Induction of α-Amylase in Barley Endosperm by Substrate Levels of Glutamate and Aspartate

Abstract

Incubation of embryoless barley (Hordeum vulgare) (4), the seeds were then washed 10 times with about 4 volumes of sterile distilled water in a sterile test tube (25 x 150 mm) covered with a Morton stainless steel closure. After the final wash they were covered with distilled water and soaked overnight at 4 C. Using aseptic technique, the seeds were cut in half perpendicular to their long axis, and the embryoless halves were used for the bioassays. Triplicates of two-half-seeds incubated in 1 ml total volume of medium in a 20-ml beaker were tested for each treatment. The medium contained 12.5 ,ug of chloromycetin per ml and, except as indicated in the text, was adjusted to pH 4.8 with either HCl or NaOH. The various substances incorporated in this medium were sterilized by filtration through 0.22 A pore diameter Millipore filters.After incubation at 30 C for various periods, the medium was removed, and the seeds and beaker were washed with 2 ml of sterile distilled water. The combined wash and incubation medium from each beaker was assayed for a-amylase by a slight modification of the procedure of Shuster and Gifford (15). The reaction mixtures consisted of 0.5 ml of enzyme plus 0.5 ml of soluble starch reagent. The starch reagent was prepared by dissolving soluble starch (1.5 mg/ml), KH.PO, (6 mg/ml), and CaCl, (0.22 mg/ml) in 0.1 M sodium acetate buffer at pH 4.8 and heating the solution until it started to boil. The enzyme-substrate mixtures were allowed to react for 1 to 10 min at 30 C, and the reaction was stopped by adding 2 mi of iodine reagent. The iodine reagent was prepared by diluting a stock I.-KI solution (60 mg of KI plus 6 mg of I, per ml of water) 100-fold with 0.05 N HCI. The tubes were held in an ice bath until diluted for absorbancy measurements by adding 5 ml of distilled water. The absorbancy of the blue starch-iodine complex was measured at 620 nm in a Gilford spectrophotometer. Dilutions of the enzyme were made so that no more than half of the starch in the reaction mixtures was hydrolyzed and under these conditions the kinetics of the reaction were zero order with respect to starch. The extent of hydrolysis was estimated from a standard curve for the starch-iodine complex, and the results are expressed as units of a-amylase released per half-seed. A unit is that amount of enzyme which hydrolyzes 100 ,ug of starch per min at 30 C. The values reported in individual experiments are the mean of the triplicate determinations.

Adenosine 3′:5′-Cyclic Monophosphate in Chlamydomonas reinhardtii: Isolation and Characterization

“…Other reports provide circumstantial evidence for the possible occurrence of cAMP in green plants. These reports describe effects of exogenously supplied cAMP (6)(7)(8)(9)(10)(11), or chromatographic similarities between authentic cAMP and a plant metabolite derived from adenine (12)(13)(14), or the presence of enzyme activities that may function in cAMP metabolism (5,(15)(16)(17).…”

Regulation of a Plant 5′(3′)-Ribonucleotide Phosphohydrolase by Cyclic Nucleotides and Pyrimidine, Purine, and Cytokinin Ribosides

“…cAMP (Ki = 3.4 ,uM) applied at millimolar levels to barley aleurone mimics the effect of GA3 on induction of a-amylase (18,19,28), secretion of soluble sugar (28), induction of phosphatase (20-22), and induction of protease (20). At these levels, cAMP also stimulates the effect of GA but not of kinetin on lettuce seed germination (25).…”

“…Elevation of cAMP levels in Avena coleoptiles occurs as a consequence of indole-3-acetic acid treatment (4). In phytohormone-replacement experiments, cAMP has been shown to apparently mimic responses to indole-3-acetic acid (8,(15)(16)(17) and GA (18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) in appropriate bioassays.…”

Evidence for modification of protein phosphorylation by cytokinins

“…In turn the protein kinases regulate the activity of various intracellular processes by phosphorylating key proteins (Jost & Rickenberg, 1971). To date there has been no compelling evidence that cyclic AMP controls such processes in plants (but see Duffus & Duffus, 1969;Galsky & Lippincott, 1969;Pollard, 1970;Kamisaka & Masuda, 1970;J6st & Rickenberg, 1971; Salomon & Mascarenhas, 1971; Kamisaka & Masuda, 1971;Woodet al, 1972). Becauseofthe possibility that cytokinins might modify either the amount or the action of cyclic AMP in plants we examined the effect of cyclic AMP on Chinese-cabbage leaf discs.…”