J. Biosci., Vol. 6, Number 1, March 1984, pp. 47-59. Printed in India.

Isolation and partial characterisation of -amylase components evolved during early wheat germination
J. P. MACHAIAH and U. K. VAKIL
Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India MS received 14 September 1983; revised 9 January 1984 Abstract. The development of -amylase (EC 3.2.1.1) activity in wheat was followed during 4 days of germination. The enzyme was purified and separated by gel chromotography into two distinct entities (-amylase I and -amylase II), with different molecular weights and isoelectric points. -Amylase I contained a much higher content of sugars than -amylase II, which decreased as the germination proceeded. The time sequence analysis of the starch degradation pattern showed that on the 4th day of germination, 15% of the total activity was present in amylase I and the rest in -amylase II. Similarly, differences in the relative rates of synthesis of their isoenzymes were observed. -Amylase I was resolved on the 4th day of germination, only into 3 isoenzymes, whereas -amylase II could separate into 4 isoenzymes. The enzyme activity was however maximal in the most electropositive isoenzyme in both the components. Keywords. Wheat; -amylases I and II; germination; development; physicochemical characterization.

Introduction -Amylase (alpha1-4-glucan 4-glucanhydrolase, EC 3.2.1.1) is a key enzyme hydrolysing reserve starch in the endosperm of germinating cereals. It is synthesized de novo in the aleurone cells, in response to gibberellic acid (Yomo and Varner, 1971). Scutellar epithelium has also been indicated as the dominant site of the enzyme formation in germinating rice seeds (Okamoto and Akazawa, 1979). Different -amylases are known to be evolved during germination (Frydenberg and Nielsen, 1965), and consist of number of isoenzymes (Jacobsen et al., 1970). However, the mechanism by which the isoenzymes are transported from the site of synthesis to the endosperm is not well understood. It was suggested that the enzyme was relased by simple diffusion (Varner and Mense, 1972) and a partial degradation of cell walls facilitated this process (Ashford and Jacobsen, 1974). A soluble mode of secretion (Jones, 1973) as well as through a secretory vesicles derived from endoplasmic reticulum (Locy and Kende, 1978) was reported. Recently, the transport of the membrane-bound enzyme across the membrane was explained by signal hypothesis (Boston et al., 1980). Poly(A)-enriched
Abbreviations used: BSA, Bovine serum albumin; SDS, sodium dodecyl sulphate; PAGE, Polyacrylamide gel electrophoresis.

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mRNA, which synthesizes polypeptide precursor of -amylase, was isolated from the aleurone layers of barley (Mozer, 1980) and wheat(Okita et al.,1979). Cereal -amylases were shown to be glycoproteins (Rodaway, 1978; Miyata et al., 1981) like other secretory proteins. In the present investigation, the formation of two different entities of -amylase during wheat germination was monitored and their relative amounts estimated. The isoenzymes were partially characterized physico-chemically. Highly glycosylated -amylase I exhibited significantly low enzymic activity compared to the activiy of -amylase II, with a low carbohydrate content. Materials and methods Seeds and chemicals Newly harvested Vijay variety of wheat samples were obtained from the Agricultural Research Station, Niphad, Maharashtra. The seeds had about 100% viability. The molecular weight standards [bovine serum albumin (BSA), ovalbumin, pepsin, chymotrypsin], polyvinyl pyrrolidone and sugar standards were from Sigma Chemicals, St. Louis, Missouri, USA. Other chemicals and solvents used were of Analar grade. Extraction of -amylase Wheat seeds (10 g lots) were washed with. 1 % NaOCl solution, rinsed and imbibed with distilled water for 16 h. Germination was carried out in the dark at 25C for 2 to 4 days on filter paper moistened with streptomycin and penicillin (5g/ml) to prevent microbial growth. The vegetative portions were discarded and the endosperm tissues were homogenised at 3Cin 001 sodium acetate buffer (pH 56)containing0.003 CaCl2 and 1% polyvinyl pyrrolidone. The extract was centrifuged at 5,000g for 10 min and the supernatant adjusted to pH 80 with 1 NaOH. The solution was heated to 70C to inactivate -amylase, cooled (4C) and centrifuged. The pellet was discarded and the supernatant fraction was used as the crude enzyme preparation. Enzyme purification The crude extract was adjusted to 45% (NH4)2SO4 saturation, kept for 16 h at 4C and centrifuged at 12,000g. The precipitate was dissolved in 0-01 acetate buffer (pH 56) containing 3mM CaCl2 and dialysed against the same buffer for 6 h. The dialysed enzyme was chromatographed on a Sepharose 4B column (2515 cm), equilibriated with 005 Tris-HCl buffer (pH 82) containing 005 Na+ and 0003MCa2+ designated as Buffer A and eluted (2 ml fractions) with the same buffer. The fractions, exhibiting -amylase activity were pooled; the enzyme was reprecipitated at 45% (NH4)2SO4 saturation and centrifuged (12,000g). The pellet was dissolved and dialysed in the 005 Tris buffer (pH 82). The diaiysate was applied on Bio-gel P-100 column (25cm
<

