Isozymes of alpha-Amylase Induced by Gibberellic Acid in Embryo-less Grains of Barley.

Yomo (8) and Paleg (5) independently fouind that the induction of a-amylase in the embryo-less grain was stimulated by gibberellin. This fact was confirmed by several investigators (1, 4, 7). Recently one of the authors and his co-workers found that helminthosporol and its air-oxidation derivative, helminthosporic acid, had the same effect as gibberellin. We have investigated isozyme(s) of a-amylase induced by gibberellic acid and helminthosporol to see whether or not the gibberellinand helminthosporol-induced a-amylase are the same. This paper reports on isozymes of a-amylase induced by GA3. Seeds of Hordeutm distichon L., Kirin-chokuichi, of the 1965 harvest were used in this experiment. Seeds were dehulled with 50 % (v/v) sulfuric acid and washed with running water. Embryos were removed from the seeds with a ra7or blade. The remaining endosperms were soaked in 75 % alcohol (v/v) for 30 seconds and sterilized by dipping in a supernatant soluition of 10 % concentration of bleaching powder solution (g/v) for 10 minutes. After rinsing with sterile redistilled water, 40 endosperms were placed in a 9 cm petri dish which contained either 10 ml of sterile redistilled water or the same volume of 2 AM Ga3 solution. 'rhe petri dishes were incubated at 250 for 4 days. One hundred endosperms were extracted with calcium acetate-sodium chloride solution [0.01 M Ca(O-CO-CH3)2 0.02 M NaCl]. The extract was precipitated by adding solid ammonium sulfate up to 0.75 saturation. The precipitate was centrifuged at 00, the supernatant fraction being discarded. The precipitate was dissolved in a small volume of 0.01 Nt Tris-HCl buffer containing 0.01 M NaCl, pH 8.5, and filtered through Sephadex G 50 coluimn, 2 X 20 cm, to eliminate low molecuilar compounds. The filtrate was adsorbed onto a 2 X 20 cm DEAE-cellulose column. The active material was eluted with 200 ml of solution, employing a linear gradient of 0.01 to 1.00 M NaCl. The NaCl solution was adjusted to pH 8.5 with 0.01 M TrisHCl buffer. Eluate was collected for each 4 ml and the amylase activity was determined as follows. But the culture medium was not used to extract, because the medium contained only a small amount of the a-amylase which was similar chromatographic pattern of the endosperm. The activity of a-amylase was measured by the Blue value method modified by Fuwa (3). Two ml of 0.1 M acetate buffer, pH 5.7, which contained 0.5 % starch, was added to 0.5 ml of enzyme solution. After 15 or 30 minuites of incubation of the combined solution at 400, 5 ml of 0.5 N acetic acid was added. One ml of this solution was added to 10 ml of iodine solution which consisted of 0.0003 M iodine, a small amount of potassium iodide and 0.03 N HCL. The optical density of this solution at 700 mu was measured at room temperature with a Hitachi-spectrophotometer. Activity unit of the enzyme was expressed as mg of hydrolyzed starch under the condition in which the optical density of starch-iodine complex at 700 m,u was decreased 10 % with 1 ml of enzyme solution for 30 minutes at 40°. The activity of -amylase was measured by the method of Schwimmer (6). Three different fractions with a-amylase activit)y were obtained. We tentatively named the fractions a-amylase-I (a1), -II (a,) and -III (a3) according to the order of elution (fig 1). The a, fraction induced by 2 ,uM GA3 showed a high activity of a-amylase. The amount of a,, was greater than that of both a1 and a3. Butt in the case of treatment with higher concentration of GA3, such as 0.1 mM helminthosporol, the amount of a3 was too small to be isolated. According to our data for 0.2 mm helminthosporol, the amotunt of a, was about twice that of a,. Each amount of a1 and a, induiced by 2 AM GA3 was more than 5 times that of the control. Although the a3 fraction was not obtained in the control. Varner showed only 1 fraction with a-amaylase activity (7). On the other hand, we obtained 3 active fractions at least. The difference between Varner's result and ours mav be dLue to the difference of incutbation period and pH valtue of elutant. GA.3 had nlo effect onl the level of fl-amaylase. Each of the 3 fractions induced by GA3 was concentrated to a small volume in an ice box and dialysed for 2 days against 0.01 M Tris-HCl buffer, pH 8.5, which contained 0.01 M NaCl. After the dialysis, each of the fractions was rechromato-