Study of α-amylase and R-enzyme contaminants in Q-enzyme preparations from cotton leaves

MICROCHEMICAL

JOURNAL

28, 363-373 (1983)

Study of cx-Amylase and R-Enzyme Contaminants Q-Enzyme Preparations from Cotton Leaves CHONG

W.

Western Cotton Research L&oratory, Keceived

in

CHANG

ARS, USDA, Phoenix, Arizona 85040 June

10, 1982

INTRODUCTION

Q-Enzyme, or plant branching enzyme ((Y-1,4-D-glucan:c-w-1,4-D-glucan 6-glucosyl transferase; EC 2.4.1.18), catalyzes the synthesis of the a-l ,6D-glucosidic linkages of the amylopectin fraction of starch. This enzyme acts by the transfer of a chain fragment, removed from a donor by breakage of a 1,4-bond, to an acceptor with the formation of a 1,6-bond (4). QEnzyme activity is most frequently assayed by monitoring the branching of glucan as measured by the decrease in absorbance (660 nm) of the glucan-iodine complex resulting from the branching (2, 3, 9, 19). With these conventional procedures, however, the determination of Q-enzyme activity from cotton leaves failed because of major enzymatic contaminants such as a-amylase and R-enzyme found in the Q-enzyme preparation. R-Enzyme, or debranching enzyme (amylopectin 6-glucanohydrolase; EC 3.2.1.9), hydrolyzes LY-1,6-D-glucosidic linkages of amylopectin (16). a-Amylase (a-l ,4-D-glucan 4-glucanohydrolase; EC 3.2.1.1) also catalyzes an essentially random hydrolysis of nonterminal a-l ,4-D-glucosidic linkages in amylose (15). Q-enzyme activities from various sources (2, 3, 9, 19), however, have been reported without investigating the influences of cY-amylase and R-enzyme contaminants on crude branching enzyme preparations. The purposes of this communication are (a) to present the conditions which overcome the interferences of a-amylase and R-enzyme with the branching action of the partially purified Q-enzyme preparation; and (b) to demonstrate the successful determination of branching activity by treating the cotton leaf Q-enzyme preparation with EDTA and alumina (r)-gel. MATERIALS

AND METHODS

Glandless cotton plants (Gossypium raised as previously described (7).

hirsutum

L., cv. Coker 100) were

363 0026-265X/83 Copyright All rights

$1 SO

0 1983 by Academic Press, Inc of reproduction in any form reserved.

364

CHONG

W.

CHANG

Preparation of Q-enzyme. Ten to twenty grams of frozen cotton leaves was homogenized in 0.05 M sodium citrate buffer at pH 5.7. The homogenate was partially purified as previously described (8) except that the concentrated protein was precipitated by bringing the solution to O-50% saturation of ammonium sulfate. The protein pellet was then dialyzed against 0.05 M sodium citrate buffer at pH 7.0 overnight. Assay of Q-enzyme. The procedure of Drummond et al. (9) was used with minor modifications. The reaction mixture, containing 124 pg of amylose, 10 p,mol of sodium citrate at pH 7.0, and 0.3 ml of Q-enzyme sample (40-80 p,g protein) in 0.8 ml total volume, was incubated at 37°C for 2 hr. The reaction was terminated by the addition of 0.8 ml of 1 M glacial acetic acid. One part of iodine reagent was added to four parts of the reaction mixture. Iodine reagent was made daily from 1.O ml of stock solution (1.0 g of IZ and 10 g of KI in 500 ml of water) by diluting 20-fold with H20. The absorbance of the Ilamylose complex was measured at 660 nm and compared to a control containing boiled Q-enzyme. The reactions also were monitored by the change in absorbance of the glucan-12 complex over the range 420-750 nm. One unit of activity is defined as a decrease in absorbance (660 nm) of 1.O per hour per milligram protein. Assay of R-enzyme. The procedure of Okita and Preiss (18) was used with minor modifications. The reaction mixture, containing R-enzyme specific substrate, pullulan (I%), 19 pmol of sodium citrate buffer at pH 7.0, and 0.3 ml of enzyme in 0.68 ml total volume, was incubated at 37°C for 90 min. Samples were tested for increases in copper-reducing power by the procedure of Nelson (17). Assay of a-amylase. The enzyme activity was assayed by the procedure of Chang (6) using o-amylase specific substrate, amylopectin-azure. Assay of protein. The procedure of Lowry et al. (14) was used. RESULTS

