Studies on Barley and Malt Amylases. Part XIX. Activation of Zymogen ƒà-amylase in vivo and Amylase. Formation in Isolated Aleurone Layers

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1 [Agr. Biol. Chem., Vol. 36, No. 3, p. 378 `382, 1972] Studies on Barley and Malt Amylases Part XIX. Activation of Zymogen ƒà-amylase in vivo and Amylase Formation in Isolated Aleurone Layers By Ryu SHINKE and Narataro MUGIBAYASHI Department of Agricultural Chemistry, Faculty of Agriculture, Kobe University, Kobe City Received July 31, 1971 Changes of salt-soluble and salt-insoluble zymogen ƒà-amylase (Z-ƒÀ-A), papain-soluble and papain-insoluble Z-ƒÀ-A were pursued during germination of barley. These four types of Z-ƒÀ-A decreased as germination progressed and correspondingly the salt-soluble active J-amylase. (A-ƒÀ-A) increased. The gibberellic acid (GA)-treatment during germination ac celerated the activation of Z-ƒÀ-A, but had no effect on the amount of total p-amylase. The incubation of isolated aleurone layers with 0.5ƒÊM GA at 25 Ž for 5 days resulted in a remarkable increase in the total saccharogenic activity, to which the contribution of ƒà-amylase was found to be much smaller than that of a-amylase. From these results it may be reasonable to conclude that the increase in A-ƒÀ-A during germination is ascribable to the activation of Z-ƒÀ-A and that the amount of A-ƒÀ-A syn thesized during germination, if any, is negligibly small in comparison with that of A-a-A activated from Z-ƒÀ-A. As reported previously,"" barley grains contain active ƒà-amylase (A-ƒÀ-A) and four types of zymogen ƒà-amylase (Z-ƒÀ-A) are syn thesized during ripening of barley. Although Z-ƒÀ-A in barley has long been regarded as insoluble, two soluble types of Z-ƒÀ-A were isolated from ground barley and investigated on some of their enzymatic properties.3,4) berellic acid (GA).7 `10 ) The purpose of the present paper is to confirm an enhanced activation of four types of Z-ƒÀ-A during germination with the GA-treat ment, and to show a direct evidence by using isolated aleurone layers that the increase in A-ƒÀ-A during germination is exclusively due to the activation of Z-p-A. Two different mechanisms of activation, pro teolytic activation and disulfide bond cleaving activation, were also found to be concerned with activation of Z-ƒÀ-A during germination." The measurement of total ƒà-amylase (T-ƒÀ-A) in barley and malt suggested that the increase in A-ƒÀ-A during germination might be ex clusively due to the activation of Z-ƒÀ-A.6) However, it seems unestablished yet whether or not de novo synthesis of A-ƒÀ-A takes place during germination of barley, in spite of many reports on de novo synthesis of a-amylase in isolated aleurone layers in response to gib MATERIALS AND METHODS Barley samples. Two-row and six-row barley sam ples grown in 1970 at various districts in Japan were used in the experiments. Ground barley samples at various stages of germination with or without the GA-treatment were prepared in the same way as described previously.s) Measurement of amylase activity. i) a-amylase ac tivity. One g of ground barley or malt was extracted with 50 ml of 0.1 M acetate buffer (ph 4.8) or 0.1% papain and each extract obtained was used as the

