硫代葡萄糖苷及其降解产物异硫代氰酸盐

硫代葡萄糖苷是一种含硫的次级代谢产物,广泛分布于十字花科植物中。不同的栽培种、不同的生理阶段、不同的组织部位以及不同的栽培条件,都会使植物中含有的硫代葡萄糖苷的含量和成分有所变化。当硫代葡萄糖苷经葡萄糖硫苷酶作用时会发生降解,生成异硫代氰酸盐等产物。采后的一系列处理会影响植物中硫代葡萄糖苷的含量。硫代葡萄糖苷的降解产物异硫代氰酸盐作为一种化学预防剂,能抑制阶段Ⅰ酶（phase Ⅰ enzyme）,诱导阶段Ⅱ酶（phaseⅡenzyme）,从而防止癌症的发生。目前对硫代葡萄糖苷的鉴定方法主要是高效液相色谱法,气相色谱法等。     

黑芥子酶研究进展

黑芥子酶又称黑芥子硫苷酸酶,广泛存在于自然界。它能催化硫代葡萄糖苷水解成为葡萄糖和不稳定的中间物配基,此配基易重排形成异硫氰化合物,硫氰化合物,腈,口恶唑烷硫酮等有毒物质。当植物受到昆虫,哺乳动物或病源伤害时,黑芥子酶与硫代葡萄糖苷混合,释放有毒的水解产物。因此认为黑芥子酶-硫代葡萄糖苷系统是植物重要的防御系统。另一方面,黑芥子酶催化硫代葡萄糖苷所生成的有毒物质,在一定程度上也影响了十字花科油料及蔬菜作物的品质,因此,引起了对黑芥子酶及硫代葡萄糖苷研究的重视。本文仅对黑芥子酶的研究概况作一综述。1　黑芥子酶的…     

植物中硫代葡萄糖苷生物代谢的分子机制

硫代葡萄糖苷是十字花科植物中重要的次生代谢物。它在内源芥子酶作用下水解为具有不同生理功能的活性物质。现从分子水平综述硫代葡萄糖苷生物合成、降解反应及其代谢调控的研究进展,为提高植物抗病性和改善营养品质等方面研究提供一定的理论依据。     

菜籽粕脱毒液中硫代葡萄糖苷提取工艺的研究

本文用两步萃取法从菜籽粕脱毒液中提取硫代葡萄糖苷,以3,5-二硝基水杨酸比色法测定硫代葡萄糖苷含量,采用单因素试验法,分别考察可能影响硫代葡萄糖苷提取效果的各种因素,确定了最佳工艺条件：第一步萃取剂为异辛烷,母液与异辛烷体积比为2：1,搅拌时间为30min,异辛烷萃取次数为2次,异辛烷单次萃取率达71．2％。硫代葡萄糖苷收率为83．7％。     

植物芥子酶研究进展

芥子酶防御系统为白花菜目植物特有．由芥子酶及其底物硫代葡萄糖苷组成．芥子酶和硫代葡糖苷分别储藏在不同的细胞中,在受到病虫侵袭时,底物和酶相遇,硫代葡糖苷被降为有毒化合物,起防御作用．对植物芥子酶防御系统研究进展进行了综述,包括基因家族的结构、基因的表达调控、芥子酶的细胞定位、植物以外其它生物的芥子酶、硫代葡糖苷／芥子酶系统起源进化以及其可能功能等．     

硫代葡萄糖苷合成核心途径与植物生长素微调

硫代葡萄糖苷是刺山柑Capparis spinosa(Capers)和十字花科(Brassicaceae)植物中一类特有和重要的次生代谢产物.在植物体内,硫代葡萄糖苷的合成包括前体氨基酸侧链延伸,核心结构的形成和次级侧链修饰3个步骤.利用核心结构合成途径上有关催化酶的基因突变体证明,后醛肟反应途径上任何催化酶基因的突变,都将导致吲哚乙醛肟结构相似的吲哚乙酸(IAA)含量变化,硫苷核心途径对生长素的动态平衡起着微调(fine-tune)作用.     