AND DISCUSSION

As shown in Fig. 1, the Q-enzyme preparation contained a-amylase and R-enzyme (refer to the figure legend for control levels of these enzyme activities in the buffer at pH 7.0). Dialysis of the sample against the buffer containing EDTA did not inhibit R-enzyme (see the constant level of this enzyme activity), but depressed a-amylase activity. The latter enzyme activity was reduced to a minimum after a few hours of dialysis and thereafter the minimum became constant. The inhibited activity, however, was restored near to the control after the sample was dialyzed against 20 rnM CaCl* for about 10 hr. This seemed to show that EDTA inactivated a-amylase activity by removing calcium cations, which are associated with the structural rigidity of the enzyme molecule required for catalytic activity (13). As shown in Fig. 2, an increasing concentration of alumina (r)-gel grad-

STUDY OF a-AMYLASE

duration

AND R-ENZYME

of

dialysis

CONTAMINANTS

365

(hr)

FIG. 1. Effects of EDTA on activities of a-amylase and R-enzyme in Q-enzyme preparations. Q-Enzyme was isolated from 20 g of fresh cotton leaves and purified as described under Materials and Methods. The enzyme solution was dialyzed against 0.05 M Na citrate buffer (pH 7.0) containing 7 mM EDTA overnight. Activities of a-amylase (6) and R-enzyme (18) were determined at various time intervals during the period of dialysis. The total activities of o-amylase and R-enzyme in 0.05 M Na citrate buffer at pH 7.0 before dialysis were assigned control values of lOO’%. An absorbance of 134 optical density at 59.5 run/2 ml sample was the control o-amylase activity. A value of 10.7 ug glucose/O.5 ml sample was the control R-enzyme activity.

ually decreased R-enzyme activity in Q-enzyme preparations. This decrease reached a minimum in 10 min when the sample was treated with about 60 mg of gel/ml sample (inset, Fig. 2). An increase in gel concentration above 40 mg/ml did not further decrease the level of R-enzyme activity. The gel treatment, however, reduced the a-amylase activity to only about 80% of the control. As shown in Fig. 3, alumina (t-)-gel decreased the total protein content to about 45% of the control. Concomitant with this decrease in protein content, the fall in total R-enzyme activity permitted an increase in the measured specific activity of Q-enzyme. The changes of these enzymatic activities and protein level occurred concurrently during the first IO-min gel treatment. Thereafter, the minimum levels of protein plus R-enzyme activity, and the maximum Q-enzyme activity, became constant. The data support the relationships that alumina (r)-gel removed R-enzyme protein by a selective absorption and protected Q-enzyme from the debranching

366

CHONG W. CHANG

<-AMYLASE

0

* 0

20 alumina(r)-gel

40

60 60 concentration(mg/ml)

I 100

FIG. 2. Effects of alumina (r)-gel on activities of R-enzyme and a-amylase in Q-enzyme preparations. Q-Enzyme was isolated and purified as described in Fig. 1. The enzyme solution (about 310 ug protein/ml) was mixed with an equal volume of Hz0 containing various amounts of alumina (r)-gel. The mixture was gently stirred with a magnetic stirrer at 4°C for 60 min. After centrifugation, a clear supernatant was assayed for R-enzyme activity (18). Inset: experimental conditions were similar to those for above experiments except that Q-enzyme preparation was mixed with an equal volume of gel suspension (60 mgiml) and activities of R-enzyme (18) plus a-amylase (6) were determined at various time intervals during the period of gel treatment. Refer to Fig. 1 for the control activity levels of R-enzyme and o-amylase.

