O2、ACC和CO2对番木瓜ACC氧化酶活性的影响

研究了番木瓜果皮l-氨基环丙烷-l-羧酸(ACC)氧化酶的部分纯化,底物(O2和ACC)浓度、辅助因子(CO2和Fe2+)和抑制剂(Co2+和α-氨基异丁酸)对体外乙烯产生速率的影响.通过DEAE-Sepharose和Phenyl-Sepharose柱层析后,番木瓜果皮ACC氧化酶被纯化了19.5倍.在乙烯产生中,ACC氧化酶对O2的Km值主要取决于ACC的浓度,随着ACC水平的增加而下降;当O2的浓度增加时,酶对ACC的Km值降低.CO2显著地增加酶的活性以及对O2和ACC的Km值.Fe2+提高酶的活性,Co2+抑制酶的活性;Fe2+能够拮抗Co2+对酶活性的抑制作用.这些动力学资料表明ACC氧化酶遵循一种顺序结合机制,首先与02结合,然后与ACC结合.     

O2、ACC和CO2对番木瓜ACC氧化酶活性的影响

研究了番木瓜果皮1－氨基环丙烷－1－羧酸（ACC）氧化酶的部分纯化，底物（O2和ACC）浓度，辅助因子（CO2和Fe^2 )和抑制剂（Co^2 和α－氨基异丁酸）对体外乙烯产生速率的影响，通过DEAE－Sepharose和Phenyl-Sepharose柱层析后，番木瓜果皮ACC氧化酶被纯化了19．5倍，在乙烯产生中，ACC氧化酶对O2的Km值主要取决于ACC的浓度，随着ACC水平的增加而下降，当O2的浓度增加时，酶对ACC的Km值降低。CO2显著地增加酶的活性以及对O2的ACC的Km值，Fe^2 提高酶的活性，Co^2 抑制酶的活性，Fe^2 能够拮抗Co^2 ，对酶活性的抑制作用，这些动力学资料表明ACC氧化酶遵循一种顺序结构机制，首先与O2结合，然后与ACC结合。     

活性氧对外源IAA诱导的ACC合酶活性的影响

本文试图从活性氧的角度阐明外源IAA诱导ACC合酶活性的机制.绿豆(Phaseolus radiatus L.)幼苗的乙烯产生及ACC合酶活性从萌发的第5天开始上升,到第10天达到高峰,接着下降.10 μmol/L的外源IAA能明显促进绿豆幼苗乙烯的产生及ACC合酶的活性,同时也促进了超氧阴离子自由基(O(-)/(*)2)、过氧化氢(H2O2)的产生.显示外源IAA诱导的ACC合酶的活性与其诱导的活性氧的产生具有某种相关性.外源O(-)/(*)2处理能明显提高绿豆幼苗的乙烯产生速率及ACC合酶的活性,而外源H2O2无论对乙烯产生或ACC合酶的活性均没有明显的作用.外加O(-)/(*)2的清除剂SOD对绿豆幼苗乙烯的产生及ACC合酶活性的提高有一定的抑制作用,而外源过氧化氢酶却没有明显的作用.为此我们可以得出结论:外源IAA诱导的绿豆幼苗ACC合酶活性的提高可能是由于其诱导的O(-)/(*)2产生的升高引起的,这可能也是高等植物中调控乙烯生物合成的机制之一;而IAA诱导的H2O2产率的升高并不是其诱导ACC合酶活性升高的原因.     

超氧阴离子自由基对绿豆黄化幼苗ACC合酶的影响

以0．5、5和50mmol／L的连二亚硫酸钠(Na2S2O4)为外源超氧阴离子自由基(O2^-)源,处理20min能明显提高绿豆黄化幼苗ACC合酶(ACC synthase,ACS,EC4．4．1．14)的活性。超氧阴离子自由基的特异性清除剂超氧化物歧化酶(superoxide dismutase,SOD)和1,4-二氮杂二环(2,2,2)辛烷[1,4-diazabicyclo(2,2,2)Octane,DABCO]能抑制Na2S2O4的这种作用,显示Na2S2O4产生的O2^-;引起了ACC合酶活性的升高。但Na2S2O4处理较长时间,ACC合酶活性开始下降,处理60min的ACC合酶活性明显低于对照,SOD和DABCO的加入有助于抑制ACC合酶活性的降低,表明超氧阴离子自由基对ACC合酶活性具有促进和抑制作用并存的“双重性”影响。研究表明,外源Na2S2O4处理20min能明显降低以S-腺苷蛋氨酸(S-adenosylmethionine,SAM)为底物的ACC合酶的Km值,处理60min反而提高了ACC合酶的Km值,表明O2^-;是通过改变ACC合酶对底物SAM的亲和力,从而影响其活性的。     

