小麦叶片暗诱导衰老期间1,5-二磷酸核酮糖羧化酶/加氧酶的降解

研究了小麦(Triticum aestivum L. cv.Yangmai 158)叶片暗诱导衰老过程中1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco EC 4.1.1.39)的降解.发现在此期间Rubisco大亚基(LSU)发生裂解,产生50 kD的降解条带,同时在自然衰老过程中也检测到这一产物.初步实验结果表明LSU发生这步裂解时Rubisco全酶没有解离.另外,在粗酶液中当温度在30～35℃,pH 7.5时,这一步裂解反应能有效进行.     

水稻叶片在自然衰老过程中1，5-二磷酸核酮糖羧化酶/加氧酶的降解(英文)

１,５二磷酸核酮糖羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）是光合碳同化的关键酶,研究其降解机理对合理调控水稻生长后期光合衰退具有重要意义。前人用人为诱导植物衰老的方法,研究了Ｒｕｂｉｓｃｏ的降解机理,认为该酶降解之前,必需发生亚基间的交联聚合和向类囊体膜转移,这样在结构和空间上有利于水解酶的作用。我们用自然衰老叶片进行研究的结果表明：Ｒｕｂｉｓｃｏ在降解过程中其比活基本保持恒定,意味着未发生酶的失活,也就是说酶结构未发生根本性改变,由此也可初步判断酶未发生亚基间的交联聚合（已证明亚基交联可导致酶失活）。接着用ＳＤＳＰＡＧＥ和蛋白印迹技术证实了上述观点：Ｒｕｂｉｓｃｏ降解之前只有极少量的大亚基聚合体,随后同未聚合大亚基一起很快降解。此外,研究结果进一步表明酶分子在降解之前有少量与叶绿体膜结合,但降解过程中并未见膜结合蛋白增加。根据上述结果我们认为,亚基间交联聚合和向膜转移并非水稻叶片自然衰老时Ｒｕｂｉｓｃｏ降解的必要条件。     

核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)

文章就核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的分布、结构、性质、分类与功能的研究进展作了介绍。     

烟草核酮糖-1,5-二磷酸羧化酶/加氧酶四级结构的研究(英文)

本文提出三种证据证明烟草核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的大亚基伸展在小亚基的外面,小亚基排列在大亚基中间的概念。证据是:1.固定化胰蛋白酶在一定条件下可水解RubisCO的大亚基但不水解小亚基,而天然胰蛋白酶水解大亚基,也水解小亚基。2.固定化抗小亚基IgG-Sepharose可与游离的小亚基相结合,但不能与全酶结合。3.低浓度尿素处理可使固定化的RubisCO-Sepharose上的小亚基解离下来,而大亚基仍结合在载体上,这说明RubisCO是通过定位在分子表面上的大亚基的ε-氨基与Sepharose共价偶联的。当RubisCO中的小亚基全部被解离后,大亚基之间的结合进一步增强,这时解离大亚基所需的尿素浓度要比小亚基存在时高。任何RubisCO的四级结构模型都应将小亚基置于大亚基中间受保护的位置,一部份小亚基可暴露于全酶分子表面。     

不同叶位鸡蛋果叶片中核酮糖-1,5-二磷酸羧化酶/加氧酶的动力学特性

随着叶龄的增加,鸡蛋果Ru BP羧化酶/加氧酶的V_(max)(CO_2)值明显减小,K_m(O_2)值提高;K_m(CO_2)和V_(max)(O_2)值则保持相对的稳定。Ru BP羧化酶活性和Ru BP加氧酶活性均随叶龄增加而下降,但前者下降的速度高于后者,致使羧化/加氧此值也随叶龄增加而减小。     

二磷酸核酮糖羧化酶／加氧酶装配研究进展

本文对Rubis CO大、小亚基在叶绿体和大肠杆菌中的合成,装配,酶的体外重组以及亚基结合蛋白的性质和作用等进行了综述,并对这个领域的研究前景作了展望。     

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

蔗糖对水稻幼苗叶片谷氨酰胺合成酶和1,5-二磷酸核酮糖羧化酶/加氧酶的影响

报道了在光照和暗处培养下,不同的浓度的蔗水稻幼苗叶片GS及其同工酶、1,5－二磷酸核酮糖羧化酶／加氧酶（Rubisco）的影响。无论是在光照或在暗处,蔗糖对GS活性均有抑制作用,尤其是在较高蔗糖下作用更为明显;虽然Rubisco及可溶性蛋白的水平在光照和暗处有显著的差别,但蔗糖对其未见明显影响。NativePAGE与活性染色表明,在光照下或在暗处,蔗糖对GS2的抑制蔗糖浓度升同而加强,但对GS1未有明显影响。这些结果提示,在水稻幼苗生长中,蔗糖不能象不光一样诱导叶水GS活性及其同工酶表达。     

