An Endogenous α-Amylase Inhibitor in Barley Kernels

Weselake, Randall J.; MacGregor, A. W.; Hill, Robert D.

doi:10.1104/pp.72.3.809

Cited by 112 publications

(60 citation statements)

References 4 publications

(9 reference statements)

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“…More recent results (25) have shown that a-amylase III is a mixture of a-amylase II and a low mol wt protein. This may explain the immunochemical identity observed previously between a-amylases II and III (20).…”

Section: Resultsmentioning

confidence: 99%

Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

MacGregor¹,

MacDougall²,

Mayer³

et al. 1984

Plant Physiol.

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Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18°C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for a-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same a-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar a-amylase was excreted into the endosperm compared to aleurone synthesized a-amylase. Aleurone cells appeared to synthesize appreciably more a-amylase than did scutellar tissue.There has been much discussion recently on the relative importance of aleurone and scutellar tissues in the production of hydrolytic enzymes during germination and early seedling growth of cereal grains (3,4,11,23,24). a-Amylase synthesis in barley has received particular attention because ofthe technological importance of this enzyme in malting and brewing and also because synthesis of the enzyme in aleurone cells from barley kernels has been used as a model system to study hormonal action on protein synthesis (7,13). Estimates on the contribution made by the scutellum to total a-amylase synthesis in barley seedlings have varied from little or none (24), to a relatively small proportion of 14% (4) direct tissue line immunoelectrophoresis (9) to determine quantitatively changes in the levels of a-amylases I and II in these different sections. These results provide information on the sites of synthesis of the two groups of a-amylase and the rates at which the enzymes move into the endosperm. MATERIALS AND METHODSGermination Conditions. The two-rowed barley (Hordeum vulgare L. cv Klages) used in this study was grown in test plots in Brandon, Manitoba in 1976. Barley kernels were dehusked by a 3-h steep in 50% (v/v) H2SO4 (8) followed by thorough washing with sterile, distilled H20. Petri dishes, each containing 25 dehusked kernels, 50 g sterile sand, and 10 ml sterile H20, were incubated in a germination chamber at 1 8°C and high humidity. After 24, 48, 72, and 96 h, Petri dishes were removed and frozen at -20°C until analyzed.Dissection. Frozen kernels were dissected on a cold plate using an Olympus dissecting microscope. Tissues were frozen immediately after they were separated from the kernel. Ten kernels from each sample were completely dissected to give the following six fractions (Fig. 1): center portion of the scutellum, crushed cell layer and subscutellar starch, strip of aleurone and adhering scutellum along the aleurone-scutellum interface (Fig. 1A), residual aleurone, sub-aleurone starch, and core starch. Care was taken to ensure that each fraction was clean and not contaminated by material from another fraction. Frozen tissues were freeze-dried, weighed, and ground to a fine powder. a-Amylase levels did not appear to be a...

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Section: Resultsmentioning

confidence: 99%

Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

MacGregor¹,

MacDougall²,

Mayer³

et al. 1984

Plant Physiol.

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“…One of these soluble seed proteins has recently been identified as an inhibitor of the endogenous barley or-amylase 2 isozyme and of the bacterial protease subtilisin and has been shown by amino acid sequencing to be a member of the soybean trypsin inhibitor (Kunitz) family (11,18,23,24). As with other seed proteins (2), the level of translatable mRNA for this bifunctional inhibitor peaks from 15 to 30 days after anthesis (MUNDY and BRANDT, unpublished data).…”

Section: Introductionmentioning

confidence: 99%

Hormonal regulation of α-amylase inhibitor synthesis in germinating barley

Mundy

1984

Carlsberg Res. Commun.

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Keywords: Abscisic acid, gibberellic acid, bifunctional a-amylase/subtilisin inhibitor, aleurone layerThe in vivo synthesis of n-amylase and of a bifunctional inhibitor of endogenous or-amylase and subtilisin was studied in embryoless barley half grains treated with abscisic (ABA) and gibberellic (GA3) acids. Fluorography of newly synthesized proteins and of immunoprecipitated u-amylase and its inhibitor separated by SDS-PAGE showed that GA~ induces the production of large amounts of ct-amylase while the synthesis of other proteins, including the or-amylase inhibitor, is reduced. In contrast, synthesis and/or accumulation of the inhibitor is induced in embryoless half seeds incubated with ABA. These results identify the u-amylase inhibitor as an ABA-induced protein in barley aleurone layers and suggest that it functions as an active mediator of amylase activity during the development and germination of barley seeds.

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“…A preliminary three-dimensional model has been constructed for the closely related wheat a-amylase/subtilisin inhibitor [4]. The strict isoenzyme specificity of BASI in its reactions with barley a-amylases has previously been established, thus BASI reacts with a-amylase 2 or high pI a-amylase (AMY2), but not with the 80% identical isozyme 1 or low pl a-amylase [5][6][7]. The BASI-AMY2 complex is tightly bound at pH 8 and shows a pH optimum more alkaline than that of the AMY2 substrate reaction [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Stopped‐flow kinetic studies of the reaction of barley α‐amylase/subtilisin inhibitor and the high pI barley α‐amylase

et al. 1995

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Key words." Reaction mechanism; Bifunctional a-amylase/ serine protease inhibitor; Stopped-flow fluorescence kinetics; Substrate-mediated inhibitor displacement; Barley; High pI a-amylase dimeric 0.19 a-amylase inhibitor from wheat [14]. No a-amylase inhibitor reaction has been thoroughly, kinetically investigated, but those of the red kidney bean a-amylase inhibitor and porcine pancreatic a-amylase have been partially characterized by Wilcox and Whitaker [15], who reported the formation of a 1 : 1 complex in a two-step reaction path.In the present study the kinetics of the formation of complexes between BASI and AMY2 have been investigated in order to expand our knowledge of the inhibition mechanism of proteinaceous a-amylase inhibitors. The results were in accordance with a two-step reaction model, the equilibrium and rate constants of which were determined from a combination of stopped-flow and substrate displacement kinetic data. The BASI-AMY2 and the red kidney bean a-amylase inhibitor--~-amylase reactions apparently differ markedly in their second reaction step, so that, in spite of similar overall dissociation constants, they seem to belong to different classes. In contrast to the red kidney bean inhibitor, which is slow reacting, BASI was found to be a fast reacting, tight-binding inhibitor. Materials and methods

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An Endogenous α-Amylase Inhibitor in Barley Kernels

Cited by 112 publications

References 4 publications

Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Hormonal regulation of α-amylase inhibitor synthesis in germinating barley

Stopped‐flow kinetic studies of the reaction of barley α‐amylase/subtilisin inhibitor and the high pI barley α‐amylase

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