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应用原紫外CD光谱、内源荧光光谱以及ANS荧光光谱探究了Ca~(2+)对钙调神经磷酸酶(CN)调节亚基(CNB)结构和功能的影响.结果显示:Ca~(2+)能够激活CN磷酸酶活性提高到约1.5倍;CD谱显示结合Ca~(2+)后CNB出现α-螺旋含量上升、无卷曲含量下降、分子有序性升高等现象;!源荧光光谱显示结合Ca~(2+)导致CNB结构更加紧凑;ANS荧光光谱显示Ca~(2+)结合导致其疏水性增强.结合Ca~(2+)后,CNB分子有序性升高,结构更加紧凑,疏水表面暴露.这些结构变化促进了其与CNA的结合并激活了CNA的磷酸酶活性. 相似文献
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肖方祥 《湖北民族学院学报(自然科学版)》1997,15(3):65-68
钙调神经磷酸酶是一种依赖Ca^2 -CaM的磷蛋白磷酸酶。主要存在于脑组织神经元中,由催化亚基A和调节亚基B1:1组成。钙调神经磷酸酶是一个侈底物的磷蛋白磷酸酶。它的活性还受到Ni^2 和Mn^2 等金属离子的调节。 相似文献
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重组人CNB高表达大肠杆菌已由本室构建成功,摇瓶发酵液表达量可达100mg·L-1. 通过改进培养基配方及摇瓶表达工艺,使CNB在摇瓶发酵液中表达量达到500mg*L-1以上.在高表达量的基础上,又进行了产业化改进,以蒸馏水添加无机盐取代了原配方中的自来水,并提高了表达量. 相似文献
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应用1,3,4,6-四氯-3α,6α二苯基苷尿(Iodogen)法对钙调神经磷酸酶B亚基进行1125I标记,获得了比活度为740MBq.mg^-1,放化纯度为95.7%的125I标记的钙调神经磷酸酶B亚基,标记率高达85%,利用该标记物研究血脑屏障对其进入脑的阻碍作用,结果显示血脑屏蔽能够阻止完整的钙调神经磷酸酶B亚进入脑,但是它的降解肽段能够透过血脑屏障进入脑组织。 相似文献
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HPLC和MALDI-TOF-MS法分析重组人钙调磷酸酶B亚基的纯度和肽图谱 总被引:3,自引:0,他引:3
用反相高效液相色谱(RP-HPLC)法对重组人钙调磷酸酶B亚基(rhCNB)的纯度和肽图谱进行了测定,以比较不同批次产品的纯度及其一级结构的一致性,对其进行质量控制.并用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)法进行验证.结果表明,运用梯度洗脱RP-HPLC法能对样品进行纯度检查和肽图谱分析,测得3批样品纯度均达到98%以上,肽图谱能达到批间一致.同时用MALDI-TOF-MS法测定,纯度结果二者相符,质谱肽图经计算机辅助分析,确证rhCNB一级结构与天然CNB一致.证明该方法灵敏、准确、可靠. 相似文献
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研究在压力负荷下阿托伐他汀对钙调神经磷酸酶介导的心肌肥大的影响.选用SD大鼠随机分为3组:假手术组(n=10)、单纯模型组(n=10)和阿托伐他汀组(n=10).大鼠通过腹主动脉缩窄建立压力超负荷模型,8周后测定左室重量指数,B超检测左室形态结构,Westernblot检测心肌CaN蛋白表达,RT-PCR法检测心肌CaNmRNA水平.结果①单纯模型组和阿托伐他汀组心肌肥厚指数明显高于假手术组,阿托伐他汀组心肌肥厚指数明显低于单纯模型组(P<0.05).②单纯模型组和阿托伐他汀组心肌CaN蛋白及CaNmRNS表达水平高于假手术组(P<0.05),阿托伐他汀组低于单纯模型组 (P<0.05).说明阿托伐他汀可能参与干预钙调神经磷酸酶介导的通路,从而抑制心肌肥厚的形成. 相似文献
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钙调磷酸酶B类似蛋白互作蛋白激酶(CIPKs)在植物生长发育和抗逆过程中发挥着重要作用。为了对桃中CIPK家族基因进行系统分析,利用桃基因组数据库,通过生物信息学手段,鉴定桃CIPK家族基因的基因结构、染色体定位和编码蛋白,通过序列比对进行进化和分类分析。结果表明,桃基因组中含有18个CIPK基因,分布于桃的6条染色体上。MEME和Pfam保守结构域分析显示,桃CIPK蛋白均含有2个保守的PKinase和NAF结构域。进化树分析表明CIPKs可分为2个亚家族。Net Phos 2.0 Server结果显示Pp CIPKs存在着大量的丝氨酸(Ser)、苏氨酸(Thr)及酪氨酸(Tyr)潜在磷酸化位点。以上结果将为今后揭示桃CIPK蛋白的功能提供重要的理论基础。 相似文献
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AceK是具有激酶与磷酸酶活性的双功能酶。本文研究了金属离子Mg~(2+)、Fe~(2+)、Ni~(2+)、Zn~(2+)对AceK激酶活性及磷酸酶活性的影响。实验表明Mg~(2+)是AceK激酶与磷酸酶激活剂,对激酶最佳激活浓度为2mmol·L~(-1),磷酸酶为5mmol·L~(-1)。但Fe~(2+)、Ni~(2+)、Zn~(2+)单独存在时,对AceK激酶、磷酸酶活性均没有明显作用,反而能较强地抑制Mg~(2+)激活的AceK酶活性,对激酶抑制作用由强到弱为Zn~(2+)、Ni~(2+)、Fe~(2+),对磷酸酶抑制作用由强到弱为Ni~(2+)、Fe~(2+)、Zn~(2+)。研究内容和结果为进一步研究AceK催化机制和结构功能打下了一定的基础。 相似文献
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探讨了不同浓度处理的无机盐CaCl2和CoNO32溶液对瓶插百合切花保鲜效果的影响.结果表明:CaCl2和CoNO32配合使用可以使百合切花花枝硬挺,花径增大,改善切花的观赏品质,延长瓶插寿命;同时,还具有保持百合切花花枝鲜重、维护花瓣细胞膜透性的稳定、维持瓶插溶液pH值保持在适合的浓度范围之间、缓解叶绿素的降解等作用.通过对各处理的各项指标的综合观测与分析可知,以0.1%CaCl2和0.05‰CoNO32溶液的配合使用对百合切花的保鲜效果最好. 相似文献
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A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis 总被引:3,自引:0,他引:3