action of R-enzyme. This resulted in an increase in the observed specific Q-enzyme activity. As shown in Fig. 4, the activity of the Q-enzyme preparation dialyzed against EDTA and treated with alumina (r)-gel (as measured by a decrement in absorbance at 660 nm) was linear with time through the 2-hr incubation period (Fig. 4A). The activities of Q-enzyme preparations containing various amounts of enzyme protein also showed that the rate of decrease in absorbance (660nm) changed with enzyme concentration over the range tested from 0 up to about 100 p.g of total protein (Fig. 4B). In experiments (Fig. 4B), each volume of enzyme sample contained not only increasing amounts of Q-enzyme but also increasing amounts of foreign proteins. The latter substances, however, did not interfere with the linearity in activity. This may be explained in part by the extremely low K, value of Q-enzyme (Fig. 5). As shown in Fig. 5, the Michaelis constant (K,) of purified Q-enzyme was 1.4 )uV. The Michaelis constants of a great many enzymes have been

STUDY OF CY-AMYLASE

c 0)

AND R-ENZYME

0

367

0 O

a .z z ii

CONTAMINANTS

Q-*Lyme

0

protein 50.

E” z 5

1

.

5 2 c z z

.

2

.

,

50

60

R-enzyme *

5 4 *

aR

Oo

10

20 alumina(rkgel

30

40 treatmenttmin)

FIG. 3. Effects of alumina @)-gel along with EDTA on R-enzyme activity, total protein, and branching action in Q-enzyme preparations. Q-Enzyme was prepared as described under Materials and Methods. The samples were further dialyzed against EDTA and treated with alumina (r)-gel as described in Figs. 1 and 2, respectively. R-Enzyme activity (18). Qenzyme activity (9), and protein content (14) were determined at various time intervals during the 60-min gel treatment. A value of 14.8 pg glucose/O.5 ml sample (the activity determined before gel treatment) was assigned a control of 100% R-enzyme activity. A value having an absorbance of 1.25 optical density at 660 nmihrimg protein (the maximum specific activity at the plateau level) was assigned a control of 100% Q-enzyme activity. A value of 298 pg (the protein content determined before gel treatment) was assigned a control of 100% protein.

reported to range between 1 x lop2 and 1 x lo5 @4 (II). Since the K, value of the present Q-enzyme is low, it appears that cotton Q-enzyme possesses a very high affinity for the substrate. As shown in Fig. 6A, the activity of the Q-enzyme preparation treated with EDTA alone (I) reached a maximum (about 45% of the control) by a nonlinear fashion and decreased thereafter. This time course of enzyme activity influenced by EDTA alone (curve I) differed from that of the enzyme preparation treated with gel alone (II). The activity of the former sample was higher than that (about 35% of the control) of the latter during the first approximately 1.5 hr of the 2.0-hr incubation period. Thereafter, a sharp decrease in measured enzyme activity occurred (I) in contrast with a gradual decrease in activity (II). The data (I and II) indicate that ol-amylase (active when treated with gel alone, II) and R-enzyme (active when treated with EDTA alone, I) influenced the branching action at two

368

CHONG W. CHANG

0.3.

.

A

6

E c ‘2 ID 0.2.

.

