THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 32, Issue of November 15, pp. 19392-19398,1989 Printed in U. S. A.

The Calcium Requirement for Stability and Enzymatic Activity of Two Isoforms of Barley Aleurone a-Amylase*

(Received for publication, May 5, 1989)

Douglas S. Bush$, Liliane Sticher, Robert van Huysteee, Doris Wagner, and Russell L. Jones From the Department of Plant Biology, University of California, Berkeley, California 94720

a-Amylases (EC 3.2.1.1) secreted by the aleurone layer of barley grains are Ca’+-containing metalloen- zymes. We studied the effect of Ca’+ on the activity and structure of the two major groups of aleurone a-amy- lase by incubating affinity purified enzyme in solutions containing Ca’+ from pCa 4 to 7. Both groups of iso- forms required one atom of Ca’+/molecule of enzyme as determined by isotope exchange, but the two groups differed by more than 10-fold in their affinity for Ca’+. Both groups of a-amylase were irreversibly inacti- vated by incubation in low Ca’+ (pCa 7). This inacti- vation was not due to changes in primary structure, as measured by molecular weight, but appeared to be the result of changes in secondary and tertiary structure as indicated by circular dichroism spectra, serology, lability in the presence of protease, and fluorescence spectra. Analysis of the predicted secondary structure of barley aleurone a-amylase indicates that the Ca’+- binding region of barley amylases is structurally sim- ilar to that of mammalian a-amylases. Our data indi- cate that micromolar levels of ca’+ are required to stabilize the structure of barley a-amylases in the en- doplasmic reticulum of the aleurone layer where these enzymes are synthesized.

Barley (Hordeum vulgare L.) grains synthesize a group of a-amylase isoforms (EC 3.2.1.1) that function during germi- nation and early seedling growth to mobilize storage carbo- hydrate for the growing embryo (1, 2). a-Amylase synthesis in the aleurone layer of barley is regulated by the plant hormone gibberellin and the calcium ion (1,2). The ability of calcium to stimulate production of a-amylase from isolated aleurone layers has been extensively studied in an attempt to understand how cell Ca2+ modulates protein synthesis and secretion in plant cells (1). Understanding the mechanism of Ca2+ action in this tissue, however, is complicated by the fact that barley a-amylases (Amy),’ like other a-amylases, are metalloenzymes that contain Ca2+ (3).

To distinguish the effects of Ca2+ on cellular processes

* This research was supported by grants from the National Science Foundation and the United States Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Present address: Dept. of Plant Sciences, The University of

Western Ontario, London N6A 5B7, Canada. The abbreviations used are: Amy, barley a-amylases; PPA, por-

cine pancreatic a-amylase; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis; BAPTA, bis-(0-aminophenoxy)- ethane-N,N,N’,N‘-tetraacetic acid; ER, endoplasmic reticulum; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethelenenitrilo)]tetraacetic acid.

leading to a-amylase production from a direct effect of Ca2+ on the activity of the enzyme it is necessary to obtain quan- titative information on the effect of Ca2+ on the activity and structure of a-amylase. The purpose of this study was to determine the amount of Ca2+ needed to activate and stabilize the two main isoform groups of barley a-amylase and to study the effects of Ca2+ depletion on the structure of the enzyme.

In the Himalaya cultivar of barley, isoforms of Amy fall into two groups that are the products of separate gene families (4). These two groups differ by 23% of their amino acid sequence, are serologically distinct, and may be separated from each other on the basis of their isoelectric points (PIS), which range from 4.4 to 5.2 for the low PI group (Amyl) and from 5.9 to 6.6 for the high PI group (Amy2) ( 5 , 6). Both groups of enzymes are presumed to contain Caz+, although only Amy2 has been shown to lose activity when incubated in solutions containing Ca2+ chelators such as EDTA (7, 8).

We here report that both groups of barley a-amylase bind Ca2+ but do so with affinities that differ by an order of magnitude. Removal of calcium from the barley amylase molecule results in the irreversible inactivation of the protein. Ca2+ stabilizes the tertiary structure of both Amyl and Amy2 as it does for porcine pancreatic a-amylase (PPA, 9-11). Our analysis of the secondary structure of Amyl and Amy2 pre- dicted by the methods of Chou and Fassman (12) shows that these enzymes are structurally similar to PPA in the NH2- terminal region, supporting the hypothesis put forward by Buisson et al. (13) that the structure of the Ca2+-binding domain of all a-amylases is similar.