挥发性香气物质和乙烯生产在"湖景蜜露"桃果实发育过程中的变化

以"湖景蜜露"水蜜桃(Prunus persica L.)为试材,检测了果实从未成熟到成熟发育过程中乙烯生成、呼吸速率及挥发性香气性物质的变化;同时对果实大小、果皮色泽、果肉硬度、可溶性固形物、可滴定酸进行了测定;对与果实乙烯产生密切相关的1-氨基环丙烷-1-羧酸(ACC)含量、ACC合成酶活性、ACC氧化酶活性也进行了测定.结果表明,随果实成熟度的增加,果实大小、果皮L*值、可溶性固形物含量增加,而果实硬度、果皮h°值、可滴定酸含量减少.在未成熟的果实中,C6的醛类(反式-2-己烯醛)和醇类(顺式-3-己烯醇)是主要的成分;乙烯生成量很低;呼吸速率较高.到跃变阶段C6～C12的内酯类物质明显增加,尤其是γ和δ-内酯类成为果实主要的香气挥发性物质.推测果实乙烯、呼吸作用等基本的生理变化可能调节着内酯类物质的生成.在乙烯跃变上升时果肉中ACC氧化酶的活性下降,ACC含量和ACC合成酶活力的变化与乙烯生成量变化的趋势一致.根据以上结果可以认为桃果实主要的香气挥发性物质的形成与乙烯、呼吸跃变的开始密切相关.香气物质形成速率动态变化可能是桃果实发育过程中成熟度的另一个生理学指标.     

活性氧对外源IAA诱导的ACC合酶活性的影响(英)

本文试图从活性氧的角度阐明外源IAA诱导ACC合酶活性的机制。绿豆 (PhaseolusradiatusL .)幼苗的乙烯产生及ACC合酶活性从萌发的第 5天开始上升 ,到第 10天达到高峰 ,接着下降。 10 μmol/L的外源IAA能明显促进绿豆幼苗乙烯的产生及ACC合酶的活性 ,同时也促进了超氧阴离子自由基 (O-·2 )、过氧化氢 (H2 O2 )的产生。显示外源IAA诱导的ACC合酶的活性与其诱导的活性氧的产生具有某种相关性。外源O-·2 处理能明显提高绿豆幼苗的乙烯产生速率及ACC合酶的活性 ,而外源H2 O2 无论对乙烯产生或ACC合酶的活性均没有明显的作用。外加O-·2 的清除剂SOD对绿豆幼苗乙烯的产生及ACC合酶活性的提高有一定的抑制作用 ,而外源过氧化氢酶却没有明显的作用。为此我们可以得出结论 :外源IAA诱导的绿豆幼苗ACC合酶活性的提高可能是由于其诱导的O-·2 产生的升高引起的 ,这可能也是高等植物中调控乙烯生物合成的机制之一 ;而IAA诱导的H2 O2 产率的升高并不是其诱导ACC合酶活性升高的原因。     

O2和CO2配比对气调贮藏梨采后褐变及相关理化因子的影响

以采后'丰水'梨果实为材料,在乐扣气调试验箱中研究了O2和CO2配比对果实褐变率、多酚氧化酶(PPO)和过氧化物酶(POD)活性、丙二醛(MDA)和总酚含量的影响,以探讨适宜减轻梨气藏褐变的气体成份.结果表明:在整个贮藏过程(150 d)中,'丰水'梨果肉未发生褐变.从贮藏60 d开始,气调处理和冷藏对照果实的果皮均出现褐变,气调处理在贮藏120 d之前对果皮褐变的影响不显著,而在贮藏120～150 d内可显著减轻果皮的褐变、抑制果皮PPO和POD活性及降低总酚含量.与冷藏对照相比,气调处理可推迟果心褐变的时间,且(8%～10%)O2+3% CO2处理可完全抑制果心的褐变;气调处理亦可降低果心PPO活性、减少总酚及MDA含量;(8%～10%)O2+1% CO2处理能够显著提高果心的POD活性,而(8%～10%)O2+3% CO2处理对果实POD活性的影响不显著.可见,气调贮藏主要是通过降低'丰水'梨果皮PPO、POD活性及总酚含量来减轻组织的褐变,并以(8%～10%)O2+3% CO2处理对果实褐变因子的控制效果较理想.     