G 0) :,

*

b 0.1. 3 :4

. /

0

0

0

1 lncubatlon

20 timdhr)

50 protein(pg)

101

FIG. 4. The effect of Q-enzyme activity on the cu-glucan-12 complex. (A) Q-Enzyme was prepared as described under Materials and Methods. The samples were further purified with EDTA and gel as described in Figs. 1 and 2, respectively. Q-Enzyme activity was determined at various time intervals during the incubation period by measuring the decrement in absorbance at 660 nm (9). (B) Conditions were similar to those of the above experiment (A) except that the variable was protein concentration.

separate times and sites. o-Amylase was able to react on its preferred substrate, amylose primer, more than branched glucan (16), mainly during the early incubation period (II). On the other hand, R-enzyme affected its specific substrate, branched glucan, mostly after it was formed at the later stage of incubation (I). These findings were further supported by the experimental results (Fig. 6B) that the level of reducing sugars as (Yamylase products was high during the early incubation period and thereafter did not increase (II). By contrast the extent of reducing power as R-enzyme products was initially low and accumulated with the subsequent incubation time (I). The untreated Q-enzyme preparation (III, Fig. 6A), however, showed only a slight increase in branching activity (approximately 15% of the control) after about 30 min of incubation. This activity then continuously decreased to a negligible level, probably because of the degradative activities of a-amylase and R-enzyme contaminants. In experiments (Fig. 7), the branching action of Q-enzyme preparations treated with EDTA and gel was tested on amylose by monitoring the reactions by the decrease in absorbance and wavelength maxima of the glucan-12 complexes. A similar approach was previously adopted to identify the property of Q-enzyme branching action in corn (4). The enzyme reduced the absorption (660 nm) of the amylosecomplex to about 35% (f, Fig. 7A) of the control (a, Fig. 7A) after 1.5 hr of incubation. With

STUDY OF a-AMYLASE

AND R-ENZYME

CONTAMINANTS

369

FIG. 5. Michaelis constant (K,) value for purified Q-enzyme. A crude Q-enzyme was prepared from about 100 g of fresh cotton leaves as described under Materials and Methods. This was purified by chromatography on a column of DEAE-cellulose according to the method of Drummond er al. (9). The enzyme was further purified with EDTA dialysis and alumina @)-gel as described in Figs. 1 and 2, respectively. The initial velocities of this enzyme were measured according to the standard method (see Materials and Methods). The Michaelis constant was calculated from the graph of I/v against l/(S). In this graph, the varying velocity values were extrapolated to the zero concentration of substrate for the determinations of the slope and the value of l/V (10). r’, Velocity as enzyme units. (S), Substrate concentration, which was expressed by the method of Fekete and Cardini (10). V, maximum velocity.

this change, the wavelength maximum also was gradually shifted from 620 to 560 nm (a, b, c, d, e, and f in Fig. 7A). Absorption spectra of glucan-12 complex of glucan formed at the end of 1S-hr incubation period (I, Fig. 7B) was similar to that of the purified cotton amylopectin (II, Fig. 7B). Both showed maximum absorbance at 560 nm. The wavelength maxima of cotton amylose and branched glucans formed from this primer were different from those of other materials reported (4, 5). The differences in wavelengths for maximum absorption and molecular extinction coefficients are known to be characteristic of the plant sources (1). In contrast, in experiments conducted with Q-enzyme preparations which were treated with EDTA alone (Fig. 7C), the absorption (660 nm) of the glucan-Iz complex was reduced to only about 70% (f, Fig. 7C) of the control (a, Fig. 7C) at the end of the IS-hr incubation period. The wavelength maximum also was shifted from 660 to only about 580 nm instead of 560 nm (a-f in Fig. 7C). The results obtained from Q-enzyme samples treated with gel alone did not greatly differ from those from experiments (Fig. 7C).

370

CHONG W. CHANG

Incubation

tlmdhr)

FIG. 6. Branching activities during a 2-hr incubation period for Q-enzyme preparations dialyzed against EDTA, treated with alumina (r)-gel, and untreated. (A) Q-Enzyme was prepared as described under Materials and Methods and further treated with EDTA alone (I), alumina (r)-gel alone (II), or was untreated (III), as described in Figs. 1 and 2. These differently treated samples were assayed for branching activity (9) during a 2-hr incubation period. (B) Conditions were similar to those for experiment (A) except that degradation products of a-amylase (II) and debranched products of R-enzyme (I) were determined by the procedure of Nelson (17) and that of Okita and Preiss (18) respectively.

