEATMENT ~~~~pH33RATMENT

5 10 15 20 5 10 15 20INCUBATION TIME IN MINUTES

FIG. 9. Effect of pH 3.3 treatment on a-amylase activities of frac-tions A and B at pH 5.0 and 6.9 (I). After pH 3.3 treatment (0 C for 24hr), a 5-,gl aliquot of each fraction was diluted to 1.0 ml and was incu-bated with 0.1% soluble starch dissolved in either 0.05 M acetate buffer(pH 5.0) or 0.02 M phosphate buffer (pH 6.9) in the presence of 5 mMEDTA. Straight lines: No pH 3.3 treatment controls.

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10 20 30 40 10 20 30 40

INCUBATION TIME IN MINUTES

FIG. 10. Effect of pH 3.3 treatment on a-amylase activities of frac-

tions A and B at pH 5.0 and 6.9 (II). Details of pH 3.3 treatment werethe same as those explained in Figure 9, except 20 ,l aliquots were usedfor experiments. Straight lines: No pH 3.3 treatment controls.

low pH treatment has often been taken as a characteristic be-havior of a-amylase (8-10, 25). It is interesting to note that f,-amylase-type activity is slightly detectable in fraction B afterthis treatment (Figs. 9 and 10). However, reaction productsproduced by the residual fraction closely resembled those pro-duced by the untreated preparation on a paper chromatography(cf. Fig. 6). It thus indicates that fraction B is a-amylase.

DISCUSSION

The synthesis of multiple forms of a-amylase in GA-treatedbarley half-seeds has been reported by a number of workers (20,21, 23, 33). Our recent experiments with rice seeds have demon-strated that the banding patterns of a-amylase isozymes are nearlyidentical in both the GA-treated embryoless and embryo-attached (no GA treatment) half-seeds, indicating a crucial roleof GA in the synthesis of a-amylase isozymes as well as in thedynamics of starch breakdown in seeds at the onset of germina-tion (28).

Extraordinarily high resolving power of the isoelectric focusingtechnique in showing heterogeneity of apparently homogeneouspreparations of a number of enzyme proteins was establishedby Rutter and his associates (27). They demonstrated the pres-

590

STARCH HYDROLYSIS

O~~~~~~~~ B

A

MALTOSE FORMATION

.A

B



a-AMYLASE ISOZYMES IN BARLEY SEEDS

ence of variants in a single a-amylase preparation isolated fromrabbit pancreas and parotid glands (18). However, in the presentinvestigation separation of individual isozyme components bypreparative isoelectric focusing was not successful, although thepresence of multiple forms in each purified fraction (A and B)was clearly detectable on isoelectrically focused gels. Therefore, itis not known at this stage if either fraction A or B consists of ho-mologous enzyme molecules. The initial investigation by Fryden-berg and Nielsen (10) dealing with the formation of a-amylaseisozymes in barley seeds was attempted to correlate the enzymepatterns with the varietal differences of plants. Thus furtherstudies are needed to explore whether or not multiple forms ofa-amylase are governed by different genes or allelic genes. Wealso cannot elirninate a possibility that some of the isozymecomponents detected might have resulted from modification ordegradation of enzyme molecules during the GA-induced en-zyme synthesis as well as in the subsequent purification steps(cf. 13, 28). These possibilities must be considered in light of theexperimental results with the enzyme fraction B shown in Fig-ure 2 as well as the results shown in Figure 4, indicating theformation of artifacts during the step of isoelectric focusing.Most explicit in the present finding, however, is the formation