2 Studies on Barley and Malt Amylases. Part XIX 379 enzyme solution. The enzyme assay was performed according to Kneen and Sandstedt method.11) ii) ƒà- Amylase activity. The enzyme assay was described in the previous paper.2) Namely, total ƒà-amylase (T-ƒÀ-A) or A-ƒÀ-A activity was measured with or without the treatment of activation by 0.2M 2-mer captoethanol (2-ME) containing papain for 2 hr at 30 Ž, respectively. Z-S-A activity was calculated by differences between T-ƒÀ-A and A-ƒÀ-A activities. Corrections were also made for the effect of co-existent a-amylase in malt samples on ƒà-amylase activity. Each activity of four types of Z-ƒÀ-A were calculated in the same way as described previously.6) Chromatography of barley and malt extracts. Twenty g of ground barley or malt was extracted with 80 ml of M phosphate buffer (ph 7.0) and centrifuged. Each extract obtained was dialyzed against the same buffer and loaded on the DEAE-Sephadex A-50 column (2.8 x 30 cm). The elution was carried out by increasing linearly the concentration of sodium chloride from 0.1 M to 0.5 M in the same buffer. Protein concentration and amylase activity of each fraction (10 ml) were measured as described previ ously.4) GA-treatment of aleurone layers. Isolation of aleurone layers was performed according to Chrispeels and Varner.121 Two-row barley seeds (Seijo No. 17) har vested in Kagoshima Prefecture were soaked in 50 0 sulfuric acid for 2 hr. The seeds dehulled were thoroughly washed with sterilized deionized water and cut in half with t razor blade. The embryoless halves having further breaks lengthwise were placed in Petri dishes (diameter: 9 cm), each containing u Toyo -o. 2 filter paper wetted with 10 ml of sterilized deionized water, and aseptically preincubated at 25 Ž for 2 days. Aleurone layers obtained from 30 halfseeds after the preincubation were transferred to a test tube containing 5 ml of an incubation medium of M acetate buffer (ph 4.8), 500 /.SM calcium chloride, 0.5ƒÊM GA and a few kinds of antibiotics, and incubated at 25 Ž for 5 days on a reciprocal shaker. The incubation liquid obtained was kept at 25 Ž for 2 hr with an equal volume of 0.2% papain solution. The aleurone layers were also mercerized in mortar with quartz sand, to which 10 ml of 0.1% papain solution was added, kept at 25 Ž for 2 hr and centrifuged. The solutions obtained in both cases were used as enzyme solutions for determination of ƒ - and ƒà-amylase activities. RESULTS Activation of Z-ƒÀ-A in vivo Changes of four types of Z-ƒÀ-A during ger mination of six-row barley grown Kagoshima TABLE I. CHANGES OF FOUR TYPES OF ZYMOGEN /j-amylase DURING GERMINATION OF BARLEY Salt-soluble Z-ƒÀ-A activity was measured after the treatment of the buffer extract of ground barley with 0.2M 2-ME at 30 Ž for 2 hr. Salt-insoluble Z-ƒÀ-A activity was expressed by deduction of A-ƒÀ-A and saltsoluble Z-ƒÀ-A activities from that of T-ƒÀ-A. Papain-soluble Z-ƒÀ-A activity was measured after the treat ment of the 0.1% papain extract of ground barley with 0.2M 2-ME at 30 Ž for 2 hr. Papain-insoluble Z- ƒà-a activity was measured by extracting the papain residue with 0.2M 2-ME containing 0.10 papain. Each activity was expressed in terms of maltose mg/one grain. The length of sprout: 0, 0.2, 0.3, 0.7 and 1.0 for normally germinated samples and 0, 0.3, 0.4, 0.9 and 1.2 for GA-treated samples, respectively.

