蛹虫草金属硫蛋白分离纯化及性质研究

研究探讨锌离子胁迫下蛹虫草Cordyceps militaris金属硫蛋白的产生及性质。蛹虫草菌丝体以15g/L Zn2+在10L发酵罐中诱导培养56h后收集,产率为每升发酵液收集12.021g菌丝体（干重）,细胞破碎取上清液通过两次凝胶柱层析,冷冻干燥得到蛹虫草金属硫蛋白纯品。利用Bradford法进行蛋白质含量测定,用银饱和分析法结合原子吸收光谱（AAS）测定MT含量,发酵终点处金属硫蛋白含量为12.876mg/g菌丝体（湿重）。用电喷雾质谱法测得金属硫蛋白的分子量为7 390Da,用Ellman’s方法和火焰原子吸收法分别测得每分子蛋白质含有14个巯基、结合5个Zn原子。氨基酸组成分析结果显示,每分子蛋白质共含57个氨基酸,其中含有13个半胱氨酸,疏水氨基酸占29.8%,且含有组氨酸。以上表明,研究中的蛹虫草金属硫蛋白与哺乳动物金属硫蛋白结构差异较大,但与酵母菌金属硫蛋白结构组成类似。     

古尼虫草与亚香棒虫草亲缘关系初探

目的：用分子生物学方法,对古尼虫草和亚香棒虫草进行研究,对其寄主昆虫COI（cytochrome oxidase subunit I,细胞色素氧化酶亚基I）和真菌ITS（Internal Transcribed Sequence,内转录间隔区）区的基因序列进行比较,以确定两者亲缘关系。方法：在古尼虫草和亚香棒虫草性状研究的基础上,对两者来源真菌ITS区和寄主昆虫COI基因进行了PCR扩增和序列测定,对序列进行比对分析,并与GenBank核酸序列数据库中的序列进行BLAST检索比对。结果：发现古尼虫草和亚香棒虫草的来源真菌ITS区和寄主昆虫COI基因序列均有较高相似度。结论：古尼虫草和亚香棒虫草有较近的亲缘关系。     

古尼虫草与亚香棒虫草亲缘关系初探

目的:用分子生物学方法,对古尼虫草和亚香棒虫草进行研究,对其寄主昆虫CO(Icytochrome oxidase subunit I,细胞色素氧化酶亚基I)和真菌ITS(Internal Transcribed Sequence,内转录间隔区)区的基因序列进行比较,以确定两者亲缘关系。方法:在古尼虫草和亚香棒虫草性状研究的基础上,对两者来源真菌ITS区和寄主昆虫COI基因进行了PCR扩增和序列测定,对序列进行比对分析,并与GenBank核酸序列数据库中的序列进行BLAST检索比对。结果:发现古尼虫草和亚香棒虫草的来源真菌ITS区和寄主昆虫COI基因序列均有较高相似度。结论:古尼虫草和亚香棒虫草有较近的亲缘关系。     

鲫鱼金属硫蛋白的提纯及性质研究

用氯化镉诱导鲫鱼(Carassius aurattus),应用层析介质 Sephacryl S 体系从鱼肝脏中提取金属硫蛋白(MT),并对其性质进行研究.实验表明鲫鱼 MT 的性质与哺乳动物 MT 的性质十分相似.分子量约10 000,含丰富的半胱氨酸,较多的赖氨酸,不含芳香族氨基酸.紫外250nm 处强吸收,280nm 处几乎没有吸收。鱼 MT 含7个金属每分子,并含有20个巯基.测得鲫鱼 MT 等电点 pI 在5.6左右.     

聚球藻类金属硫蛋白的纯化及部分性质的研究

单细胞蓝藻（Ｓｙｎｅｃｈｏｃｏｃｕｓｓｐ．ＰＣＣ７９４２）以５０μｍｏｌ／ＬＺｎ２＋诱导８ｄ后收集,破碎取上清液经凝胶过滤、离子交换层析及反相ＨＰＬＣ纯化得到类金属硫蛋白,产率为每升培养液收集１．５ｇ鲜藻,得２．５ｍｇ纯品．其单体分子量为８７５０,Ｎ未端测定为缬氨酸,氨基酸组成分析得每分子（５６个氨基酸）含１０个半胱氨酸,疏水氨酸较多,且含有芳香族氨基酸,原子吸收光谱测得每分子蛋白结合４个二价金属．以上表明,该种类金属硫蛋白与哺乳动物金属硫蛋白结构差异很大,可能只是一种进化上的趋同．     

