Amylases in Developing Barley Seeds

Abstract

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and-starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with a-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of 8-amylase. Amylases produced by the mature aleurone layers of barley have been investigated extensively (1,3,4,7,8,12,17,18 The starch solution for the amylase assay was prepared by adding 150 mg of nonsolubilized potato starch, 600 mg of KH2PO4, and 151 mg of CaClh to a 100-ml volume of water.The starch suspension was boiled for 1 min, cooled, and centrifuged at 10,000g for 15 min. The clear supernatant was used for the enzyme assay. An aliquot of enzyme (0.1 ml) was added to 0.5 ml of the starch solution in a 18-X 150-mm test tube, and the reaction was allowed to proceed for a suitable time (2-10 min). Starch hydrolysis was stopped with 1.0 ml of iodine reagent. The iodine reagent was made by adding 0.15 ml of an iodine stock solution (6 g of KI and 600 mg of L. in 100 ml of water) to 50 ml of 0.05 N hydrochloric acid. After the reaction had been stopped, water was added to the reaction mixture to give a final volume of 3 ml, and the absorbance was read at 620 nm with a Coleman colorimeter. The change in absorbance was proportional to the amount of amylase present.Seed imbibition and isolation and incubation of aleurone layers from mature seeds were performed as described by Chrispeels and Varner (3).Acrylamide Electrophoresis. Methods for the electrophoresis of amylase were followed as described by Scandalios (16) Enzyme samples from the incubation medium or from tissue extracted with 0.2 M NaCl were absorbed into small paper wicks. These wicks were inserted into a slit 4 cm from one edge of the tray. Electrophoresis was accomplished with a voltage gradient of 12 to 13 v/cm for 22 hr at 5 C. The wicks were removed after 0.5 hr. The gel was covered during electrophoresis with cellophane (Handiwrap) to retard desiccation. After electrophoresis, the gel was incubated in a starch solution (I % Lintner starch in 40 mm sodium phosphate or tris-malate buffer, pH 7.0) at 25 C for 5 hr. Bands of amylolytic activity were visualized by immersing the incubated gel in a I2-KI solution for 20 to 30 min.Amylose Hydrolysis. For end product analysis of amyla...

EFFECT OF pH, TEMPERATURE, AND CALCIUM IONS ON BARLEY MALT α-AMYLASE ISOENZYMES

“…After heat treatment for 5 min at 70°C the pH was adjusted to 5-4 and the activity measured at 30°C. Stability at low pH and i(TC.-To 1 ml enzyme solution (2-1 mg/ml of a-amylase I or 0-75 mg/ml of a-amylase 11) was added 0-5 ml 003 m acetate buffer pH 3-5 (giving a final pH of [3][4][5][6]. At suitable time intervals, 0-1 ml aliquots were taken for activity measurements.…”

Distinction between the Responses of Developing Maize Kernels to Fluridone and Desiccation in Relation to Germinability, α-Amylase Activity, and Abscisic Acid Content

“…13), also play a role in the aleurone layer. Drying, when prematurely imposed on developing grains, enables the aleurone layer of wheat and barley to respond to GA and produce a-amylase (1,2,5,17), a starch-hydrolyzing enzyme which is the predominant protein synthesized by this tissue (6). Drying, it is argued, renders the tissue sensitive to gibberellin (1,5).…”

Amylases from Aleurone Layers and Starchy Endosperm of Barley Seeds

“…The soluble starch substrate was prepared in 0.05 M sodium acetate buffer, pH 4.8, according to the method outlined by Bilderback (2). An aliquot of the column fraction (0.5-1.0 ml) was added to 0.5 ml of the starch solution in a test tube, and the reaction was allowed to proceed for a suitable time (5 min-24 hr.).…”

“…An aliquot of the column fraction (0.5-1.0 ml) was added to 0.5 ml of the starch solution in a test tube, and the reaction was allowed to proceed for a suitable time (5 min-24 hr.). Starch hydrolysis was stopped with 1.0 ml of an iodine reagent prepared according to the procedure of Bilderback (2). Absorbance was read at 620 nm.…”

“…The incubation medium for the aleurone layers was 2 mm acetate buffer, pH 5.0, with and without 20 mM CaCl2 and 1 .Mm gibberellic acid. The colorimetic assay for amylase was performed as described by Bilderback (2).…”