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The catalytic mechanism of glutamyl-tRNA synthetase of Escherichia coli. Evidence for a two-step aminoacylation pathway, and study of the reactivity of the intermediate complex. 大肠杆菌谷氨酰胺- trna合成酶的催化机制。两步氨基酰化途径的证据,以及中间复合物反应性的研究。
Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1980.TB06004.X
D. Kern, J. Lapointe
The sequence of substrate binding and of end-product dissociation at the steady state of the catalytic process of tRNAGlu aminoacylation by glutamyl-tRNA synthetase from Escherichia coli has been investigated using bisubstrate kinetics, dead-end and end-product inhibition studies. The nature of the kinetic patterns indicates that ATP and tRNAGlu bind randomly to the free enzyme, whereas glutamate binds only to the ternary enzyme · tRNAGlu· ATP complex. Binding of ATP to the enzyme hinders that of tRNAGlu and vice versa. After interconversion of the quaternary enzyme · substrates complex the end-products dissociate in the following order: PPi first, AMP second and Glu-tRNA last. In addition to its role as substrate and as effector with ATP for the binding of glutamate, tRNAGlu promotes the catalytically active enzyme state. Whereas at saturating tRNAGlu concentration the catalysis is rate-determining, this conformational change can be rate-determining at low tRNAGlu concentrations. The results are discussed in the light of the two-step aminoacylation pathway catalyzed by this synthetase.
利用双底物动力学、终端和终端产物抑制研究,研究了大肠杆菌谷氨酰胺- trna合成酶催化tRNAGlu氨基酰化过程中底物结合和终端产物解离的顺序。动力学模式的性质表明,ATP和tRNAGlu随机结合到游离酶上,而谷氨酸只结合到三元酶·tRNAGlu·ATP复合物上。ATP与酶的结合阻碍了tRNAGlu的结合,反之亦然。在季酶·底物复合物相互转化后,最终产物按以下顺序解离:PPi首先,AMP其次,Glu-tRNA最后。除了作为底物和与ATP结合的效应物外,tRNAGlu还促进了催化活性酶的状态。而在饱和tRNAGlu浓度下,催化作用是速率决定的,在低tRNAGlu浓度下,这种构象变化可以是速率决定的。根据该合成酶催化的两步氨基酰化途径对结果进行了讨论。
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引用次数: 23
ADP-ribosylated histone H1. Isolation from Ehrlich-ascites-tumor-cell nuclei and partial characterization. adp核糖化组蛋白H1。埃利希-腹水-肿瘤细胞核分离及部分鉴定。
Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1981.TB06173.X
H. C. Braeuer, P. Adamietz, U. Nellessen, H. Hilz
Conjugates between histone H1 and (ADP-ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion-exchange and boronate-cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein-bound ADP-ribose residues, and about 1% of the histone H1 population. Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP-ribose residues. The neighboring bands appeared to differ from cach other by a single ADP-ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer. Studies on the lability of the (ADP-ribose)n protein linkage showed that about 20% of the ADP-ribose was linked by an NH2OH/NaOH-sensitive bond, 70% by an NH2OH-resistant/NaOH-sensitive bond, and the residual 10% apparently by an additional, NaOH-resistant bond. Cleavage of the (ADP-ribose)n- histone H1 conjugates by N-bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.