EXPERIMENTAL PROCEDURES

Plant Material-Barley grains (Hordeum vulgare L. cv. Himalaya, 1985 harvest, Department of Agronomy, Washington State Univer- sity, Pullman, WA) were de-embryonated and allowed to imbibe water as described (14). Aleurone layers were isolated (15) and incubated in 5 PM GA, (Sigma) and 10 mM CaC12 for 18 h (20 layers/l ml).

a-Amylase Purification-a-Amylase was purified from the incu- bation medium of aleurone layers by affinity chromatography on a cycloheptaamylose-Sepharose 6B column (14). Affinity purified am- ylase was precipitated in 80% ethanol at 4 “C and the precipitate resuspended in 500 FM CaClz and 5 mM HEPES, pH 7.4. The cycloheptaamylose was removed by dialysis against the same buffer used to resuspend the protein. a-Amylase that was affinity purified from aleurone-layer incubation medium was free of contaminating proteins. The amount of the amylase/subtilisin inhibitor, in particu- lar, was below the level of detection in affinity purified a-amylase. For Caz+-binding experiments, only amylase that contained no de- tectable level of amylase/subtilisin inhibitor was used. The presence of the inhibitor was assayed in amylase preparations by isoelectric focusing, SDS-PAGE, and immunoblotting using antibodies to the inhibitor obtained from Dr. A. W. MacGregor (Grain Research Lab- oratory, Winnipeg, Manitoba, Canada; 16). Amyl and Amy2 were separated on the basis of their PI using a 33 X 1-cm chromatofocusing column according to the manufacturer’s directions (Pharmacia LKB Biotechnology Inc.). The chromatofocusing column was equilibrated at room temperature with 25 mM imidazole-HC1 buffer, pH 4.7,

19392

Calcium Requirement of Barley a-Amylase 19393

containing 0.1 mM CaCl2. Affinity purified a-amylase was eluted with Polybuffer 74 (Pharmacia LKB Biotechnology Inc.), pH 7.4 diluted 1% and containing 0.1 mM CaCl2, at a flow rate of 30 ml/h, and 3-ml fractions were collected. a-Amylase was assayed in each fraction, and isozymes were identified by isoelectric focusing.

Proteolytic degradation of purified Amy (3.6 pg) was performed with chymotrypsin (8 pg, Sigma) at room temperature for 30 min. The reaction was stopped by the addition of phenylmethylsulfonyl fluoride and the products were examined by electrophoresis.

Assay and Electrophoresis of a-Amylase-a-Amylase (EC 3.2.1.1) was assayed using the starch-IsKI procedure (17). Visualization of separated a-amylase isozymes after isoelectric focusing followed the procedures described by Jones and Jacobsen (14). SDS-PAGE was performed in a mini-gel system (6 X 9 X 0.08 cm) using 12.5% acrylamide or on a 12-20% acrylamide gradient gel using the condi- tions described by Laemmli (18). Laurel1 rocket immunoelectropho- resis was performed according to Weeke (19), using rabbit serum prepared against total barley a-amylase (20).

Determination of the Calcium Content of a-Amylase-The amount of calcium associated with a-amylase was determined after isotopic exchange with 45Ca2+. Calcium-binding experiments were performed under conditions that would approximate the presumed ionic strength and pH of the endoplasmic reticulum where Amy is synthesized. Native a-amylase was incubated for 2 h in solutions containing 100 mM KCl, 50 mM HEPES, pH 7.4, 100 pM CaC12 containing 300 &i/ mM of 45CaC1z, and a sufficient amount of the calcium chelator, bis- (0-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid (BAPTA), to set the Ca2' concentration in a range from pca 4 to 7 (i.e. lo" M to

M Ca2+). After equilibration the protein was concentrated and separated from the supernatant by centrifugation of the incubation mixture through a Centricon-10 ultrafiltration membrane (Amicon, Danvers, MA). Since BAPTA does not completely buffer Ca2+ over the entire range of Ca2+ concentrations used, incubation and centrif- ugation of the protein was repeated twice to insure that a-amylase had equilibrated at the desired Ca2+ concentration. After centrifuga- tion, approximately 93% of the a-amylase was present in the reten- tate. Differences in the amount of '%a in aliquots of the retentate and filtrate, as measured by liquid scintillation counting, were attrib- uted to calcium retention by a-amylase.

The amount of BAPTA needed to set the Ca2+ concentration in the incubation solutions was determined using a computer program we developed based on a computational strategy described by Westall (21). This program uses the total concentration of the components of the solution (Ca2+, BAPTA, etc.), the stoichiometry of components to form species (e.g. CaH,BAPTA, CaHBAPTA, etc.) and the disso- ciation constant for these species to calculate, by iteration, the concentration of each species. The concentration of BAPTA stock solutions was determined by potentiometric titrations, using a Ca2+- sensitive electrode (Radiometer, USA), against a calcium standard prepared from oven-dried CaC03. BAPTA concentration was com- puted by the method of Bers (22). This method involves measurement of electrode response in the absence of chelator between pCa 3.5 and 2. Estimates of the amount of free and bound CaZ+ in the presence of a fixed amount of chelator with varying amounts of CaZ+ are then calculated from the electrode reading using the response measured in the absence of chelator. By these means we determined that the purity of BAPTA was 98% of the nominal value with an apparent binding constant under the conditions we used of 399 nM.