变色酸显色法同时测定核酮糖-1,5-二磷酸羧化酶/加氧酶活性

提出一个用变色酸-硫酸显色浊同时测定核酮糖-1,5-二磷酸(RuBP)羧化酶/加氧酶活性的方法:RuBP羧化酶/加氧酶与底物作用后,用碱性磷酸酯酶将其产物水解生成乙醇酸和甘油酸,然后与变色酸试剂在1:5的体积比下,沸水浴中显色反应90min,乙醇酸与变色酸反应生成红紫色化合物,甘油酸生成淡棕色化合物,分别在573nm,745nm各有一特征吸收峰。根据A_(573),A_(745)与乙醇酸和甘油酸浓度间的函数关系式,求出RuBP羧化酶/加氧酶活性。     

Degradation of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50 kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco. This fragment could also be detected in natural senescence. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50 kD. And LSU could be fragmented to 50 kD at 30-35 ℃ and at pH 7.5 in crude enzyme extracts of wheat leaves dark-induced for 48 h, which suggested that maybe LSU was degraded to 50 kD by an unknown protease in chloroplast.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

小麦叶片暗诱导衰老期间内肽酶的特性

The characterization of senescence-associated endopeptidase (EP) isoenzymes in wheat (Triticum aestivum L. var. Yangmai 158) leaves during dark-induced senescence was performed. It was found that there was much higher endoproteolytic activity in dark-induced wheat leaves than in control. Six endopeptidase isoenzymes (EP1-EP6) were identified by natural gradient杙olyacrylamide gel electrophoresis (PAGE) co-polymerized gelatin in the gel, five of which (EP1, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Treatment with 6-benzylaminopurine (6-BA) delayed the expression of these EP isoenzymes and abscisic acid (ABA) accelerated it. The activity of EP3 could be detected at a wider range of pH and temperature levels while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30 45 ℃, EP1 and EP2 at pH 3 5 and 30 ℃ 45 ℃. All of the EP isoenzymes showed high thermal stability, especially EP3, EP5 and EP6 which still had activities even by incubation at 55 ℃ for 1 h. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine-proteases, EP4 as a serine-protease.     

小麦叶片暗诱导衰老期间内肽酶的特性

研究了小麦(Triticum aestivum L.cv.Yangmai 158)叶片暗诱导衰老期间内肽酶同工酶的变化及其部分生化特性,发现叶片衰老期间,内肽酶活性升高,同时出现5种新的内肽酶同工酶(EP1、EP2、EP4、EP5、EP6)。6-苄氨基嘌呤(6-BA)处理能延缓这些同工酶的出现,而脱落酸(ABA)处理则加速它们的表达。衰老期间小麦叶片内的6种内肽酶同工酶(EP1-EP6)中的EP1、EP2、EP4、EP5、EP6呈现活性的pH及温度范围较窄,而EP3有活性的pH范围和温度范围均较宽,且EP3在嫩叶、老叶中均有活性。另外,EP3、EP5、EP6对热不太敏感。蛋白酶抑制剂实验表明,EP1、EP2是需金属离子的半胱氨酸型内肽酶,EP4是丝氨酸型内肽酶。     

小麦类核糖核酸酶基因（WRN1）cDNA在自然衰老和黑暗诱导衰老条件下的表达

从普通小麦(Triticum aestivum L.)中分离了一个类核糖核酸酶(WRN1)基因的cDNA。WRN1的转录受自然衰老和黑暗诱导衰老的负调控。在幼嫩组织中WRN1也有表达。由于在两个保守的位置上组氨酸被替换,WRNl很可能已经失去了核糖核酸酶的活性。Southern分析表明,在普通小麦基因组中,WRN1以一个小基因家族的形式存在。     

小麦类核糖核酸酶基因(WRN1)cDNA在自然衰老和黑暗诱导衰老条件下的表达

从普通小麦(Triticum aestivum L.)中分离了一个类核糖核酸酶(WRN1)基因的cDNA.WRN1的转录受自然衰老和黑暗诱导衰老的负调控.在幼嫩组织中WRN1也有表达.由于在两个保守的位置上组氨酸被替换,WRN1很可能已经失去了核糖核酸酶的活性.Southern分析表明,在普通小麦基因组中,WRN1以一个小基因家族的形式存在.     

水稻叶片中过氧化氢与核酮糖-1，5-二磷酸羧化酶/加氧酶降解的关系

甲基紫精(MV)处理水稻植株能快速诱导核酮糖-1,5-二磷酸羧化酶／加氧酶(Rubisco,EC4．1．1．39)及其它可溶性蛋白的降解。MV浓度越高,降解速率越高。MV能诱发叶片内源H