EDTA dialysis and alumina (r)-gel treatment employed in this work successfully controlled a-amylase and R-enzyme contaminants, respectively, in crude Q-enzyme preparations. The procedures for treating enzyme preparations with EDTA and alumina (r)-gel were simple and efficient. This eliminated the conventional time-consuming enzyme purification steps with DEAE-cellulose, which required large amounts of starting materials. This proposed method therefore made the determination of Qenzyme activity possible from many small cotton leaf samples in our laboratory. The well-known anion-exchange DEAE-cellulose separated a-amylase, but failed to isolate R-enzyme from Q-enzyme preparations. The activities of Q-enzyme and R-enzyme eluted together from a DEAEcellulose column. Enzymes such as a-amylase and R-enzyme are widely distributed in plants (2, 6, 15, 18). The steps to control these enzyme contaminants in Q-enzyme preparations were found to be essential for an accurate determination of branching action. These essential steps, however, were not

STUDY OF cx-AMYLASE

560

0

,,,,580 560

AND R-ENZYME

620

560 750

660

580

h

A (nm)

371

750

A (nm)

660 620

CONTAMINANTS

750

(nm)

FIG. 7. Absorption spectra of a-o-glucan+ complexes of n-glucan formed by Q-enzyme preparations. (A) Q-Enzyme was prepared as described under Materials and Methods. The samples were further treated with EDTA and gel as described in Figs. 1 and 2, respectively. The reactions in the assay mixtures were monitored by the change in absorbance of the glucan-I2 complex over the range of 430-750 nm as described under Materials and Methods except that the time of incubation was 1.5 hr. (B) (I) absorption spectrum of glucan-12 complex of glucan formed at the end of the 1S-hr incubation period (from experiment (A)). (II) absorption spectrum of glucan-12 complex of cotton amylopectin. which was purified by the procedure of Chang (5). (C) Conditions were similar to those for the above experiment (A) except that Q-enzyme preparations were dialyzed against EDTA alone. Incubation time (minutes) in (A) and (C): a, 0; b, 10; c, 30; d, 50; e, 70; and f, 90.

fully taken into account in the previous studies (2, 3, 9, 19) involving the determinations of Q-enzyme activity. Alumina or aluminum oxide (AlzOs) has been used for the separation of many contaminants from crude enzyme preparations (12). Most chromatographic aluminas are of r-brand. The nature of adsorption sites on alumina (r)-gel seems to be attributed to Al-O- bonds, which are responsible for most of the polar and unsaturated molecules (12). The use of this gel in conjunction with EDTA for a successful determination of Qenzyme activity from cotton leaves has not been previously reported. The technique should also be applicable to other plants. SUMMARY Q-Enzyme is responsible for branching during the biosynthesis of amylopectin, the major form of starch in leaves. Its activity, however, cannot be determined accurately in the presence of the degradative enzymes, cY-amylase and R-enzyme (the debranching enzyme). Q-Enzyme activity was measured by incubating with amylose primer and measuring changes in absorbance of the glucan-11 complex. Maximum interference by a-amylase occurred early

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CHONG W. CHANG

because it attacked the amylose primer. Maximum interference by R-enzyme occurred later during the incubation period, because R-enzyme affected branched glucan which was formed at the later stage of incubation. Therefore, these enzymatic interferences occurred in sequence at two separate times and sites. Dialysis of Q-enzyme preparation against EDTA greatly decreased the activity of cr-amylase, probably by removing calcium. Addition of calcium to the dialyzed enzyme preparation restored most of the amylase activity. R-Enzyme was removed from the reaction mixture by complexing it with alumina (r)-gel. The insoluble complex was then removed by centrifugation. Dialysis of the enzyme extract against EDTA, followed by treatment with alumina (r)-gel, effectively overcame interferences by a-amylase and R-enzyme, and permitted accurate measurement of Q-enzyme activity.