镉诱导家兔肝脏含锌金属硫蛋白的纯化及鉴定

预先给家兔皮下注射CdCl_2四次,取肝脏经匀浆、离心、乙醇沉淀后,通过Sephadex G-50凝胶过滤层析,DEAE Sepharose Fast Flow离子交换层析和Sephadex G-25层析柱脱盐,得到MT的两个“亚型”—MT-1和MT-2,对其进行鉴定,其紫外扫描图谱示在280和250nm处无吸收峰,而最大吸收在220nm处。MT-1和MT-2均不含镉和铜,而每分子结合6个锌。用简化巯基试剂测定,MT-1含18.8个巯基,MT-2含19.1个巯基;HPLC分析MT主要以单体存在,也有少量二聚体。氨基酸组成与Cd-MT一致。整个纯化过程采用示波极谱法和火焰原子吸收光谱法测锌含量。结果表明前者可代替后者。     

酵母类金属硫蛋白抗氧化作用的研究

酿酒酵母ＹＢＤ１０１菌株的类金属硫蛋白能清除羟基在。含类金属硫蛋白的活性的干酵母饲喂７个半月龄昆明小鼠,可提高肝中ＳＯＤ活性,降低ＬＰＯ含量,对脑中ＭＡＯ－Ｂ活性有一定抑制作用,对肝中ＭＡＯ－Ｂ活性几乎不抑制。含类Ｃｕ－ＭＴ活怀干酵母对上述衰老有关三项生化指标的作用,与兔肝Ｚｎ－ＭＴ纯品效果一流,提示酵母菌类Ｃｕ－ＭＴ在预防衰老和保护人体健康方面有良好的应用前景     

金属硫蛋白的表达调控及其与锌的关系

金属硫蛋白是普遍存在于人和动物组织中的一种金属结合蛋白,它属于机体抗氧化系统的一部分．在氧化应激导致的细胞病理改变中具有重要的保护作用。金属硫蛋白可由多种因子诱导产生,而且它与金属锌密切相关,二者之间的平衡对维持机体正常的生理功能起着重要的作用。     

金属硫蛋白的生物学特性及生理作用

金属硫蛋白的生物学特性及生理作用朱赓伯（苏州医学院生化教研室,苏州２１５００７）关键词金属硫蛋白金属硫蛋白又名金属硫组氨酸甲基内盐（ｍｅｔａｌｌｏｔｈｉｏｎｅｉｎｓ,ＭＴ）。三十年前美国哈佛大学的Ｂ．Ｌ．Ｖａｌｌｅｅ首次在马的肾脏中发现Ｃｄ－ＭＴ和Ｚ...     

蝉虫草子实体与发酵菌丝体胞内多糖成分分析

比较蝉虫草子实体、孢梗束、虫体以及蝉虫草发酵菌丝体的胞内多糖含量、单糖组成和分子量差异,结果表明,蝉虫草子实体与发酵菌丝中胞内多糖的含量和组成均存在差异。葡萄糖、半乳糖和甘露糖是蝉虫草发酵菌丝体和子实体粗多糖中主要的3种单糖,阿拉伯糖和木糖是子实体粗多糖中的特征性单糖,而果糖属于菌丝体的特征性单糖。子实体粗多糖中高分子量组分（>1×10 ⁶Da）的相对含量明显高于菌丝体粗多糖。本研究对提升蝉虫草发酵多糖产品品质、促进蝉虫草开发应用具有参考价值。     

冬虫夏草菌丝体γ-谷氨酰转肽酶的探测和部分性质研究

本研究首次发现冬虫夏草发酵菌丝体含有较高活力的γ-谷氨酰转肽酶（简称CSGT）,并且通过硫酸铵分级沉淀、疏水层析、凝胶过滤层析、阴离子交换层析和制备电泳的提取纯化程序,将CSGT纯化了2300倍,然后对CSGT的基本酶学性质进行了研究。CSGT的稳定pH范围和温度范围分别为pH8-11和0-20℃, 当pH 9-10 、30℃并且以L-谷氨酸-对-硝基苯胺（简称GpNA）和双甘肽为底物时CSGT的活力达到最大值。几种还原剂均能激活CSGT,说明其活性中心含有巯基。Zn2+, Cu2+, Hg2+ , Mn2+ 等金属离子均强烈抑制CSGT活性,而K+, Ca2+, Mg2+ 和Na+等对CSGT活性没有影响。     

