几种固氮蓝藻的固氮酶活性及其某些特性

对三种固氮蓝藻：固氮鱼腥藻(水生686)、柱孢鱼腥藻和鱼腥藻7120的整细胞及无细胞抽提液的固氮酶活性,进行了比较研究。水生686的整细胞酶活虽然不低(51．9毫米乙烯峰高／光密度／30分),仅次于柱孢鱼腥藻,但其无细胞抽提液的酶活却最低。这可能与它含有大量藻胶有关。研究了Mn^ 、Fe^ 对蓝藻固氮酶的作用,以及测定其在不同酶浓度下的反应动力学表明：柱孢鱼腥藻中不存在象深红螺菌中所看到的那种激活因子。用甲苯-乙醇溶液处理藻细胞,对固氮酶作原位测定,探索了它的氧损伤及氧保护机理。     

硫酸铜强化固氮蓝藻生长的研究

培养基中添加0.005—0.03ppm 铜能强化固氮蓝藻生长。混合藻种(鱼腥藻属混合藻种)、固氮鱼腥藻 Anabaena azotica(水生686)和多变鱼腥藻 Anabaena variabslis(水生1058)最佳铜浓度分别为0.01ppm 和0.03ppm。池底铺土进行培养固氮蓝藻,铜浓度的范围略宽。     

柱孢鱼腥藻固氮酶防氧的呼吸保护

柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO2)是0．5atm．,而有氮生长细胞的半抑制浓度为0．35atm．。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。     

柱孢鱼腥藻固氮酶防氧的呼吸保护

柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO_2)是0.5atm.,而有氮生长细胞的半抑制浓度为0.35atm.。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。     

固氮蓝藻与棕色固氮菌固氮酶组分的交叉互补功能

本文报告了藻菌之间固氮酶组分的交叉互补试验。初步结果证明：固氮蓝藻(Anabaena azotica水生686)的钼铁蛋白与棕色固氮菌(Azotobacter vinelandii)的铁蛋白之间存在着明显的互补功能。但这种蓝藻的铁蛋白在非细胞形态下很不稳定,易于失活。本实验为不同生理类型和不同进化程度的固氮生物之间固氮酶组分的交叉互补研究提供了新的资料。     

固氮蓝藻与棕色固氮菌固氮酶组分的交叉互补功能

本文报告了藻菌之间固氮酶组分的交叉互补试验。初步结果证明:固氮蓝藻(Anabacnaazotica水生686)的钼铁蛋白与棕色固氮菌(Azotobacter vinelandii)的铁蛋白之间存在着明显的互补功能。但这种蓝藻的铁蛋白在非细胞形态下很不稳定,易于失活。本实验为不同生理类型和不同进化程度的固氮生物之间固氮酶组分的交叉互补研究提供了新的资料。       

水稻田中固氮蓝藻固氮活力的昼夜变化

蓝藻的固氮和光合作用密切联系,为了解固氮蓝藻在田间固氮活力的变化,我们对田间的蓝藻在不同生境下的固氮活力和昼夜变化进行现场(in situ)测定发现: 1.在不同生境中生长的各种固氮蓝藻的固氮活力的昼夜变化节律不尽相同,一般为一天出现一个活力高峰,在薄膜复盖培养池中高峰出现在中午(13∶00),而露田则在9∶00—12∶00之间;个别还出现两峰现象。2.不同种类的固氮蓝藻在田间的固氮活力有明显差别,混合藻种的酶活力最高,类颤鱼腥藻次之。3.不同生长期的类颤鱼腥藻的酶活力有明显不同。生长旺盛的活力高,衰老的活力低。4.稻田中不同小生境对蓝藻固氧酶活力有显著的影响,在水底生长的类颤鱼腥藻移至水面,其活力明显降低。根据蓝藻在田间的固氮活力的昼夜交化以及生境改变和生长状态对活力的影响,依估算生长良好的蓝藻在水田的固氮量最高可达285克氮/天/1000斤藓藻,最低51克氮,平均163克氮。假如在田间生长连续10天,即可固定1630克氮,相当于16.3斤的硫酸铵。但和测定的较高固氮活力(=258克氮/天相当于25.8斤硫铵)相比,固氮活力的潜力是相当大的。如能提高固氮蓝藻在田间的产量, 则其固氮量可大大增加。     