Microsome Isolation and Incubation-One-hundred aleurone layers were chopped as described previously (23, 24), and microsomes were isolated on a discontinuous sucrose density gradient (24). The turbid band at the interface between the 13% (w/w) and 50% (w/w) sucrose solutions was collected, 250 pl of this fraction was added to 4.75 ml of a solution containing 0.25 M sucrose, 20 mM HEPES, pH 7.3, 200 mM EGTA, 50 mM KCl, and 208 p M CaCl2 (for pCa 5) or 128 PM CaClz (for pCa 7). Microsomes were incubated in this solution for 30 min at room temperature (25 "C). Where indicated, 5 p~ ionomycin (Behring Diagnostics) or 1 p~ A23187 (Behring Diagnostics) was added. Following incubation, the microsomes were pelleted at 150,000 X g for 45 min in an SW 50.1 rotor (Beckman Instruments) and resuspended in 150 pl of 10 mM HEPES, pH 7.3, containing 1 mM CaC12, and a-amylase activity and protein content were measured.

Spectroscopy-The amount of affinity purified Amy was deter- mined from absorbance measurements at 280 nm using a Beckman Acta CIII double-beam spectrophotometer. Removal of Ca2+ from Amy had no effect on its absorbance at 280 nm. This indicates that Caz+-depleted protein was fully soluble under the experimental con- ditions we used and allowed us to use a single extinction coefficient

for native and Caz+-depleted Amy (280 nm) of 1.25 X lo6 I mol" cm". Circular dichroism spectra were measured on native and Ca2+-

depleted forms of Amy using an AVIV circular dichroism spectropho- tometer (model 60DS, AVIV, Lakewood, NJ). Prior to measurement, both forms of Amy were desalted by gel-filtration using a PD-10 column (Pharmacia LKB Biotechnology Inc.) equilibrated with 2 mM sodium phosphate, pH 7.0. Circular dichroism was expressed as mean residue ellipticity (25) using a residue weight of 110 g mol". a-Helix content of Amy was calculated from CD measurements according to the method of Greenfield and Fasman (26). The fluorescence emission spectra of the tryptophan residues in Amy were measured using a Spex fluorolog fluorimeter (Spex, Edison, NJ) and protein samples prepared as described for CD measurements.

Calculation of Secondary Structure and Hydrophobicity of Amy and PPA-Primary structures of the mature protein (i.e. not including the signal peptide, Ref. 4) deduced from cDNA clones of Amyl (clone E, Ref, 27) and Amy2 (clone pM/C, Ref. 28) and from the amino acid sequence of PPA (29) were used in structural calculations. Hydro- phobicity was calculated using the InteliGenetics program PEP with the Kyte and Doolittle (30) values as installed at the University of California, Berkeley. Predictions of a-helix and @-sheet regions were made using the Chou and Fassman rules and the revised probability values (12). Briefly, the probability of an amino acid segment forming an a-helix and P-sheet was calculated as the numerical average of the probability values for a11 the amino acids in the segment. The initia- tion and termination of each segment was determined using the Chou- Fassman probability tables for initiation and termination (12).

RESULTS

The Activity of Amy Is Correlated with Its Ca2+ Content- The effect of Ca2+ on the activity of Amy was investigated by incubating purified Amy containing a mixture of all isoforms in solutions whose free Ca2+ concentration varied from pCa 7 to 4. Potassium chloride (100 mM) was included in the incu- bation solutions to minimize the amount of nonspecific Caz+ binding to Amy and to minimize Donnan effects across the ultrafiltration membrane used to concentrate Amy after in- cubation. The calcium content of Amy was determined by ion exchange with 45CaC1z. Exchange of 45Ca between solution and protein occurred in less than 2 h at room temperature, and the amount of Ca2+ exchange was the same after 2,4, and 6 h (data not shown). The amount of Ca2+ bound to Amy varied as a function of the concentration of Ca2+ in solution, reaching a maximum of 1 atom of Ca2+/molecule of protein (Fig. 1). a-Amylase (5 PM) was completely saturated at Ca2+ concentrations greater than 100 p M (calculated ratio of Ca2+/ Amy, 20:l) and half-saturated at 800 nM Ca2+ (Ca2+/Amy, 0.16:l). The affinity of Amy for Ca2+ was not significantly affected by the presence of 20 mM MgC12 indicating a high degree of specificity for binding of Ca2+ over M e (data not shown).

The activity of Amy was closely correlated to the amount of Ca2+-binding (Fig. 1): the fraction of maximal activity after incubation was nearly equal to the fraction of the protein that retained Caz+ (Fig. 1 and inset). This strong correlation al- lowed the fraction of Amy molecules that bind Ca2+ to be estimated directly from enzyme activity. Moreover, it indi- cates that both groups of Amy require Ca2+ for activity.