ACKNOWLEDGMENTS The author thanks Dr. G. Guinn, Dr. I. P. Ting, Dr. D. L. Hendrix, and Dr. A. C. Bartlett for their critical reviews of the manuscript and Mrs. M. Eidenbock for her technical assistance.

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9.

10.

II. 12. 13. 14.

Bailey, E M., and Whelan, W. J., Physical properties of starch. I. Relationship between iodine stain and chain length. J. Biol. Chem. 236, 969-973 (1961). Baun, L. C., Palmiano, E. P., Perez, C. M., and Juliano, B. O., Enzymes of starch metabolism in the developing rice grain. Plant Physiol. 46, 429-434 (1970). Borovsky, D., Smith, E. E., and Whelan, W. J., Purification and properties of potato 1, 4-or-D-glucan; 1, 4-D-glucan-6-glucosyl transferase. Evidence against a dual catalytic function in amylose-branching enzyme. Eur. J. Biochem. 59, 615-625 (1975). Boyer, C. D., and Preiss, J., Multiple forms of a-l, 4-D-glucan, a-l, 4-D-glucan-6glycosyl transferase from developing Zea mays L. Kernels. Carbohydr. Res. 61, 321-334 (1978). Chang, C. W., Isolation and purification of leaf starch components. Plant Physiol. 64, 833-836 (1979). Chang, C. W. Determination of a-amylase activity from cotton leaves with amylopectinazure. Microchem. J. 24, 50-55 (1979). Chang, C. W., Starch depletion and sugars in developing cotton leaves. P/ant Physiol. 65, 844-847 (1980). Chang, C. W., Enzymic degradation of starch in cotton leaves. Phytochemisrry 21, 1263-1269 (1982). Drummond, G. S., Smith, E. E., and Whelan, W. J., Purification and properties of potato a-l, Cglucan. o-1, 4-glucan 6-glycosyl transferase (Q-enzyme). Eur. J. Biochem. 26, 168-176 (1972). Fekete, M. A. R. de, and Cardini, C. E., Mechanism of glucose transfer from sucrose into the starch granules of sweet corn. Arch. Biochem. Biophys. 104, 173-184 (1964). Fruton, J. S., and Simmonds, S., Kinetics of enzyme action. In “General Biochemistry” (J. S. Fruton and S. Simmonds, eds.), pp. 244-283. Wiley, New York, 1958. Heftmann, E., Adsorption. In “Chromatography” (E. Heftmann, ed.), pp. 43-57. Reinhold, New York, 1967. Hsiu, J., Fischer, E. H., and Stein, E. A., u-Amylase as calcium-metalloenzyme. II. Calcium and the catalytic activity. Biochemistry 3, 61-66 (1964). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., Protein measurement with the Folin phenol reagent. J. Biol. Chem. 192, 265-275 (1951).

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15. Manners, D. J., Enzymic synthesis and degradation of starch and glycogen. In “Advances in Carbohydrate Chemistry” (M. L. Wolfrom, ed.), Vol. 17, pp. 371-430. Academic press, New York, 1962. 16. Manners, D. J., Starch and inulin. In “Phytochemistry” (L. P. Miller, ed.), pp. 176197. Van Nostrand-Reinhold, New York, 1973. 17. Nelson, N. Photometric adaptation of the Somogi method for the determination of glucose. .I. Bid. Chem. 153, 375-380 (1944). 18. Okita, T. W., and Preiss, J., Starch degradation in spinach leaves. Isolation and characterization of the amylases and R-enzyme of spinach leaves. Plant Physiol. 66, 870-876 (1980). 19. Walker, G. J., and Builder, J. E., Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of branching enzyme, and its effect on the activity of glycogen synthetase. Eur. J. Biochem. 20, 14-21 (1971).