The effect of Zn on the Zn accumulation and biosynthesis of amino acids in mycelia of Cordyceps sinensis

Zinc is an essential trace element in the human body and it participates in various pathways of metabolism. Cordyceps sinensis is a well-known traditional Chinese medicine that contains cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, and many other biological active materials. In this study, we cultured C. sinensis in liquid medium containing Zn ions and then analyzed the biomass, the ratios of total Zn accumulation, and the organic Zn content in the mycelia. The results showed that when the media contain 150 mg/L Zn, the biomass of mycelia of C. sinensis reached 10.7 g/L and the Zn content present in the mycelia reached 4875.1 μg/g. The percentage of Zn accumulated in the mycelia was 34.05% of the total Zn in the media, of which the organic Zn accounted for 76.33%. The results also revealed that the content of total amino acid (TAA) and essential amino acid (EAA) in the mycelia were increased substantially TAA and EAA in the Zn-treated mycelia were 11.1% and 15.2% higher, respectively, than that in the mycelia without Zn treatment. These results will be useful for elucidation of the physiological mechanism of Zn effects on the biological metabolism in the mycelia of C. sinensis.     

A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H(2)O(2)-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H(2)O(2)-induced injury. H(2)O(2)-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H(2)O(2) treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells. Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.     

Four major basic proteins of barley grain. Purification and partial characterization

Four major basic proteins termed C, K, N and Q, which are synthesized very late in grain development, have been isolated from barley ( Hordum vulgare L.) mutant Bomi 1508. Immunoelectrophoretic monitoring assured a high degree of purity after a few ion exchange and gel filtration steps. Charge microheterogeneity of two of the four antigenically distinct proteins was observed. Some physico-chemical properties were determined, including molecular mass (C ∼ 28 000; K ∼ 30 000; N ∼ 11 000; Q ∼ 60000), isoelectric point(s) (C ∼ 9.7; K ∼ 10.1–10.3; N ∼ 9.3; Q ∼ 8.9–9.1 at 25°C), and amino acid composition. In total, the four proteins represent ∼ 5% of the salt-soluble protein in grains of some cultivated barleys. The most basic protein K is rich in lysine (∼ 7.9 mol %) and may account for ∼1% of the grain lysine content in these barleys.     

虫草的抗菌抗肿瘤活性研究进展

虫草代谢产物丰富,很多具有抗菌和（或）抗肿瘤活性,有关其生物活性物质的研究受到广泛关注。对有抗菌和（或）抗肿瘤活性研究报道的虫草作了系统总结。     

Purification and characterization of an acid deoxyribonuclease from the cultured mycelia of Cordyceps sinensis

A new acid deoxyribonuclease (DNase) was purified from the cultured mycelia of Cordyceps sinensis, and designated CSDNase. CSDNase was purified by (NH(4))(2)SO(4) precipitation, Sephacryl S-100 HR gel filtration, weak anion-exchange HPLC, and gel filtration HPLC. The protein was single-chained, with an apparent molecular mass of ca. 34 kDa, as revealed by SDS-PAGE, and an isoelectric point of 7.05, as estimated by isoelectric focusing. CSDNase acted on both double-stranded (ds) and single- stranded (ss) DNA, but preferentially on dsDNA. The optimum pH of CSDNase was pH 5.5 and its optimum temperature 55. The activity of CSDNase was not dependent on divalent cations, but its enzymic activity was inhibited by high concentration of the cation: MgCl(2) above 150 mM, MnCl(2) above 200 mM, ZnCl(2) above 150 mM, CaCl(2) above 200 mM, NaCl above 300 mM, and KCl above 300 mM. CSDNase was found to hydrolyze DNA, and to generate 3-phosphate and 5-OH termini. These results indicate that the nucleolytic properties of CSDNase are essentially the same as those of other well-characterized acid DNases, and that CSDNase is a member of the acid DNase family. To our knowledge, this is the first report of an acid DNase in a fungus.