The affinities of Amyl and Amy2 for Ca2+ were determined by measuring the rates of inactivation of the purified isoforms at pCa 5.5 (Fig. 2) and by inactivation of the enzymes in differing concentrations of Caz+ from pCa 6.5 to pCa 3.5 (Fig. 3). The time course of inactivation shows that when each isoform group is incubated at pCa 5.5, Amy2 isoforms are more rapidly inactivated than the Amyl enzymes (Fig. 2). A more quantitative estimate of the affinity of the two Amy groups for Ca2+ was obtained by measuring the activity of each as a function of Ca2+ concentration (Fig. 3). These data confirm that Amyl is more stable than Amy2 at low Ca2+ concentrations. Analysis of this data by a double reciprocal

19394 Calcium Requirement of Barley a-Amylase

> I-

-400 2 I- o

v, W

-

a

-200 J a > 2 a

- 0 I 1 I I

7.0 6.0 5.0 4.0 Ca2+ CONCENTRATION ( PC0 )

FIG. 1. The effect of Ca2+ concentration on the calcium con- tent and enzymatic activity of barley aleurone a-amylase and (inset) the resulting correlation between Ca2+ content and enzyme activity. The calcium content and enzymatic activity of Amy were determined after equilibrium dialysis of affinity purified Amy (a mixture of both isoforms) against solutions containing 100 mM KC1, 20 mM HEPES, pH 7.4, 100 p M CaC12 with 300 pCi/mM '5CaC12, and sufficient BAPTA to set the Ca2+ concentration between pCa 4 and 7. After 4 h of incubation in these solutions the calcium content and amylase activity of the protein were measured.

1 I I I I 0 1 2 3 4

TIME (h)

FIG. 2. The effect of low Ca2+ on the time course of inacti- vation of two isoforms of Amy. Amyl and Amy2 purified by chromatofocusing were incubated as in Fig. 1 at pCa 6.

plot shows that the two forms of Amy differ by a factor of 10 in their affinity for Ca2+: the concentration of Ca2+ required for half-maximal activity (estimated by the slope of the line) is 300 nM for Amyl and 3 /IM for Amy2. These data also indicate that the number of Ca2+ atoms required for each mole of protein (estimated by the intercepts of the double reciprocal plot) is the same for Amyl and Amy2, a result that is consistent with the estimate of 1 Ca2+/molecule of Amy obtained from analysis of %a2' exchange in a mixture of isoforms (Fig. 1).

a-Amylase activity was irreversibly lost when purified na- tive enzyme was incubated in suboptimal concentrations of Ca2+ since activity could not be restored when the protein was incubated in high Ca2+ (Table I). a-Amylase that was Ca2+- depleted (and therefore inactive) also irreversibly lost its ability to bind Ca2+ (Table I).

The irreversible loss of enzyme activity and Ca2+ binding

l I I J 5 4 3 2

Co2'CONCENTRAT10N ( pCa I

FIG. 3. The effect of Ca2+ concentration on the enzymatic activity of two isoforms of Amy and a double reciprocal plot of these data (inset). Amyl and Amy2 were incubated in solutions containing 100 mM KCl, 50 mM HEPES, pH 7.0, 100 pM CaC12, and sufficient BAPTA to set the Ca2+ concentration between pCa 6.5 and 4. Amylase activity was measured after 4 h of incubation by the standard method.

TABLE I Effect of calcium removal from a-amylase on enzyme activity

and Ca-binding capacity measured in 5 mM CaCl2 a-Amylase

form Activitf Ca binding

unitslmg mol/mol Native 1387 & 231 111 Ca depleted' 180 & 200 0.0511

Activity and Ca-binding capacity were assayed after 2 h of incu- bation in 5 mM CaC12.

* Calcium was removed by incubation in 0.2 mM BAPTA.

could not be explained by proteolytic degradation of Amy during Ca2+ removal. a-Amylase incubated at pCa 7 until 90% of the enzyme was inactivated (1 h) was indistinguishable on SDS-PAGE from active enzyme molecules incubated at pCa 4 for 1 h (Fig. 4A). Storage of Amy in solutions of pCa 7 for several weeks resulted in small amounts of protein degrada- tion. SDS-PAGE of Amy incubated for 2 weeks at pCa 7 shows the presence of low molecular weight polypeptides that were absent from enzyme preparations incubated at pCa 6 and above (Fig. 4B). These peptides were recognized by a polyclonal antibody raised against total Amy confirming that they were Amy fragments (data not shown). Nevertheless these data indicate that Ca2+-depleted amylase may be stored in solution without precipitation or substantial proteolytic degradation.