高O2或高CO2浓度气调贮藏对"那翁"甜樱桃采后生理和贮藏性的影响

研究了甜樱桃品种"那翁" ( Prunus avium L. cv. Napoleon)在1 ℃的高O2 浓度气调(CA-I: 70% O2+0% CO2)、高CO2 浓度气调 (CA-II: 5% O2+10% CO2)、自发气调 (modified atmosphere package, MAP) 和普通冷藏条件下果实生理、品质、耐藏性的变化.结果表明:与其他处理相比,高O2 浓度的气调可以抑制果实腐烂、减少果肉中乙醇含量,但果实的丙二醛(MDA)含量迅速上升、褐变严重.高CO2浓度的气调能有效抑制MDA含量上升的速率和多酚氧化酶(PPO)活性,保持果实硬度和维生素C含量,减少果实腐烂和褐变,延长贮藏寿命.不同处理对果实可溶性固形物含量的影响不大."那翁" 甜樱桃在5% O2+10% CO2气调中贮藏80 d能保持果实固有的风味品质.在MAP下, 70% O2+0% CO2和普通冷藏中的适宜贮藏期分别为40 d、20 d和30 d.     

白腐菌产漆酶的纯化及部分酶学性质

对白腐菌W 1产生的漆酶粗酶液通过超滤浓缩、分子筛和离子交换层析进行纯化 .用SDS PAGE证明该酶的分子量大约为 6 2 4kD .等电聚焦电泳显示该酶的等电点为 3 5 .酶反应的最适温度为 5 0℃ ,最适pH值为 4 5 .此酶氧化DMP的Km 值为 3 84× 10 -5mol L .金属离子对酶活的影响很大 ,其中K+ 、Mn2 + 、Ag+ 对酶活有促进作用 ,Fe2 + 、Fe3 + 、Hg2 + 、Co2 + 、Ba2 + 等对酶活有明显的抑制作用 .酶对部分染料也有一定的脱色效果     

甘蔗ACC氧化酶基因片段的克隆与序列分析

1- 氨基环丙烷-1-羧酸（ACC）氧化酶是植物乙烯合成的一个关键酶,乙烯作为一种内源激素,对植物生长、老熟过程有多方面的调节作用。根据报道的各种植物ACC氧化酶氨基酸序列上前后两个保守区设计两个简并引物,以甘蔗总DNA为模板,通过PCR扩增到一个940bp的基因片段。将片段序列在MCBI的BLAST软件上进行同源性搜寻,显示的63个序列全部是ACC氧化酶基因,因而认为克隆到的片段就是甘蔗ACC氧化酶基因的一个成员。经对不同植物来源的ACC氧化酶基因家族进行比较分析,去除一个103bp的“内含子“后,推导的氨基酸序列为279个残基,占推测全长氨基酸残基总数的86％左右。经同源性分析,序列与毛竹和水稻ACC氧化酶的同源率达到86％。系统进化分析表明,该序列最先与水稻、其次和香蕉的ACC氧化酶聚类,然后再与双子叶植物的ACC氧化酶聚类,符合按形态特征分类的血缘关系。基因的获得对下一步了解乙烯的合成表达与甘蔗生长、成熟过程之间的关系奠定了基础。     

RFLP analysis of 1-aminocyclopropane-1-carboxylate synthase ACC2 and ACC4 genes from Polish cultivars of tomato

An important trait of tomato is the rate of fruit ripening, strongly dependent on ethylene production. The ripening-related ethylene synthesis in tomato is controlled mainly by 1-aminocyclopropane-1-carboxylate synthase LE-ACS2 and LE-ACS4 isoenzymes (Rottmann et al., 1991, J. Mol. Biol. 222: 937; Lincoln et al., 1993, J. Biol. Chem. 268: 19422; Barry et al., 2000, Plant Physiol. 123: 979). In spite of numerous reports on the LE-ACS2 and LE-ACS4 gene expression, only ones considered the genomic organisation each of these genes (Rottmann et al., 1991; Lincoln et al., 1993) reported one copy of each of these genes in tomato cv VF36. In this article we suggest that the genomic organisation of LE-ACS2 and LE-ACSS4 genes may depend on tomato cultivars and may differ from that described by the above authors. The results of Southern analyses of genomic DNAs from 17-day old seedlings (cultivars Jaga, Halicz, Betalux, New Yorker) imply that the genomic organisation of LE-ACS2 and LE-ACS4 genes in Polish cultivars differs from that reported for cv VF36.     