The refined 1.9-A resolution structure of the periplasmic D-galactose-binding protein (GBP) reveals a calcium ion surrounded by seven ligands, all protein oxygen atoms. A nine-residue loop (amino-acid positions 134-142), which is preceded by a beta-turn and followed by a beta-strand, provides five ligands from every second residue. The last two ligands are supplied by the carboxylate group of Glu 205. The entire GBP Ca2+-binding site adopts a conformation very similar to the site in the 'helix-loop-helix' or 'EF-hand' unit commonly found in intracellular calcium-binding proteins, but without the two helices. Structural analyses have also uncovered the sugar-binding site some 30 A from the calcium and a site for interacting with the membrane-bound trg chemotactic signal transducer approximately 45 A from the calcium. Our results show that a common tight calcium binding site of ancient origin can be tethered to different secondary structures. They also provide the first demonstration of a metal-binding site in a protein which is involved in bacterial active transport and chemotaxis. 相似文献
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Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase 总被引:13,自引:0,他引:13
Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module. 相似文献
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Structure of the 30S ribosomal subunit 总被引:83,自引:0,他引:83
Wimberly BT Brodersen DE Clemons WM Morgan-Warren RJ Carter AP Vonrhein C Hartsch T Ramakrishnan V 《Nature》2000,407(6802):327-339
Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography. 相似文献
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In Dictyostelium discoideum, carbohydrate binding proteins (CBPs) or lectins have been implicated in the molecular basis of cellular cohesion. To determine the role of these CBPs, we have attempted to isolate structural gene mutants in which the CBPs have a defective affinity for carbohydrate ligands. We now report the isolation of a spontaneous, cross-reacting material (CRM) mutant which is non-cohesive and fails to develop. The mutant seems to have a defect in the structural gene for one of the two developmentally regulated carbohydrate binding proteins (CBP-26), which renders it unable to bind to galactose-containing ligands. The fact that wild-type cells interact with the mutant and carry it through development strongly supports a model of cell-cell interaction in which cohesion is mediated by complementary molecules. 相似文献
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The guanosine binding site of the Tetrahymena ribozyme 总被引:43,自引:0,他引:43
The self-splicing Group I introns have a highly specific binding site for the substrate guanosine. Mutant versions of the Tetrahymena ribozyme have been used in combination with guanosine analogues to identify the nucleotide in the ribozyme that is primarily responsible for recognition of the guanine base. 相似文献
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Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy 总被引:21,自引:0,他引:21
J Kallen C Spitzfaden M G Zurini G Wider H Widmer K Wüthrich M D Walkinshaw 《Nature》1991,353(6341):276-279
The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs. 相似文献
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