3 380 R. SHINKE and N. MUGIBAYASHI Prefecture were pursued. The effect of GAtreatment on their activations during germi nation was also examined. As the results are shown in Table I, the activities of four types of Z-ƒÀ-A in both samples with and without the GA-treatment decreased as germination proceeded. The decreasing rates of the saltsoluble Z-ƒÀ-A and the papain-insoluble Z-ƒÀ-A in the samples with the GA-treatment were about two times as great as those in the sam ples without the GA-treatment; both types of Z-ƒÀ-A disappeared in the 8th day sample with the GA-treatment. These decreases in the and that A-a-A activity in E-II was largely due to a salt-soluble heteropolymer type of ƒà-amylase as described in the previous paper." The decreasing rate of the salt-soluble Z-ƒÀ-A in E-II was greater in the samples with the GA-treatment and the salt-soluble Z-ƒÀ-A disappeared in the 8th day sample with the GA-treatment. Similar results were reported by LaBerge and Meredith"' about chromato graphic properties of barley and malt amylases on the CM-cellulose column. These chro matographic changes of Z-ƒÀ-A shown in Figs. 1 and 2 were consistent with those in Table I. Z-ƒÀ-A activities corresponded to the increases in the salt-soluble A-ƒÀ-A activities, while the activities of T-ƒÀ-A in both samples with and without the GA-treatment remained almost constant during the whole period of germination. Chromatographic behavior of salt-soluble Z-ƒÀ-A For further investigation into changes of Z- ƒà-a during germination, chromatography of barley and malt extracts on the DEAE- Sephadex A-50 column was performed. As the results obtained shown in Figs. 1 and 2, the barley extract was found to contain two distinct enzyme components (E-I and E-II). By determination of their activities it was found that E-I was composed of A-ƒÀ-A alone and E-II, of salt-soluble Z-ƒÀ-A and A-ƒÀ-A, Amylase formation of isolated aleurone layers Isolated aleurone layers were found to con tain no a-amylase and a small amount of ƒàamylase (2.71) which was about 12% of the T-ƒÀ-A activity in the one whole grain (23.17). The isolated aleurone layers were incubated in the presence of 0.5ƒÊM GA at 25 C for 5 days and amylase activities increased were determined. After the 5 day incubation a remarkable amount of a-amylase was detected, 90% of which was found in the incubation liquid. As shown in Table II, the saccharogenic activity by de novo synthesized ƒ -amylase (38.39) accounted for about 73 0 of the total saccharogenic activity (52.89) in the incubation liquid and aleurone layers. On the other hand, the saccharogenic activity by ƒà-amylase TABLE II. AMYLASE FORMATION OF ISOLATED ALEURONE LAYERS The enzyme activity was expressed in terms of maltose mg/aleurone layers isolated from 30 half-seeds. ƒà- and ƒ -Amylase activities of the one whole ungerminated grain were and 0, respectively. The 1000 kernel weight of the barley sample was g.

4 Studies on Barley and Malt Amylases. Part XIX 381 FIG. 1. Chromatography of Barley and Malt Ex tracts on the DEAE-Sephadex Column. A six-row barley grown in Kagoshima Prefecture was germinated without the GA-treatment and chromatography was performed as described in the text. Measurement of salt-soluble Z-p-A was carried out in the same way as in Table I. (A) barley (length of sprout: 0), (B) 4-day malt (0.3), (C) 6-day malt (0.7), (D) 8-day malt (1.0). FIG. 2. Chromatography of Barley and Malt Ex tracts on the DEAE-Sephadex Column. The same barley sample as in Fig. 1 was ger minated with the GA-treatment. The conditions of chromatography were the same with those in Fig. 1. (A) barley (length of sprout: 0), (B) 4- day malt (0.4), (C) 6-day malt (0.9), (D) 8-day malt (1.2). in the incubation liquid and aleurone layers after 5 day incubation was calculated by subtracting the saccharogenic activity by a- could be calculated to be 0.79, which was about 3.4% of the T-ƒÀ-A activity in the one whole grain. amylase from the total saccharogenic activity by ƒ - and ƒà-amylase.11) The saccharogenic activity by ƒà-amylase calculated was Accordingly, the activity increased during 5 day incubation was As this value was due to the activity in aleurone layers isolated from 30 half-seeds, the saccharogenic activity by ƒà-amylase in one intact aleurone layer DISCUSSION As shown in Table I, Figs. 1 and 2, the GA-treatment during germination of barley accelerated the activation of four types of Z- p-a, especially salt-soluble Z-ƒÀ-A and papaininsoluble Z-ƒÀ-A, but had no influence on the