G-protein-coupled alpha2A-adrenoreceptor agonists differentially alter citrus leaf and fruit abscission by affecting expression of ACC synthase and ACC oxidase

Temporal and spatial expression patterns of genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS1 and ACS2) and ACC oxidase (ACO), ACC concentration, and ethylene production in leaves and fruit of 'Valencia' orange (Citrus sinensis [L.] Osbeck) were examined in relation to differential abscission after treatment with 2-chloroethylphosphonic acid (ethephon) alone or in combination with guanfacine or clonidine, two G-protein-coupled alpha(2A)-adrenoreceptor selective agonists. Guanfacine and clonidine markedly reduced ethephon-enhanced leaf abscission, but had little effect on ethephon-enhanced fruit loosening. Ethephon-enhanced fruit and leaf ethylene production, and ACC concentration in fruit abscission zones, fruit peel, leaf abscission zones, and leaf blades were decreased by guanfacine. Guanfacine reduced ethephon-enhanced expression of ACS1 and ACO genes in leaf abscission zones and blades, but to a lesser extent in fruit abscission zones. The expression pattern of the ACS2 gene, however, was not associated with abscission. The results demonstrate that differential expression of ACS1 and ACO genes is associated with reduction of ethephon-enhanced leaf abscission by guanfacine, and suggest a link between G-protein-related signalling and abscission.     

New synthetic AICAR derivatives with enhanced AMPK and ACC activation

5-Aminoimidazole-4-carboxamide riboside (AICAR) has an important role in the regulation of the cellular metabolism showing a broad spectrum of therapeutic activities against different metabolic processes. Due to these proven AICAR properties, we have designed, synthesized and tested the biological activity of two ribose-modified AICAR derivatives, named A3 and A4, in comparison to native AICAR and its 5′-phosphorylated counterpart ZMP. Our findings have shown that A3 and A4 derivatives induce the phosphorylation of 5′-AMP activated protein kinase α (AMPKα), which leads to the inhibition of acetyl-CoA carboxylase (ACC), and down-regulate the activity of the extracellular signal-regulated kinases (ERK1/2). Cytotoxicity tests demonstrated that A3 and A4 do not significantly reduce cell viability up to 24?h. Taken together our results indicate that A3 and A4 have a comparable activity to AICAR and ZMP at 0.5 and 1?mM suggesting their potential use in future pharmacological strategies relating to metabolic diseases.     

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.     

Genetic Profiling Reveals Cross-Contamination and Misidentification of 6 Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM,ACCNS, ACCS and CAC2

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells.     

Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK

Acetyl-CoA carboxylases (ACCs) have been highlighted as therapeutic targets for obesity and diabetes, as they play crucial roles in fatty acid metabolism. ACC activity is regulated through the short-term mechanism of inactivation by reversible phosphorylation. Here, we report the crystal structures of the biotin carboxylase (BC) domain of human ACC2 phosphorylated by AMP-activated protein kinase (AMPK). The phosphorylated Ser222 binds to the putative dimer interface of BC, disrupting polymerization and providing the molecular mechanism of inactivation by AMPK. We also determined the structure of the human BC domain in complex with soraphen A, a macrocyclic polyketide natural product. This structure shows that the compound binds to the binding site of phosphorylated Ser222, implying that its inhibition mechanism is the same as that of phosphorylation by AMPK.     

番茄果实ACC合成酶的性质

测定了经4步纯化、比活性达20000U／mg蛋白质以上的番茄果实伤诱导ACC合成酶的一些酶学性质。酶反应最适PH值为9．5;酶在pH8．0下最稳定,pH7．5－10短时间处理不会使酶发生不可逆变性;酶在pH8．0和9．5的Km值分别为23和4Dμmol／L;根据酶反应不同时间的产物累积量,得出反应速度随时间的变化符合函数关系式Vt＝V0e－kt,并根据此式求出酶的半寿期为107min。光照对酶活性有抑制作用。酶的DTNB化学修饰结果表明,在酶活性中心的PLP结合部位很可能有半胱氨酸残基存在。