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81.
James Kang William Low Thomas Norberg Jill Meisenhelder Karin Hansson Johan Stenflo Guo-Ping Zhou Julita Imperial Baldomero M Olivera Alan C Rigby A Grey Craig 《European journal of biochemistry》2004,271(23-24):4939-4949
The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc. 相似文献
82.
Preparation and properties of pure, full-length IclR protein of Escherichia coli. Use of time-of-flight mass spectrometry to investigate the problems encountered. 下载免费PDF全文
L. J. Donald I. V. Chernushevich J. Zhou A. Verentchikov N. Poppe-Schriemer D. J. Hosfield J. B. Westmore W. Ens H. W. Duckworth K. G. Standing 《Protein science : a publication of the Protein Society》1996,5(8):1613-1624
IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue). Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene. 相似文献
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85.
二氢叶酸还原酶结合底物的去除 总被引:1,自引:1,他引:0
分析了应用氨甲蝶呤(MTX-Agarose)亲和层析法提纯的鸡肝二氢叶酸还原酶的组成和性质.建立了用平面粒度胶等电聚焦法去除与酶紧密结合底物的方法.讨论了结合底物对酶构象研究的影响,并指出,用未完全去除结合底物的酶研究酶在变性过程构象变化会得到错误的结论. 相似文献
86.
哺乳动物透明带糖蛋白及其生物活性 总被引:1,自引:0,他引:1
透明带在哺乳动物的受精过程中发挥着重要作用,透明带化学成分及其生物活性的研究对于了解精卵识别、顶体反应的诱导等过程的分子机理具有重要意义.近10年来的研究表明透明带主要由糖蛋白(ZPGPs)组成,是在细胞质内合成后转移至透明带的.大多数动物的ZPGPs为4~5种,不同种类动物的ZPGPs的氨基酸序列存在高度同源性.在受精过程中,ZPGPs具有精子受体和诱导顶体反应的双重作用,ZPGPs含有N-连接和O-连接寡聚糖,这些寡糖链是ZPGPs行使生物活性不可缺少的部分. 相似文献
87.
Morphometric Analysis of Hepatocellular carcinomaLaiMao-de(来茂德);ChenPei-hui(陈培辉)andZhouShui-yun(周水云)(DepartmentofPathology,Zh... 相似文献
88.
利用RT-PCR技术分离低密度脂蛋白受体基因cDNA,将长为2605bp的cDNA插入质粒pJN6,并经全序列测定证实,该序列与已报道序列相比,存在两个变异碱基,即第754位C→T,和1654位的G→A,这两个变异碱基并不改变编码的氨基酸,利用LDL受体基因cDNA中的ClaⅠ片段作为探针,检测到LDL受体基因上外显子8的StuⅠ位点是个限制性片段长度多态性位点。所克隆的cDNA含有可译框架的全部密码,因此可作为基因表达材料。 相似文献
89.
用化学修饰、内源荧光和荧光淬灭等方法研究了油麻藤凝集素(MSL)的溶液构象变化和微环境的构象特征。研究发现MSL分子中总共有9个色氨酸(Trp)残基,它们的荧光能被丙烯酰胺淬灭,但不易为KI接近而淬灭,MSL经N-溴代琥珀酰亚胺(NBS)修饰后,其内源性荧光发射谱发生相应变化,结果表明MSL分子中部分Trp残基埋藏于分子内部,而位于分子表面的Trp残基可能处于分子的疏水袋中。 相似文献
90.
木瓜蛋白酶PPAⅢ自水解作用 总被引:1,自引:0,他引:1
木瓜蛋白酶PPAⅢ自水解作用王秀艳,周慧,葛玉斌,李惟(吉林大学分子生物学系,长春130023)PPAⅢ、papain、pepsine、stembromelain等都属于以Cys-SH为活性中心的酶,同种分子之间存在着一种相互切割的趋势,称为酶自水解... 相似文献