在埃利希腹水肿瘤细胞的分离细胞核中形成的组蛋白H1和(adp -核糖)n之间的偶联物,使用高氯酸萃取、离子交换和硼酸-纤维素色谱法纯化了未修饰的H1。分离的偶联物占总蛋白结合adp核糖残基的0.6%,约占组蛋白H1群体的1%。酸/尿素凝胶的电泳分析显示,多个组分的迁移速度比未修饰的组蛋白H1慢。它们可以通过蛋白质染色或[3H] adp核糖残基的荧光成像可见。邻近的条带似乎因一个adp核糖残基而彼此不同。在大多数制剂中发现平均链长为2-3。用碱或磷酸二酯酶处理去除adp -核糖基,将条带移至未修饰的组蛋白H1的位置。在高分辨率的凝胶上,能带分裂成双峰,代表不同程度的磷酸化。在非adp核糖基化的对照组中也观察到相同的微观异质性。这表明组蛋白H1的磷酸化对adp -核糖基转移的受体性质没有显著影响。对(adp -核糖)n蛋白连锁的稳定性研究表明,约20%的adp -核糖由一个NH2OH/ naoh敏感键连接,70%由一个耐NH2OH/ naoh敏感键连接,剩余的10%显然由一个额外的耐naoh键连接。用n-溴琥珀酰亚胺切割(adp -核糖)n-组蛋白H1偶联物,并对这两个片段进行凝胶电泳分析,结果显示到目前为止,大多数adp -核糖残基(可能在多个位点)连接到c端片段。此外,大部分缀合物只在c端片段或n端片段上携带adp -核糖基。
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引用次数: 16
A cysteine-rich collagenous protein from bovine placenta. Isolation of its constituent polypeptide chains and some properties of the non-denatured protein. 从牛胎盘中提取的富含半胱氨酸的胶原蛋白。其组成多肽链的分离及非变性蛋白的一些性质。
Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1981.TB06165.X
R. Jander, J. Rauterberg, B. Voss, D. von Bassewitz
A high-molecular-weight collagenous protein was isolated, after limited pepsin digestion, from the endometrial villi of bovine placenta; it resembled the protein isolated from human aortas by Chung et al. [Chung, E., Rhodes, R. K. and Miller, E. J. (1976) Biochem. Biophys. Res. Commun. 71, 1167–1174] and from human placenta by Furuto and Miller [Furuto, D. K. and Miller, E. J. (1980) J. Biol. Chem. 255, 290–295]. After denaturation and reductive cleavage of disulfide bridges three chains with Mr of 40000–55000 were liberated. Two of the chains could be separated by ion-exchange chromatography; the third chain aggregated under non-denaturing conditions and could be isolated by sequential molecular-sieve chromatography in denaturing and non-denaturing buffers. Their amino acid composition resembled that of basement membrane collagens with respect to the high content of completely glycosylated hydroxylysine but differed with respect to their relatively low hydroxyproline content and high values of cysteine, tyrosine and glucosamine. A relatively low glycine content indicated regions of non-collagenous structure. Reduction under non-denaturing conditions and another pepsin treatment removed non-collagenous regions containing mainly hydrophobic amino acid residues. The denaturation temperature of the component was considerably reduced by this treatment, indicating stabilization of triple-helical structures by disulfide bridges in the native molecule. Fibrillar aggregates were precipitated with ATP which showed in the electron microscope a typical crossstriation pattern different to those of segment-long-spacing crystallites obtained from other collagen types. Immunofluorescence tests demonstrated the protein in blood vessels and interstitial areas of the renal medulla but not in basement membranes of glomeruli and tubuli.