四唑（Tetrazolium）对固氮鱼腥藻固氮和氢代谢的影响

固氮鱼腥藻(Anabaena azotica Ley)细胞能还原无色的TTC和NBT分别成为红色或蓝色的甲zan(formazan)沉淀。异形胞还原TTC的速率高于营养细胞。前异形胞及异形胞附近的营养细胞对NBT的还原作用最强。而异形胞对NBT不起还原作用。无论在异形胞形成红色甲zan或在营养细胞形成蓝色甲zan后都抑制固氮酶活性。NBT甲zan对固氮酶活性的抑制作用大于TTC甲zan,因为NBT氧化还原电位低于TTC。TTC和NBT两者都明显地抑制固氮鱼腥藻完整细胞的放氢。因鱼腥藻的放氢是由固氮酶催化的结果。四唑抑制放氢推想是由于它截取了固氮酶催化系统中的电子的缘故。固氮微生物(包括蓝色细菌和根瘤菌)对四唑还原与吸氢酶之间有无相关是一个争论的问题。一些学者认为分离豆科植物体的一些根瘤菌株培养于含有TTC的琼脂培养基,如还原,便可证明这些根瘤菌株能氧化氢;换言之,应用TTC的还原可作为一些根瘤菌的菌落具有吸氢酶的验证。相反,我们发现固氮鱼腥藻还原TTC和NBT之后,都没有影响吸氢的能力。因此,我们推想固氮鱼腥藻对四唑之还原与吸氢酶是没有直接的关系。     

谷氨酰胺合成酶在固氮鱼腥藻固氮调节中的作用

在MSX(methlonine sulfoximine,谷氨酰胺合成酶的不可逆抑制剂)存在下,固氮鱼腥藻(Anabaena azotica)所分泌的氨量和谷氨酰胺合成酶(GS)活力有较好的负相关性,证明谷氨酰胺合成酶-谷氨酸合成酶(GS-GOGAT)是固氮鱼腥藻氨同化的重要途径。在蛋白质合成受到氯霉素拟制时,NH_4~ 对固氮酶的失活是迅速的,同时GS活力有较大下降,表明NH_4~ 的调控酶的失活或降解。在氮固定条件下,固氮酶活力半衰期小于4小时,GS活力半衰期大于10小时,则GS并不是固氮酶的正调节因子。NH_4~ 和谷氨酰胺(gln)对固氮酶的失活作用随它的浓度增加而提高,但GS并没有这种相关性,低浓度NH_4~ (0.1—0.5mmol/L·NH_4Cl)对GS活力没有抑制作用,高浓度gln(1.0—2.0mmol/L)也没有抑制GS活力,说明GS并不直接调控固氮酶。MSX能消除NH_4~ 和gln对固氮酶的抑制作用,并与藻龄有关。     

鱼腥藻(Anabaena)对氧敏感的固氮突变种

用紫外线和亚硝基胍,诱变筛选出两株具异形胞而不能在空气中固定分子氮的鱼腥藻Anabaena)突变种。突变表型是稳定的。在微氧条件下,能固定分子氮而生长,但固氮酶活很弱。酶活的高低与藻蓝素含量和光照强度成反相关。突变种细胞的固氮酶对氧非常敏感,反应气相中加入1%的氧对乙炔还原活力已有明显抑制,20%的氧完全阻抑固氮作用。除去氧后固氮酶重新合成,其过程受 NH_4~+和氯霉素阻遏。通过氮饥饿使藻蓝素含量降低,或降低光照强度,或加入二氯苯基甲基脲抑制光合放氧时,均可显著地提高突变种的固氮酶活力。突变种细胞还原氯代三苯四氮唑为甲(?)结晶的能力低于野生种。暗示突变种固氮对氧敏感,可能是由于细胞中清除氧的酶系统有缺损,以致营养细胞产生的活性氧对其本身固氮过程有抑制作用。     

分子氢和氧对受高温胁迫蓝藻固氮活性的影响

Ａｎａｂａｅｎａ７１２０经高温处理后,固氮活性下降,对氧的敏感度增大,增大程度随氧浓度增高而递增。高温胁迫下,分子氢与对正常条件下生长的蓝藻一样可以削弱或消除氧对固氮的伤害,氢的此种行为在光照下和黑暗中表现相似,其良好作用比正常生长蓝藻显著,添加光合抑制剂。ＣＯ２或Ｎ２时亦如何。有外源蔗糖时,氢的良好作用不表现。经ＣＯ或Ｃ２Ｈ２处理的蓝藻,氢在其固氮活性受氧伤害时的良好作用消失。     

甘露醇对氯化钠胁迫下蓝藻Anabaena 7120固氮活性的增强效应

营养液中添加外源的甘露醇后,蓝藻Ａｎａｂａｅｎａ ７１２０固氮受ＮａＣｌ胁迫的程度减弱。在光合作用不能进行或受到削弱,能量供应受阻、厌氧环境中和分子氧条件下,甘露醇的良好作用减小或消失。改善能涛和碳架供应,增强氢的利用或满足合成固氮酶蛋白所需物质需求时,甘露醇缓解ＮａＣｌ胁迫蓝藻固氮的程度明显增大。     

Modes of reduction of nitrogen in heterocysts isolated from Anabaena species.