5 382 R. SHINKE and N. MUGIBAYASHI amount of T-p-A. Chromatographic changes of ƒà-amylase during germination also showed that the activation of salt-soluble Z-ƒÀ-A into A-ƒÀ-A was accelerated by the GA-treatment. These results may indirectly indicate that de novo synthesis of A-p-A hardly occurs during germination of barley and that the increase in A-ƒÀ-A activity during germination is attri butable to the activation of Z-p-A synthesized during ripening of barley. In order to confirm this directly, it was examined whether or not isolated aleurone layers could produce A-p-A in response to GA. It is clear from Table II that the total sac charogenic activity in the incubation liquid The aleurone layer in an isolated state, on the other hand, must have the capacity to produce both ƒ - and ƒà-amylase because the isolated aleurone layer may be out of a natural controlling system of amylase forma tion in barley. It may be, therefore, concluded that the amount of ƒà-amylase synthesized during ger mination is negligibly small in comparison with that of A-p-A activated from Z-ƒÀ-A, even if ƒà-amylase is synthesized by the iso lated aleurone layers, and that the increase in A-ƒÀ-A activity during germination is at tributable to the activation of Z-p-A which is accelerated by the GA-treatment. and aleurone layers increased about 19 times the initial activity before the incubation. However, it was found that the contribution of ƒà-amylase to the increased saccharogenic activity was less than one third that of a- amylase and that the amount of ƒà-amylase increased by the isolated aleurone layers might not exceed 4% of the T-p-A of the one whole grain. Jacobsen et al." reported that in response of isolated aleurone layers to GA a-amylases arised by synthesis de novo, while ƒà-amylases seemed to arise by activation of pre-existing Z-ƒÀ-A and suggested the possibility that some of a-amylases synthesized might be hybrids of ƒ - and ƒà-amylase. Tanaka and Akazawalo, also reported that a-amylase resistant to low ph treatment, which is typical of ƒà-amylase, was synthesized de novo by GA-treated em bryoless half-seeds. In view of these results on multiforms of amylases synthesized by aleurone layers and the results described in this paper, it seems that aleurone layers may show different aspects of secretion of amylases either in an inact state or in an isolated state. The aleurone layer in an intact state of germinat ing barley may not synthesize A-p-A de novo. REFERENCES 1) R. Shinke and N. Mugibayashi, Sci. Rep. Hyogo Univ. Agr., 6, 41 (1963); ibid., 7, 29 (1966). 2) N. Mugibayashi and R. Shinke, Nippon Nogeikagaku Kaishi, 41, 295 (1967). 3) R. Shinke and N. Mugibayashi, ibid., 43, 802 (1969). 4) R. Shinke and N. Mugibayashi, Agr. Biol. Chem., 35, 1381 (1971). 5) R. Shinke and N. Mugibayashi, Nippon Nogeikagaku Kaishi, 43, 556 (1969). 6) R. Shinke and N. Mugibayashi, Agr. Biol. Chem., 35, 1391 (1971). 7) Y. Momotani and J. Kato, Plant Physiol., 41, 1395 (1966); Plant Cell Physiol., 8, 439 (1970). 8) R. L. Jones, Planta, 87, 119 (1969); ibid., 88, 73 (1969). 9) J. V. Jacobsen, J. G. Scandalios and J. E. Varner, Plant Physiol., 45, 347 (1970). 10) Y. Tanaka and T. Akazawa, Plant Physiol., 46, 586 (1970). 11) E. Kneen and R. M. Sandstedt, Cereal Chem., 18, 237 (1941). 12) M. J. Chrispeels and J. E. Varner, Plant Physiol., 42, 398 (1967). 13) D. E. LaBerge and W. 0. S. Meredith, J. Inst. Brew., 75, 19 (1969).

Core practical 14: Investigate the effect of gibberellin on the production of amylase in germinating cereals using a starch agar assay

Core practical 14 Teacher sheet Core practical 14: Investigate the effect of gibberellin on the production of amylase in germinating cereals using a starch agar assay Objectives To investigate the effect