The effect of low Ca2+ on the activity of Amy sequestered in microsomal vesicles isolated from barley aleurone layers was examined to determine whether low CaZ+ affects the activity of Amy in vivo. Microsomal membranes isolated from aleurone layers contain vesicles derived from the endoplasmic reticulum (ER) and Golgi apparatus that are high in Caz+ and Amy (24). a-Amylase activity in microsomal membranes from aleurone cells is unaffected by incubation of membranes in pCa 7 or 5 for 30 min at 25 "C (Fig. 5). In the presence of the Ca ionophores A23187 or ionomycin, however, the amylase activity of microsomes incubated in pCa 7 for 30 min declines by 50% (ionomycin) and 67% (A23187) relative to microsomes incubated in ionophore at pCa 5 (Fig. 5). These data show that Amy sequestered in microsomal vesicles is also sensitive to low Ca2+.

The Effect of Ca2+ on the Structure of Amy-Because the

Calcium Requirement of Barley a-Amylase 19395

I

Amy + + + + - - / Chym - - + + + - pCa 4 7 4 7 / /

8

m

pCa / 7 6 5 4

FIG. 4. The effect of Ca2+ removal from Amy on its suscep- tibility to proteolytic degradation by chymotrypsin ( A ) and on its stability in the absence of added protease ( B ) . A, affinity purified Amy was incubated in high (pCa 4 ) or low (pCa 7) levels of Ca2+ for 1 h in the presence (+) or absence (-) of chymotrypsin (Chyrn) a t 25 "C. The products of the reaction were then analyzed by SDS-PAGE. The arrow marks the position of native Amy. Molecular mass markers are shown in the rightmost lane and were (from top to bottom, in kDa): 97.4, 66.2, 42.5, 31.0, 21.5, and 14.4. R, Amy was incubated in a range of Ca2+ concentrations, as described in Fig. 1, and stored for 2 weeks a t 4°C in the presence of 0.05% sodium azide. Amy was then analyzed by SDS-PAGE with equal amounts of protein added to each lane except for pCa 7 where half the amount of protein was added. The molecular mass markers in the leftmost lane are the same as in A.

* O 7

O L

p c o : 7 7 7 5 5 IJJM IONOMYCIN - + - - +

1flM A23187 - - + " FIG. 5. The effect of Ca2+ and Ca2+ ionophores on the activ-

ity of Amy sequestered in microsomal vesicles. Microsomes isolated from 100 aleurone layers were incubated a t pCa 5 (shaded bars) or pCa 7 (open bars) in the presence (+) or absence (-) of the calcium ionophores ionomycin or A23187. After incubation, the mi- crosomes were pelleted by centrifugation, resuspended, and amylase activity/milligram membrane protein was measured.

data on the irreversible inactivation of Amy suggest a struc- tural role for Ca2+ in the Amy molecule, the effects of Ca depletion on the secondary and tertiary structure of the enzyme were studied. Information about secondary structure

was obtained from CD spectra and, by this method, Ca2+ depletion had a slight effect on the secondary structure of the Amy molecule (Fig. 6). CD spectra of Ca"-depleted and Ca"- saturated molecules were very similar showing a broad band of negative ellipticity between 208 and 240 nm typical of many globular proteins (Fig. 6). The a-helix content of both forms of the protein was estimated from the mean residue ellipticity a t 208 nm (26) to be 27%. Ca2+ depletion did cause a signifi- cant decrease in mean residue ellipticity of Amy between 200 and 208 nm (Fig. 6), a spectral region where the random coil conformation has a large negative ellipticity (25,26).

The effects of Ca2+ depletion on the tertiary structure of Amy were inferred from serology, fluorescence spectra, and the susceptibility of the protein to proteolysis. The ability of polyclonal antibodies raised against native Amy to recognize the Ca-depleted form of the enzyme was studied using Laurel1 rocket eletrophoresis. Removal of Ca2+ decreased the amount of Amy that was recognized by the antibody (Fig. 7 ) . At pCa 7, where both isoforms are inactive, 27% of Amyl and 19% of Amy2 are recognized by the antibody (Fig. 7 ) .

Ca2+ depletion also induced large changes in the tryptophan fluorescence spectra of purified Amy. Tryptophan fluores- cence in the Ca2+-depleted protein was shifted to slightly lower wavelengths and was reduced compared with Ca2+- saturated Amy (Fig. 8), indicating that Ca depletion causes changes in the chemical environment of tryptophan residues.

The susceptibility of Ca2+-depleted Amy to proteolytic deg- radation by chymotrypsin also indicates a change in tertiary structure following Ca2+ removal (Fig. 4A). Incubation of Ca2+-depleted Amy for 30 min a t 25 "C in the presence of chymotrypsin resulted in substantial degradation of the en- zyme as shown by the disappearance of the 44-kDa protein and the appearance of low molecular mass fragments on SDS- PAGE (Fig. 4A). Incubation of the native protein under identical conditions, however, showed much less Amy degra- dation (Fig. 4A).