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55cm) and eluted (14ml/h) with the same buffer. Fractions (3 ml each) showing enzyme activity were seperated into two distinct protein peaks. These were pooled seperately and the enzyme protein in each peak was precipitated and dialysed as described above. The molecular weights of the individual enzymes designated as -amylases I and II, were determined by calibrating Bio-gel P-100 column with the following standard proteins: albumin (68,000), ovalbumin (45,000), pepsin (35,000) and chymotrypsin (22,500). The purified enzyme samples could be stored at 5C up to three months, without any appreciable loss of activity. Isoelectric focusing The enzyme was applied on to an isoelectrofocusing column (LKB 8101, 110 ml) containing 5 to 50% sucrose gradient and 1% ampholite (pH 4 to 7) as described by Machaiah and Vakil (1981). The run was continued for 66 h at 4C with a constant voltage of 500 volts. The enzyme activity and pH were measured in each fraction (2 ml). pH was determined with a EMCO digital pH meter type EE 330 A, equipped with a reference glass electrode. Enzyme assay The enzyme preparation (1 ml) was incubated with 015% starch in 001 Na acetate buffer (pH 56) containing 0003 Ca2+ for 10 min at 37C (Jones and Varner, 1967). The reaction was stopped by addition of 1 ml of I2/KI reagent. The absorbance was measured at 620 nm in Bosch and Lomb Spectronic 20. The activity of the enzyme required for reduction of 10% in absorbance at 620 nm, was expressed as one unit. Polyacrylamide gel electrophoresis (PAGE) -Amylase isoenzymes were separated by electrophoresis on 7% Polyacrylamide gels with 001 Tris-glycine (pH 82) buffer (Davis, 1964). The protein bands were stained with Coomassie brilliant blue. The enzyme activity in different bands were detected by incubating the gel for 1 h at 37C in 1% starch solution in acetate buffer (pH 56) and then stained with 0003 I2 solution. In another experiment, the enzyme sample (C. 200g protein) was mixed thoroughly with 1% sodium dodecyl sulphate (SDS), 5% 2-mercaptoethanol and 0001% bromophenol blue (Laemmli, 1970). A drop of glycerol was added and the sample loaded on 10% Polyacrylamide gel and electrophoresed at 25C for 25h. Excess of SDS was removed (Leach et al., 1980), the gels were stained separately for proteins and carbohydrates with 01% Coomassie brilliant blue and fuschine sulphite reagent, respectively. The bands were scanned in Shimadzu spectrophotometer at 620 nm. Molecular weights were determined by comparing their mobilities with those of the marker proteins. Total proteins were estimated using crystalline BSA as standard (Miller, 1959).
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Carbohydrate analysis Total sugars were estimated colourimetrically (Dubois et al., 1956). For individual sugar analysis, the enzyme sample was hydrolysed in 3 HCl for 4h at 100C and treated with Dowex-18 (HCO3) to raise the pH to 70. The filtrate was then passed through a Dowex-50 H+ column (9cm1cm). The neutral sugars were eluted with distilled water, concentrated and separated by descending paper chromatography technique with n-butanol:pyridine:water (6:4:3) as the solvent system. Sugars were detected by a dipping method with silver nitrate-sodium hydroxide reagents (Trevelyan et al., 1950). Acidic sugars were eluted with 2 HCl; acid was evaporated and the residue was taken in citrate buffer (pH 22). Glucosamine and galactosamine were analysed by a Beckman automatic amino acid analyser using reference standards.