in GA-treated half-seeds of two forms of ca-amylase, evidentlydiffering in their properties and reaction patterns. A detailedcomparison of the kinetic properties between two a-amylasegroups will be a subject of another paper. From their distinctlydifferent nature, we are inclined to speculate that a-amylaseisozymes represented in fractions A and B may function nor-mally in germinating barley seeds, and that the complete break-down of starch reserve is accomplished by their cooperativeaction. It appears that the fraction A enzyme constitutes a rela-tively minor component in total a-amylase present in the seedextract. However, its unique nature, (a) activation by heatingand (b) enhanced activity at the optimum pH 5.0, is clearly dis-tinguishable from that of fraction B. It would be of interest tostudy whether or not similar isozyme patterns occur in othervarieties of barley seeds or in other cereal seeds. Recently Jacob-sen et al. (12) have reported that the addition of GA to isolatedaleurone cells of barley seeds causes the de novo production andsecretion of four a-amylases, which are somewhat different intheir enzymic nature from ours. It has been well established thata-amylases originated from different plant sources exhibit dif-ferent reaction properties (8). Among several properties, heatlability and optimal pH at the acidic side are thought to betypical in distinguishing f3-amylase from a-amylase. On the otherhand, the inactivation upon standing at low pH (3.3) for pro-longed periods is typical of at-amylase. Therefore, based on thesecriteria, our present experimental results show that fraction Aand B are mostly composed of a-amylase. The presence of a-amy-lase resistant to low pH treatment as recorded in results of Fig-ures 9 and 10 accords with the findings of Jacobsen et al. (12).

Evidence for the synthesis of a-amylase de novo in barleyaleurone ceJls in response to GA addition came from experi-ments of Varner and his associates (7, 13, 30-32). Although it isdifficult to compare experimental results from different labora-tories, our study indicates that caution is needed in concludinghomogeneity of a newly synthesized enzyme molecule.

Acknowledgment-The authors wish to record their most sincere thanks to Dr. T.Kosuge, Department of Plant Pathology, University of California, Davis, for his kindhelp in preparing the manuscript.

LITERATURE CITED

1. AKAZAWA, T., K. SAIO, AND N. SUGIYAMA. 1965. On the structural nature of frac-tion-I protein of rice leaves. Biochenm. Biophys. Res. Commun. 20: 114-119.

2. BERNFELD, P. 1955. Amylase, a and ,B. In: S. P. Colowick and N. 0. Kaplan, eds.,Methods in Enzymology, Vol. 1. Academic Press, New York. pp. 149-158.

3. BRIGGs, D. E. 1963. Biochemistry of barley germination: Action of gibberellic acidon barley endosperm. J. Inst. Brew. 69: 13-19.

4. CHRISPEELS, M. J. AND J. E. VARNER. 1967. Gibberellic acid-enhanced synthesisand release of a-amylase and ribonuclease by isolated barley aleurone layers.Plant Physiol. 42: 398-406.

5. CHRISPELS, M. J. AND J. E. VARNER. 1967. Hormonal control of enzyme synthesis:On themode of action of gibberellic acid and abscisin in aleurone layers of barley.Plant Physiol. 42: 1008-1016.

6. DAVIS, B. J. 1964. Disc electrophoresis. II. Method and application to human serumproteins. Ann. N. Y. Acad. Sci. 121: 404 447.

7. FILNER, P. AND J. E. VARNER. 1967. A test for de novo synthesis: Density labelingwith H20s of barley a-amylase induced by gibberellic acid. Proc. Nat. Acad.Sci. U.S.A 58: 1520-1526.

8. FISCHER, E. H. AND E. A. STEN. 1960. a-Amylases. In: The Enzymes, Vol. 4. Aca-demic Press, New York. pp. 313-343.

9. FRENCH, D. 1960. ,-Amylases. In: The Enzymes, Vol. 4. Academnic Press, New York.pp. 345-368.

10. FRYDENBERG, 0. AND G. NIEESEN. 1966. Amylase isozymes in germinating barleyseeds. Hereditas 54: 123-139.

11. HoTrcKuss, R. D. 1948. A microchemical reaction resulting in the staining of poly-saccharide structures in fixed tissue preparations. Arch. Biochem. 16: 131-141.

12. JACOBSEN, J. V., J. G. SCANDALIOS, AND J. E. VARNER. 1970. Multiple forms ofamylase induced by gibberellic acid in isolated barley aleurone layers. PlantPhysiol. 45: 367-371.

13. JACOBSEN, J. V. AND J. E. VARNER. 1967. Gibberellic acid-induced synthesis of pro-tease by isolated aleurone layers of barley. Plant Physiol. 42: 1596-1600.

14. JoNES, R. L. 1969. Gibberellic acid and the fine structure of barley aleurone cells. 1.Changes during the lag-phase of a-amylase synthesis. Planta 87: 119-133.

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