The secondary structures of Amyl and Amy2 computed using the Chou-Fassman (12) rules were then compared with the secondary structure of PPA determined by crystallogra-

$5 y -15 190 200 210 220 230 240 250 260

WAVELENGTH ( nm )

FIG. 6. The effect of Ca2+ removal on the circular dichroism spectrum of Amy. Amy was incubated for 4 h in 100 mM KCI, 25 mM HEPES, pH 7.4,lOO mM EDTA, and either 100 mM CaC12 (+Ca) or 0 mM CaC12(-Ca). Amy was then desalted by gel filtration through a Pharmacia PD-10 column that had been equilibrated with 2 mM sodium phosphate, pH 7.0, and 0.5 mM CaCI,. Amy was then concen- trated and the CD spectra measured.

19396 Calcium Requirement of Barley cu-Amylme r -I

100 - 9 I

s? - 50+

LAURELL ROCKET ELECTROPHORESIS

c / /

_L 1.-1- L 7 6 5 4

caz+ CONCENTRATION (pCa)

0- "- ~I ~ ~~ I ." 1 - 7 0 6 0 5 0 4.0

ca2+ CONCENTRATION ( p c a )

FIG. 7. The effect of Ca2+ on the antigenicity of Amy. Affinity purilied Amy containing hoth Amyl and Amy2 was incubated at different Ca" concentrations. as described in Fig. 1, and its antige- nicity was assayed by Laurel1 rocket electrophoresis (inset, arrow denotes the Amyl rocket; the unmarked rocket is Amy2). Antigenicity was determined from rocket height and expressed as percent of maximum height.

0 ' J 300 325 350 375 400 425

WAVELENGTH (nrn )

FIG. 8. The effect of Ca2+ on the tryptophan fluorescence spectra of Amy. (la"-containing (+Ca) and Ca"-depleted (-C'a) Amy was ohtained as descrihed in Fig. 6. Tryptophan fluorescence was then measured hy excitation at 2995 nm.

phy (Fig. 9). The computed secondary structures for both Amyl and Amy2 are very similar to PPA and to each other at the NH, terminus (roughly residues 1-250, Fig. 9). The best alignment was obtained when PPA was shifted 18 amino acid residues with respect to Amyl and Amy2 (Fig. 9). The NH,-terminal 410 amino acid residues of PPA have two domains: domain A consisting of eight segments, each with an n-helix and @-sheet region, that form a barrel and domain R consisting of eight @-sheet regions inserted between the third [j and third n regions of domain A (13). A similar organization of amino acid residues is seen at the NH2 ter- minus of the computed secondary structure of Amy. Like the A domain of PPA, the NHy-terminal residues (1-85) of Amyl and Amy2 show an initial alternation of n and @ segments, interrupted by a large hydrophobic coil (residues 40-65), ending with a helix in a hydrophilic region. An extended @- sheet region in Amy corresponding to domain B of PPA and consist,ing largely of hydrophobic residues is computed for residues 100-140 (Amyl). Termination of this @-sheet region is not clear from the computed structure. In PPA, the B domain terminates in /%sheets made of hydrophilic residues

(13). Based on hydrophobicity, the corresponding region in Amyl and Amy2 would lie between residues 130 and 150, but helix or coil regions rather than @-sheets are predicted in this region. Although the termination of the @-sheet region is unclear in Amy, alternating n and @ regions are predicted starting around residue 180 (Amyl) and continuing to the COOH terminus. This segment corresponds to the large sec- ond segment of the A domain of PPA.

The four Ca"-binding amino acids in PPA lie at the inter- face of the A and R domains (13). Three of these amino acids can be found in structurally similar regions of Amy (Fig. 9). Asn-92 of Amy appears to correspond to Asn-100 of PPA, the most NH2-terminal Ca"-binding residue of PPA, since both are found in a hydrophobic region between two @-sheets. Similarly, Asp-138 and Asp-149 of Amy which span a hydro- phobic region in the B domain appear to correspond to Asp- 159 and Asp-167 of PPA. The Ca"-binding His-201 residue of PPA does not appear to have a structural analog in Amy. The degree of correspondence in structure between PPA and Amy declines toward the COOH-terminus. The C domain at the COOH terminus of PPA, an eight-stranded @ barrel, appears to be missing in Amyl and Amy2 (Fig. 9).

Another important difference between Amy and PPA is in the number of Cys residues and disulfide bonds. PPA contains 12 Cys residues and five disulfide bridges (Fig. 9 and Refs. 13 and 29). One of these bridges, Cys-70-Cys-115, links domains A and B of PPA thereby stabilizing the Ca"-binding region (13). In contrast, Amyl contains only 4 Cys residues and Amy2 only 3. None of these Cys residues appear to be in a position to link domains A and B of the Amy molecule (Fig. 9).

DISCUSSION

The Ca'+ Content of Barley n-Amylase-The aim of this work was to provide quantitative data on Ca'+ binding to barley n-amylase. Our results show that both Amyl and Amy2 bind one atom of Ca'+/molecule of protein, although their affinities for Ca'+ differ by an order of magnitude. Calcium binding to Amy is an irreversible process and is required to maintain both the activity and the stability of the enzyme. These data confirm previous observations showing that cereal n-amylases, like n-amylases from animals, fungi, and bacteria, are Ca-containing metalloenzymes whose activity and stabil- ity depend on Ca'+ (3).