Results Purification of -amylase Though glycogen precipitation method (Loyter and Schramm, 1962) is widely used for the isolation of -amylase, complete removal of glycogen after the dissociation of the enzyme from the complex could not be achieved. Hence, the modified purification procedure (table 1) was followed. On addition of (NH4)2SO4 some interfering phenolic compounds were coprecipitated along with the enzyme. Incorporation of 1% Polyvinyl pyrolidone in the extraction buffer and two subsequent (NH4)2SO4 precipitations removed these compounds. Further, by employing Sepharose 4B chromatography,

Table 1.

Purification of -amylases from germinating wheat.

Endosperms from 4 days old wheat seedlings were used for the isolation of the enzyme. Calculated by combining the values of total protein and -amylase activities in peaks I and II after Bio-gel P-100 chromatography.

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Figure 1. Separation of -amylases I and II by Bio-gel P-100 column chromatography, Amylase was isolated from 2 to 4 days old wheat seedlings and subjected to gel filtration on Bio-gel P-100 column. In each fraction the protein content () and -amylase activity (---) were determined.

repeated dialysis and by gel filtration, the enzyme could be purified about 55-fold. This was resolved by Bio-gel P-100 column chromatography into two major distinct enzyme protein peaks referred to as -amylase I and -amylase II (figure 1). It was observed (table 2) that the enzyme activity was mainly confined to -amylase II. -Amylase I activity was reduced from about 22 to 15% during 4 days of germination with concomitant increase in -amylase II activity from 77 to 85%. Molecular weights, calibrated from Bio-gel P-100 column revealed that -amylase I had higher molecular weight (43,000) compared to that of -amylase II (22,500). Isoelectric point Isoelectric focusing patterns of -amylases I and II, are shown in figure 2. The single peak obtained for both the isoenzymes indicated that each component was purified to near homogeneity. These results further support the validity of comparing the physicochemical properties of the enzymes. -Amylase I had initially higher isoelectric

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Table 2. Distribution of -amylases I and II activity in germinating wheat seedlings.

Distribution of the total activity in -amylases I and II was calculated (from figure 1), by measuring the peak area with planimeter. Specific activities ( units/mg protein) are given in parenthesis.

point and more acidic component (pH 58) appeared only on the fourth day germination. Whereas, in the electrophoretic mobility of -amylase II did not vary significantly during 4 days of germination. Separation of -amylase by SDS-PAGE Separation pattern of -amylases I and II (from 2 to 4 days old seedlings) by SDSPAGE and the densitometric scannings of the bands are shown in figure 3. Both were resolved into 3 subunits (stained for proteins) of varying intensities with molecular weights of 22,500,44,000 and 55,000 (peaks 1,2 and 3, respectively). Whereas -amylase II from 4th day sample had an additional band (76,000, peak 4). However, in all the samples, maximum amount of protein as well as the enzyme activity were confined in the fast moving low molecular weight (22,500) band; though other bands were almost inactive. However, high molecular weight bands (55,000 and 76,000) of -amylase II from 3 and 4 days old seedlings, exhibited some enzyme activity (data not shown). Separation of -amylase isoenzymes -Amylases were separated into isoenzymes by simple PAGE and stained for proteins (figure 4A). Densitometric scanning of the gels are shown in figure 4B. Both -amylases I and II showed nearly the same zymogram patterns. However, significant differences in, their appearance during germination were observed. -Amylase I isoenzymes (A, 1,2) were not readily distinguishable upto 3 days. However, on the 4th day, it was separated into 3 isoenzymes, (A,3; B,4I). On the other hand, -amylase II could be separated into four isoenzymes but with very low intensity on the 2nd day (A,4; B,2II). It showed a progressive increase during germination (A,5,6; B,3II, 4II). The typical distribution of total proteins and the enzyme activities in different isoenzymes on the fourth day of germination are shown in figure 5. Quantitative analysis revealed that maximum amount of proteins and enzyme activities could be recovered from the most electropositive and fastest moving 4th band of -amylase II (2) sample. Other isoenzyme bands had low activity.

