藓羽藻(Bryopsis hypnoides)核酮糖-1,5-二磷酸羧化酶/加氧酶的分离与活性测定

ISOLATION AND ACTIVITY ASSAY OF RIBULOSE-1, 5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM MARINE ALGA BRYOPSIS HYPNOIDES

采用硫酸铵分部沉淀与凝胶过滤的方法 ,进行藓羽藻Rubisco的分离研究。结果表明 ,分离的藓羽藻Rubisco经SDS 聚丙烯酰胺凝胶电泳检测呈两条清晰条带 ,分别为Rubisco大亚基与小亚基 ;与菠菜相比 ,藓羽藻Rubisco大亚基分子量与菠菜基本相同 ,而小亚基较之稍大一些。藓羽藻Rubisco活力测定结果表明 ,Rubisco分离过程中用硫酸铵分部沉淀后活力降低许多 ,分离后活力有所上升 ,但仍比粗提液活力弱 ;在Rubisco活力测定过程中 ,藓羽藻Rubisco的活化温度与其它物种Rubisco活化的温度不同 ,在低温下活化效果较好。这些结果说明Ru

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) plays an important role in photosynthetic carbon fixation by controlling the initial stage of the Calvin cycle and possessing the functions of both CO 2 fixation and ribulose-1,5-bisphosphate (RuBP) oxygenation. Rubisco is commonly composed of eight large and eight small subunits. The large subunit is encoded by chloroplast genosome and the small subunit by nuclear genosome. Rubisco is the most abundant enzyme in nature and comprises up to 50% of the soluble protein in the leaf of C 3 plants. Rubisco from high plants has been studied in many plant species and the isolation method on alga is similar to that of high plants. Regarding marine alga, many studies have previously been undertaken on Ulva and Kelp etc. In this paper some physiological characteristics of Bryopsis hypnoides, a multi-nuclear unicellular green marine alga are discussed. Activity of Rubisco isolated from B. hypnoides was measured at different stages of the isolation. The isolation method was similar to higher plants but for the preparation of crude extract. Chloroplasts of B. hypnoides were extracted in 50mmol/L HEPES-KOH buffer (pH 8.0) containing 1mmol/L EDTA, 20mmol/L MgCl 2, 10mmol/L NaHCO 3, 10mmol/L β-mercaptoethanol and disintegrated by sonication, then centrifuged and the precipitate was discarded. The supernatant was fractionated with ammonium sulfate. The precipitate between 35% and 50% saturation was collected and loaded onto a Sephadex G-200 column equilibrated with extract buffer. The first peak monitored at 280nm absorbance comprising mostly of Rubisco was collected. SDS-PAGE showed that enzymes from spinach eluted from the gel column were not purified but those from B. hypnoides purified. The molecular weight of the large subunit of Rubisco from B. hypnoides was the same as that from spinach, while the small subunit of Rubisco from B. hypnoides was larger. Enzyme activity was also measured using the spectrophotometric assay at three steps (crude extract, fractioned with ammonium sulfate and eluted from the gel column) during the period of isolation. Rubisco was activated at different temperatures before the determination and the assay mixture contained 100mmol/L Tris (pH 8 0), 10mmol/L MgCl 2, 5mmol/L ATP, 5mmol/L DTT, 0.2mmol/L NADH, 10mmol/L NaHCO 3 and 0 1mmol/L EDTA. After,that 5 units of G 3PDH/PGK were added to the mixture and absorbance at 340nm (A 340) was recorded every 20 seconds for 3 minutes to get a native rate of NADH oxidation, then the reaction was initiated by adding RuBP to a final concentration of 0.3mmol/L and the determination was continued with A 340 being recorded for 3 minutes. Rubisco activity was calculated according to the formula and the native rate of NADH oxidation should be considered. Compared with spinach, results of the assay indicated that the activity of Rubisco from B. hypnoides was weak and exhibited obvious decline during the period of isolation. It was also found that enzyme activity value in crude extract from B. hypnoides varied according to different pre-incubated temperatures before the enzyme assay. All results showed that activity of Rubisco was affected by ammonium sulfate and that Rubisco from B. hypnoides was less stable than that from higher plants.