Quantitative aspects of our data differ from previous pub- lished reports on Ca binding to cereal n-amylases. While our experiments show that Ca binding to affinity purified Amy is saturated a t one atom/molecule of protein (Fig. I), other determinations of the Ca content of the enzyme by atomic absorption spectrophotometry indicated a stoichiometry of 2:l for Ca binding (31). The higher estimate may be due to nonspecific binding which we have minimized by including 100 mM KC1 in the reaction mixtures. Estimates of Ca binding to various animal and fungal n-amylases also indicate a range in Ca content from one atom/protein molecule for mammalian n-amylase (10) to up to 10 atoms/mole for fungal and bacterial a-amylases (3).

While both major groups of Amy bind one atom of Ca, their affinities for this element differ (Figs. 1 and 3). Barley n- amylases coded by genes on chromosome 1 and designated Amyl have a high affinity for Ca2+, whereas a-amylases coded by genes on chromosome 6 and designated Amy2 have a low affinity for Ca?+ (Figs. 2 and 3). These data are consistent with the observations of Jacobsen et al. (5) that under the conditions they used, EDTA inactivated Amy2 but not Amyl. Therefore, our data support their proposal that Amyl iso-

Calcium Requirement of Barley a-Amylase 19397

FIG. 9. A comparison of hydro- phobicity ( H ) and predicted a-helix (a, open horizorttal bars) and 8- sheet (8, closed horizontal bars) re- gions of Amyl and Amy2 with hy- drophobicity and known a-helix and &sheet regions of PPA (13). Amyl and Amy2 were aligned with PPA by an 18-amino acid residue offset with respect to PPA. The location of the three struc- tural domains of PPA (A and C, light regions of hydropathy plot, and B, dark region of hydropathy plot) are shown with our prediction of the corresponding regions in Amy. Disulfide bridges in PPA (C-C) and cysteine residues in Amy ( C ) where potential disulfide bridges could occur are also indicated. The 4 amino acid residues known to participate in Ca2+ binding in PPA ( N , D, D, H ) are indicated with our prediction of the cor- responding residues in Amy ( N , D, D ) . Amino acid sequence numbers for Amyl, Amy2, and PPA start with the mature peptide and do not include the signal sequence.

H - I Ij - 2

z I

H O - I

-2

I

AMYI, AMY2 0

PPA 0 i-

forms bind Ca2+ strongly but do not support their alternate interpretation, that Amyl is not a Ca2+-containing metallo- enzyme.

The affinity of both forms of Amy may vary as a function of their chemical environment. We have measured Ca2+ bind- ing to Amy under conditions which approximate intracellular pH and ionic strength. Thus, the values we report represent those necessary for enzyme activity in the endomembrane system where Amy is synthesized and processed. The impor- tance of Ca2+ in stabilizing Amy in vivo is indicated by our experiments with isolated microsomal membranes. These in- dicate that the activity of newly synthesized Amy sequestered in the lumen of aleurone microsomes is sensitive to Ca2+ concentration (Fig. 5 ) . The level of Ca2+ that is necessary to stabilize Amy under other conditions, such as the mildly acidic extracellular environment into which it is secreted, may differ from the values we report here. However, the role of Ca2+ in stabilizing the secondary and tertiary structure appears to be fundamentally important and is unlikely to change in acidic conditions.

The Structural Role of Ca2+ in Amy-The irreversible in- activation of barley amylase caused by ea2+ removal suggests that Ca2+ is necessary for maintenance of the structure of the active site. The decline in activity of Amy associated with Ca2+ depletion cannot be attributed to changes in primary structure, since the molecular weight of the Ca2+-depleted form is the same as that of the native enzyme (Fig. 4) . However, some change in the secondary structure caused by Ca2+ depletion can be inferred from CD spectra (Fig. 6). Although the amount of a-helix does not appear to change, the increased negative ellipticity around 200 nm indicates an increase in the amount of random coil. Changes in tertiary structure of Amy following ea2+ depletion are indicated not only by altered enzyme activity (Table I), but also by reduced antigenicity (Fig. 7), by increased susceptibility to proteolysis (Fig. 4A), by the inability to bind Ca2+ (Fig. I), and by the change in the tryptophan fluorescence spectrum (Fig. 8).

Since Ca2' in solution readily exchanges with Ca2+ bound to Amy, we speculate that there is an apoenzyme of Amy that

50 loo 150 200 250 300 350 4p0 450 590 I ' I ' I I I I ! I

5b 100 150 ZOO d o 300 350 400 450 500

AMINO ACID RESIDUE

lacks Ca2+ but retains its tertiary structure. The high corre- lation between enzymatic activity and Ca2+ content indicates that in barley this putative apoenzyme must spontaneously change its tertiary structure into a form which is inactive and cannot bind Ca2+. This proposal is summarized by the follow- ing equation:

A*-Ca?+ a Ca2+ + A+ 4 A,

where A*-Ca2+ is the Ca'+-containing native enzyme, A* the apoenzyme that lacks ea2+, and A the inactive enzyme that cannot bind Caz+. This scheme would explain the irreversible inactivation of Amy caused by Ca2+ depletion as a balance between the rate at which Ca2+ binds to Amy and the rate at which A* undergoes conformational change.