N2 fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, D-glucose 6-phosphate, 6-phosphogluconate and DL-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

蓝藻Anabaena 7120光漂白细胞固氮的研究

蓝藻Anabaena 7120经光漂白后固氮活性明显下降,转入正常光照下又恢复活性。此种经光漂白的蓝藻细胞,其固氮活性对氧敏感度小,受分子氢的促进大些,而忍受CO_2和N_2抑制的浓度相对高些。其固氮活性为弱光和光合抑制剂减弱,而加入外源的碳水化合物则能提高它的固氮活性。当碳水化合物和光合抑制剂一起加入反应系统时,蓝藻光漂白细胞的固氮活性并不能受到促进。     

D-erythrose supports nitrogenase activity in isolated Anabaena sp. strain 7120 heterocysts.

Among organic compounds tested for their ability to support nitrogenase activity in isolated heterocysts of Anabaena sp. strain 7120 under argon, D-erythrose (5 mM) was unique in supporting acetylene reduction at 10 times the control rates. Higher concentrations of D-erythrose exhibited substrate inhibition. At 50 kPa of H2, all concentrations of D-erythrose inhibited H2-supported acetylene reduction. The effects of D-erythrose on nitrogenase activity were explored. Erythrose enhanced 15N2 incorporation by heterocysts, but NADP+ did not enhance erythrose-supported acetylene reduction. H2 protected nitrogenase from O2 inactivation, but erythrose did not; erythrose did not counter protection by H2. Tests with inhibitors of electron transport showed that erythrose-supported acetylene reduction requires electron flow through ferredoxin, a b-type cytochrome, and a 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone-sensitive transfer agent whose electron flow is not mediated through the plastoquinone and Rieske iron protein.     

低温对氯化钠胁迫下蓝藻固氮活性的影响

低温加剧氯化钠对蓝藻固氮的抑制,营养液中氯化钠浓度增高时,抑制程度更甚.能源受限(暗处理和加抑制剂时的光合受抑,N_2和Ar的厌氧下呼吸代谢受阻)和氧下固氮酶受到伤害时,低温处理使氯化钠对蓝藻固氮的抑制进一步加剧.在能源和还原剂供应,合成固氨酶蛋白的物质基础(如CO_2和N_2的加合).光合作用正常进行的条件得到改善和保证,以及供应CO_2、外源蔗糖和氮氧加合时,低温加剧氯化钠对蓝藻固氮的抑制程度明显变小.     

硝酸钾缓解氯化钠胁迫蓝藻Anabaena 7120固氮的生理基础

营养液中添加适量ＫＮＯ３可在一定程度上缓解ＮａＣｌ对鱼腥藻固氮活性的抑制作用。暗处理或加光合抑制剂时,ＫＮＯ３对ＮａＣｌ胁迫的缓解作用便消失。供给外源蔗糖、提高ＣＯ２浓度、同时供给ＣＯ２和Ｎ２时,ＫＮＯ３对缓解ＮａＣｌ胁迫的作用则增高．同时供给Ｏ２和Ｈ２对ＫＮＯ３的缓解作用增高影响较小,而在厌氧（Ａｒ或Ｎ２中）或单加氧下,ＫＮＯ３的缓解效应则明显减弱或消失．     

Nitrogenase Activity in Cell-Free Extracts of the Blue-Green Alga, Anabaena cylindrica

Cell-free extracts with high nitrogenase activity were prepared by sonic oscillation and French press treatment from the blue-gree alga Anabaena cylindrica. Extracts were prepared from cells grown on a 95% N(2)-5% CO(2) gas mixture followed by a period of nitrogen starvation under an atmosphere of 95% argon-5% CO(2). No increase in the specific activity of extracts was achieved by breaking heterocysts. Activity (assayed by acetylene reduction) was found to be dependent on adenosine triphosphate (ATP), an ATP-generating system, and a low-potential reductant. Na(2)S(2)O(2) employed as reductant supports higher rates of nitrogenase activity than reduced ferredoxin. The activity is associated with a small-particle fraction that can be sedimented by ultracentrifugation. In contrast to the particulate nitrogenase of Azotobacter, which is stable in air, the A. cylindrica nitrogenase is an oxygen sensitive as nitrogenase prepared from anaerobic bacteria.     

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.