| Abstract: | The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiolated rice (Oryza sativa L. subsp, japonica var. nongken 58) seedlings. Approximately l kg of 6d old seedlings (the first 2 days at 33℃, the last 4 days at 27 ℃ in darkness) were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer, at final pH 8.45 (4 ℃). After polyethylenimine precipitation, phytochrome in extract was converted to Pfr by irradiation of the resulting supernatant for 10 min with red light. The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ratio of 10 ml per phytochrome unit. The pellet precipitated with ammonium sulfate at 42% saturation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8 ml instead of 0.65 ml per phytochrome unit. Then it was washed successively with 200 mmol/l and 100 mmol/1 phosphate buffer (0.85 ml per phytochrome unit). Native phytochrome (120 kDa) in 12% yield was dissolved in 2 mmol/l EHPES buffer (2.2 ml per phytochrome unit, pH 7.8, containing 5 mmol/l EDTA and 14 mmol/l 2-mercaptoethanol) was proved to be pure in SDS- polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome. |