The irreversible inactivation of Amy by ea2+ depletion contrasts with animal and bacterial a-amylases. Human sal- ivary and Bacillus subtilis a-amylase incubated in 10 mM EDTA can be completely reactivated by addition of 20 mM ea2+ (10). Stein and Fisher (32) have shown that irreversible loss of activity in these a-amylases was the result of protease activity and they proposed a reaction scheme similar to Equa- tion l where inactivation was catalyzed by trypsin. Inactiva- tion of Amy, however, does not appear to require the action of proteases. Affinity purified Amy contains only very low amounts of protease activity. The Ca2+-depleted, inactive form of Amy may be kept for weeks in low Ca*+ before evidence of proteolysis may be seen on SDS-PAGE (Fig. 4 B ) . This contrasts markedly with the rapid degradation of puri- fied Amy in the presence of added protease where even at 4°C degradation is almost complete in 30 min (Fig. 4A). These data indicate that Ca2+ is responsible for maintaining struc- tural features as well as the activity of the Amy molecule.

The ability of Ca2+ to stabilize the secondary and tertiary structure of Amy without influencing its primary structure can be explained by the model proposed by Buisson et al. (13) for the structure of PPA. They show that in PPA, ea2+ holds two P-sheet regions together (one in domain A and one in domain B), thereby creating and stabilizing the active site (13). Structural similarities between Amy and PPA are indi-

19398 Calcium Requirement of Barley a-Amylase

cated by their many common chemical and physical properties and by amino acid sequence homology. Both Amy and PPA are acidic proteins with molecular weights close to 50,000, are heat stable and protease resistant, and contain a high propor- tion of aromatic amino acids (29, 33-35). In addition Rogers (36) has identified three regions of homology between the amino acid sequence of PPA and Amy. These segments are found in three short regions between residues 65 and 305 of Amy and are offset an average of 12 amino acids from the homologous regions in PPA. Thus, the 18-residue offset that is required to align the structurally homologous regions of PPA and Amy (Fig. 9) also aligns some weakly conserved regions of amino acid sequence.

Although the correspondence between the structure of PPA and that computed for Amy is not exact, the organization of the NH2 terminus of PPA into two domains (A, with alter- nating a and /? segments, and B, with eight /?-sheets) can be clearly seen in the predicted structure for Amy (Fig. 9). This homology is particularly significant since it is the interaction of the A and B domains of PPA that produces the Ca2+- binding site (13). Three of the four amino acids involved in Ca2+ binding in PPA can be found in structurally similar regions of Amy. We propose that in Amy Ca" could link the /?-sheet region predicted between residues 80 and 160 (corre- sponding to domain B of PPA) with the predicted /?-sheet region between residues 160 and 175 (corresponding to the NHz-terminal region of the second segment of domain A of PPA) .

The importance of Ca2+ for maintenance of the structure and activity of Amy is relevant to the long-standing question of how Ca2+ stimulates amylase secretion from the aleurone layer (2). The large difference in the affinity of the two groups for Ca2+ that we observed is consistent with the observation that high levels of Ca2+ stimulate the production of Amy2, the low affinity amylase (Fig. 3), far more than Amyl, the high affinity amylase (Fig. 3 and Ref. 2). This leads US to speculate that in order to insure the stability of AmyZ, Ca2+ levels in the endoplasmic reticulum may be of central impor- tance for regulating amylase production. Since structural changes in Amy induced by Ca2+ removal appear to be irre- versible in isolated microsomal vesicles (Fig. 5), it may be necessary for the aleurone to maintain Ca2+ in the ER at a level that is 5-10 times greater than in the cytoplasm. In contrast, Amyl would be stable if ER Ca2+ levels were only equal to cytoplasmic levels. We have recently measured Ca2+ levels in ER isolated from aleurone cells actively synthesizing Amy2 and have found them to be at least 3 pM (25). Since cytoplasmic Ca2+ in the aleurone cell is in the range of pCa 7-6.5 (38, 39), these elevated ER Ca2+ levels require active transport. We have identified a Ca" transporter on the ER membrane whose activity is stimulated during amylase syn- thesis.

Acknowledgments-We wish to thank Xiao-Wen Guo and Dr. J. Kirsch for help in making CD measurements, Dr. F. DuPont for help with fluorescence measurements, and Eleanor Cmmp for help in preparing the manuscript. We wish also to thank Drs. A. Glazer and F. Wilt for critical reading of the manuscript.

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