西兰花中β-硫代葡萄糖苷总量的测定

通过内源和外源芥子酶酶解硫代葡萄糖苷产生的葡萄糖的测定,间接测出西兰花中β-硫代葡萄糖苷总量,并对测定的影响因素进行了研究,与吡啶滴定法对比实验表明,该法准确可靠,操作简便。     

乙烯拮抗剂—硫代硫酸银的生理作用

近年来,对乙烯在植物体内的代谢和作用,以及乙烯靶细胞和结合位点的研究十分活跃。在这些研究中,有不少是以硫代硫酸银(STS)作为“实验工具”的。本文就STS在研究乙烯生理作用中的主要优点及其抗乙烯的生理效应和可能的生理机制作简要介绍。     

异硫代氰酸盐的抗癌机理及其相关研究

本文概述了异硫代氰酸盐抗癌作用机理的研究进展以及一些相关研究,包括各种采后因子对芸薹属蔬菜活性ITCs产生的影响,ITCs在体内的代谢,以及芥子油苷生物合成的研究及其应用等。     

植物活性成分协同抗生素消减细菌耐药性的研究进展

细菌耐药性问题给人类生命安全带来了前所未有的挑战。常用抗生素对某些耐药性细菌的治疗作用逐渐减弱甚至无效,开发新型化学合成抗菌剂成本高、耗时长、难度大。以植物活性成分如生物碱类、多酚类、萜类、凝集素、皂甙类、硫代葡萄糖苷等化合物为基础的抑菌剂不仅在抗菌等方面具有巨大的潜力,还可以通过多种途径与抗生素发挥协同抗菌作用。本文综述了细菌耐药现状以及植物活性成分协同抗生素对耐药菌的作用及其机制,以期恢复或增强耐药菌对现有抗生素的敏感性,为植物活性成分在消减细菌耐药性的应用领域提供理论基础。     

Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases

The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a beta-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-beta-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant beta-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant beta-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant beta-O-glucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant beta-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position.     

Purification and characterisation of a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.)

Plant myrosinases and glucosinolates constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa+/-500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the Brassica specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase.     

Spatial organization of the glucosinolate-myrosinase system in brassica specialist aphids is similar to that of the host plant.

Secondary metabolites are important in plant defence against pests and diseases. Similarly, insects can use plant secondary metabolites in defence and, in some cases, synthesize their own products. The paper describes how two specialist brassica feeders, Brevicoryne brassicae (cabbage aphid) and Lipaphis erysimi (turnip aphid) can sequester glucosinolates (thioglucosides) from their host plants, yet avoid the generation of toxic degradation products by compartmentalizing myrosinase (thioglucosidase) into crystalline microbodies. We propose that death, or damage, to the insect by predators or disease causes disruption of compartmentalized myrosinase, which results in the release of isothiocyanate that acts as a synergist for the alarm pheromone E-beta-farnesene.     

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore.

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 degrees C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mM, and the kcat as 36 s(-1). With glucotropaeolin the Km and kcat values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degrees C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to beta-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.     

IAN-bildung aus glucobrassicin: pH-Abhängigkeit und wachstumsphysiologische Bedeutung

Summary Indole auxin synthesis during enzymatic hydrolysis of glucobrassicin by myrosinase proved to be strictly dependent on pH. Neither IAN nor other indole compounds with auxin activity are synthesized at pH values higher than 5.2. An in vivo function of the indole glucosinolates as auxin precursors in Cruciferae is therefore of low probability.     

Revised determination of free and complexed myrosinase activities in plant extracts.

The enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147, formerly EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates after tissue damage in plants of the order Brassicales. The various myrosinase isoforms occur either as free soluble dimers or as insoluble complexes. We propose a reliable method for determination of both soluble and insoluble myrosinase activity concentrations in partially purified plant extracts. The procedure requires the removal of endogenous glucosinolates through ion-exchange columns previous to enzyme measurements. Myrosinase activity was assayed in continuous mode by photometric quantification of the released glucose using glucose-oxidase with peroxidase and colorimetric indicators. The measurement of the colored product at 492nm has a favorable signal to noise ratio both in clear extract solutions (free dimers) and in turbid pellet suspensions (insoluble complexes). No interferences by ascorbic acid were found in continuous analyses. With the recommended sample preparation methods and assay conditions potential activities in damaged plant tissues can be characterized which are involved in plant defense mechanisms.     

Induction of plant responses by a sequestering insect: Relationship of glucosinolate concentration and myrosinase activity

Induction of plant allelochemicals is of particular ecological importance for interactions with herbivores that can make use of induced metabolites by incorporating them for their own defence. Induction patterns in white mustard, Sinapis alba, were investigated following herbivory of the turnip sawfly, Athalia rosae, which sequesters plant glucosinolates. Larvae of different age were allowed to feed for 24 h on young leaves of premature, non-flowering plants. Changes in primary and secondary metabolites were recorded in the damaged leaves (local) and in the adjacent leaves and stems (systemic) for several days. Organ- and time-specific patterns were evident. Local responses included increases in glucosinolate concentrations, soluble and insoluble myrosinase activity and glucose levels, while systemic responses in leaves were restricted to increases in myrosinase activities and glucose. All effects were strongest immediately after feeding and declined mostly within a day. Stems had overall lower constitutive levels of glucosinolates and myrosinase activities than leaves. Feeding by one large larva had a greater impact on the plant's physiology than feeding by three small ones, even though both treatments resulted in quantitatively similar leaf destruction. Local increase in glucosinolate concentration could be beneficial for larvae, while conspecifics feeding on induced adjacent leaves might be negatively affected due to higher myrosinase activity levels. The results are discussed in the context of the ‘optimal defence theory’ and the ‘lethal plant defence paradox’.     