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Figure 2. Isoelectric focusing of -amylases I and II purified from germinating wheat, Amylase (200g) was electrofocused as described in the text. After 66h run, 1ml fractions were collected and the enzyme activity () and pH (..) determined in each fraction.

Carbohydrate content of -amylases It was observed that -amylase bands, separated by SDS-PAGE, were positively stained for carbohydrates (figure 3, bottom gel). In both, -amylases I and II, the maximum staining was in 22,500 band (number 1), whereas other bands of higher molecular weights were faintly stained. Total sugar content of 22,500 dalton bands was always higher in -amylase I than in II, but decreased as the germination proceeded

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Figure 3. SDS-PAGE of -amylases I and II from germinating wheat. Electrophoresis was carried out and the gels were stained for proteins (top) and for carbohydrates (bottom) as described in the text. These were then scanned for proteins () and carbohydrates (.....) and absorbancy at 620 nm was measured.

(table 3). In all -amylase I samples, carbohydrates, were confined, almost 100% to 22,000 protein. However, in -amylase II,22,000 protein contained 905,938 and 813% of total sugars from 2, 3 and 4 days old wheat seedlings, respectively. This gradual decrease was reflected in concomitant increase in carbohydrate contents in 44,000 (25 to 52%) and 55,000 (37 to 4.3 %) proteins from 2nd and 3rd days. -Amylase II samples similarly, 76,000 polypeptide from the 4th day sample contained about 185% carbohydrate. Further, carbohydrates: protein ratio was calculated. On the fourth day, when the enzyme activity was maximum (table 2), the ratio was 131 in -amylase I and
<

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Figure 4. Separation of -amylase isoenzymes by PAGE. -Amylase isoenzyme bands (1 to 4 on gels) were stained for proteins as described in the text. 1,2,3: -amylase I zymograms and 4,5,6: amylase II zymograms at 2, 3 and 4 days germination, respectively. Densitometrie scanning (B) of -amylase I gel at 4 days' (4 I). -Amylase II gels (2 II; 3 II and 4 II) at 2,3 and 4 days' germination, respectively.

only 014 in -amylase II, the most active enzyme form. This indicates an inverse relationship between this ratio and the enzyme activity. Further, the enzyme samples from wheat seedlings were hydrolysed and separated into individual sugars by paper chromatography (figure 6). Since xylose and fucose were cochromatographed by the method employed, the corresponding spot was tested chemically for fucose using cysteine-sulphuric acid reaction (Dische arid Shettles, 1948). Fucose was not detected in any of the samples analysed. The quantitative analysis of neutral sugars (table 4) revealed that xylose and mannose were the main sugars. They contributed about 25 to 30% and 45 to 50%, of the respective content of total sugars in -amylases I and II. It was interesting to note that on the fourth day in -amylase II, galactose was not detected but glucose content (25%) was higher than that in -amylase I (13%). Galactosamine was not present in any of the samples. Glucosamine was present only in the 4th day samples; -amylase II contained higher amount (10 g/mg protein) compared to that in -amylase I (45g/mg protein).

Discussion Total -amylase activity as well as protein content in wheat seedlings were maximum on the fourth day. However, this was reflected mainly by a marked increase in -amylase II

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Figure 5. Electrophoretic patterns of amylases at 4 days of germination, -Amylases I (A) and II (B) were separated by gelelectrophoresis. Gels were incubated with starch solution as described in the text. Four bands with -amylase activity were obtained.

Table 3. Determination of carbohydrate: protein ratio of -amylases (22,000 band).

The areas of peaks related to carbohydrate and proteins of 22,500 bands (figure 3) were measured and their ratio calculated. The values were averages of two independent experiments in duplicates. The figures in the parenthesis total carbohydrates (mg/100 mg of protein).

activity compared to -amylase I (figure 1). Similar differences were also observed when sequential occurrence of their isoenzyme patterns were examined by PAGE (figure 5). The low enzyme activity in -amylase I could be attributed to a delayed appearance of the most electropositive fast-moving band; which appeared earlier in -amylase II. Molecular weights were estimated as 43,000 and 22,500, respectively for -amylase I

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Figure 6. Separation of neutral sugars of -amylases. Purified enzyme preparations from 2, 3 and 4 days old wheat seedlings were subjectud to paper chromatography. Neutral sugars were detected as described in the text.

Table 4. Carbohydrate composition of wheat -amylases I and II.

Neutral sugars were separated from acid hydrolysate of the enzyme samples by paper chromatography and quantified colorimetrically, using glucose as standard. The values are mean of three experiments in duplicates. Figures in the parenthesis represent % of the total sugars present.

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and II. They showed a tendency to aggregate in the presence of SDS. However, these high molecular weight bands may not be artifacts, since high molecular weight bands of -amylase II (figure 3, bands 3 and 4) were enzymically active. The anomalous behaviour of glycoproteins in the presence of SDS is known, and the glycopeptide-SDS complexes yield abnormally high molecular weight estimates (Leach et al., 1980.). Similarly, isoelectric points of -amylases I and II, separated from 2 and 3 days old seedlings were different. Such differences in isoelectric pH of -amylase components at different stages of barley germination had been reported (MacGregor, 1978). Further, a correlation between the enzyme activity and the sugar content was observed. Further, -amylases I and II were heterogenous in their sugar content (table 4). Such heterogeneity with respect to sugar content in barley -amylases had also been shown (Rodaway, 1978). Noticeably, -amylase I contained very high proportion of mannose units. Several secretary glycoproteins from animal and plant cells contain high mannose type oligosaccharides (Bailey et al., 1980). The rapid disappearance of carbohydrate moieties of -amylase II as the germination proceeds, also suggests the rapid turnover of sugar residues than that of the polypeptides of the enzyme. It has been shown that -amylase is synthesised on rough endoplasmic reticulum (Jones and Chen, 1976), transported to the lumen of endoplasmic reticulum and glycosylated (Czichi and Lennars, 1977). This is followed by their accumulation in the secretory vesicles (Gibson and Paleg, 1975). Miyata et al. (1981) demonstrated that an oligosaccharide chain is added to the cleaved polypeptide precursor of the enzyme in germinating rice to produce the mature and final secretory form of the enzyme. Since only deglycosylated protein exhibits maximum activity, we are tempted to suggest that -amylase I represents the secretory protein synthesized and fully glycosylated; and -amylase II is the secreted active form of the enzyme, variously deglycosylated on different days of germination.

References
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Miller, G. L. (1959) Anal. Chem., 31, 964. Miyata, S., Okamoto, K., Watanabe, A. and Akazawa, T. (1981) Plant Physiol., 68, 1314. Mozer, . J. (1980) Plant Physiol., 65, 834. Okamoto, . and Akazawa T. (1979) Plant Physiol., 63, 336. Okita, . W., Decaleya, R. and Rappaport, L. (1979) Plant Physiol., 63, 195. Rodaway, S. J. (1978) Phytochemistry, 17, 385. Trevelyan, W. E., Proctor, D. D. and Harrison, J. S. (1950) Nature (London), 166, 444. Varner, J. E. and Mense, R. M. (1972) Plant Physiol, 49, 187. Yomo, . and Varner, J. E. (1971) in Current Topics in Developmental Biology, eds A. A. Moscena and A. Monray (New York: Academic Press) Vol. 6, p. 21.