Characterisation of aphid myrosinase and degradation studies of glucosinolates

Myrosinase from Brevicoryne brassicae was purified by ammonium sulfate fractionation, dialysis, and chromatography on a DEAE column. The chromatography yielded a single peak and a 115.6-fold purification. Further FPLC gel filtration gave a single peak at 120 kDa. Denaturing SDS/PAGE of the protein revealed a single band at 60 kDa, indicating that the native B. brassicae myrosinase is a dimer. Kinetic parameters towards 8 glucosinolates were calculated. Strong differences of V(max) and K(m) were observed depending on the substrate. Degradation products of each glucosinolate were identified and quantified by GC-MS and GLC-FID, respectively. Using both crude aphid homogenates and purified myrosinase, two unique hydroxyglucosinolates, 3-butenyl- and benzyl-isothiocyanates were identified from progoitrin ((2S)-2-hydroxybut-3-enyl-glucosinolate) and sinalbin (4-hydroxybenzyl-glucosinolate) degradation respectively. Addition of ascorbic acid to the reaction mixtures containing sinalbin and progoitrin caused the production of hydroxylated degradation products usually associated with plant myrosinase metabolisation. The occurrence of the myrosinase system in B. brassicae is discussed in terms of similar allelochemical adaptation between the herbivore and its host plant.     

Sulphate can induce differential expression of thioglucoside glucohydrolases (myrosinases)

Thioglucoside glucohydrolase (EC 3.2.3.1; myrosinase) hydrolyses glucosinolates and thereby liberates glucose and sulphur and nitrogen compounds. To examine the hypothesis that the myrosinase-glucosinolate system is influenced by environmental factors, the effect of sulphate on the expression of myrosinases was examined. On examining different plant organs at various stages, it was observed that sulphate induces a differential expression of myrosinase polypeptides in plants ofSinapis alba L. (white mustard). Specific myrosinase polypeptides, dependent on sulphate in the growth medium, were detected on immunoblots. Without sulphate a maximum of three polypeptides was detected in buds, two in cotyledons and one in stems and roots. In plants cultured on medium with sulphate up to four polypeptides could be observed in cotyledons, five polypeptides in buds, two in stems and one in roots. Expression of myrosinases was, in general, high in plants cultured on a medium supplemented with sulphate. In floweringS. alba plants, sulphate-starved plants showed a higher expression of myrosinase in cotyledons and stems compared to plants fed with sulphate. Sulphate-fed plants had a high expression in inflorescences and roots. The organ- and time-specific induction of the myrosinase expression is discussed in relation to sulphate metabolism and availability of sulphate under normal conditions of cultivation and in relation to protection of Brassicaceae species. This is the first evidence for a specific induction of individual myrosinase proteins.     

Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense

In Arabidopsis and other Brassicaceae, the enzyme myrosinase (beta-thioglucoside glucohydrolase, TGG) degrades glucosinolates to produce toxins that deter herbivory. A broadly applicable selection for meiotic recombination between tightly linked T-DNA insertions was developed to generate Arabidopsis tgg1tgg2 double mutants and study myrosinase function. Glucosinolate breakdown in crushed leaves of tgg1 or tgg2 single mutants was comparable to that of wild-type, indicating redundant enzyme function. In contrast, leaf extracts of tgg1tgg2 double mutants had undetectable myrosinase activity in vitro, and damage-induced breakdown of endogenous glucosinolates was apparently absent for aliphatic and greatly slowed for indole glucosinolates. Maturing leaves of myrosinase mutants had significantly increased glucosinolate levels. However, developmental decreases in glucosinolate content during senescence and germination were unaffected, showing that these processes occur independently of TGG1 and TGG2. Insect herbivores with different host plant preferences and feeding styles varied in their responses to myrosinase mutations. Weight gain of two Lepidoptera, the generalist Trichoplusia ni and the facultative Solanaceae-specialist Manduca sexta, was significantly increased on tgg1tgg2 double mutants. Two crucifer-specialist Lepidoptera had differing responses. Whereas Plutella xylostella was unaffected by myrosinase mutations, Pieris rapae performed better on wild-type, perhaps due to reduced feeding stimulants in tgg1tgg2 mutants. Reproduction of two Homoptera, Myzus persicae and Brevicoryne brassicae, was unaffected by myrosinase mutations.