从牛胎盘的子宫内膜绒毛中分离出一种高分子量的胶原蛋白,经胃蛋白酶有限消化;它类似于Chung等人从人类主动脉中分离出的蛋白质[Chung, E., Rhodes, R. K. and Miller, E. J. (1976) Biochem]。Biophys。Furuto, D. K. and Miller, E. J. (1980) J. Biol。化学学报,1999,19(2):1 - 8。二硫化桥经过变性和还原裂解后,释放出Mr为400000 ~ 55000的3条链。其中两条链可以通过离子交换色谱分离;第三链在非变性条件下聚集,可以在变性和非变性缓冲液中通过顺序分子筛色谱分离。它们的氨基酸组成与基底膜胶原相似,全糖基化羟赖氨酸含量高,但羟脯氨酸含量相对较低,半胱氨酸、酪氨酸和氨基葡萄糖含量高。相对较低的甘氨酸含量表明非胶原结构区域。非变性条件下的还原和另一种胃蛋白酶处理去除了主要含有疏水性氨基酸残基的非胶原区域。通过这种处理,组分的变性温度大大降低,表明天然分子中的二硫桥稳定了三螺旋结构。纤维聚集体被ATP沉淀,在电子显微镜下显示出不同于从其他胶原类型中获得的片段长间距晶体的典型交叉条纹模式。免疫荧光试验显示,该蛋白存在于肾髓质的血管和间质区,但不存在于肾小球和肾小管的基底膜。
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引用次数: 62
Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4. 遗传过程的能量供应研究。T4噬菌体感染大肠杆菌对膜电位的要求。
Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1983.TB07126.X
E. Kalasauskaĭte, D. Kadisaite, R. Daugelavičius, L. Grinius, A. Jasaitis
In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.
在本研究中,考虑到T4噬菌体DNA注射到大肠杆菌细胞中需要电化学质子梯度的假设得到了实验验证。噬菌体引起了细胞膜的可逆去极化,而噬菌体“幽灵”则引起了不可逆的去极化。当噬菌体/细胞比大于5时,噬菌体感染严格依赖于大肠杆菌膜电位值。当该比值接近1时,膜电位下降至-100 mV时,对噬菌体感染几乎没有影响。当膜电位低于该“阈值”时,观察到感染抑制。另一方面,在促进跨膜pH梯度值同时增加的条件下,膜电位的降低对噬菌体感染没有影响。通过关闭呼吸链(在这些突变细胞中唯一产生原动力的细胞),通过atp酶缺陷细胞的膜的噬菌体DNA转移被可逆地抑制。在噬菌体DNA进入细胞期间,膜应保持在通电状态。噬菌体在大肠杆菌上吸附后,呼吸控制因子可逆释放。因此,本文的结果表明,噬菌体T4 DNA注入大肠杆菌需要跨质膜的电化学质子梯度。他们支持宿主细胞代谢能量消耗的概念,噬菌体T4 DNA通过膜转移。
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引用次数: 40
Chicken-gizzard actin. Interaction with skeletal-muscle myosin. 鸡肫肌动蛋白。与骨骼肌肌球蛋白的相互作用。
Pub Date : 2005-03-03 DOI: 10.1111/J.1432-1033.1980.TB06024.X
E. Próchniewicz, H. Strzelecka-Gołaszewska
Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.
通过测定鸡胗肌动蛋白和兔骨骼肌肌动蛋白与兔骨骼肌肌球蛋白的超沉淀率、肌球蛋白和重肌球蛋白的mg -ATP酶活性和k -ATP酶活性的抑制,以及测定在没有ATP的情况下重肌球蛋白的结合情况,比较鸡胗肌动蛋白和兔骨骼肌肌球蛋白与兔骨骼肌肌球蛋白的相互作用。混合肌动蛋白的超沉淀率和肌动蛋白对肌动蛋白atp酶的激活率均低于骨骼肌肌动蛋白。两种肌动蛋白对肌球蛋白mg -ATP酶的激活也表现出对底物浓度的不同依赖性:对于砂眼肌动蛋白,底物抑制始于较低的ATP浓度。重肌凝蛋白的mg - atp酶活性与肌动蛋白浓度的双倒数图得出两种肌动蛋白在无限肌动蛋白浓度(V)下的外推atp酶活性值相同,而对于砂眼肌动蛋白,则几乎是产生半最大激活(Kapp)所需的肌动蛋白浓度的两倍。在没有二价阳离子的情况下,两种肌动蛋白抑制重肌球蛋白k - atp酶活性的能力也存在相应的差异。讨论了砂囊肌和骨骼肌肌动蛋白氨基酸序列的改变对它们与肌球蛋白相互作用的影响。
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引用次数: 20
Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana. 拟南芥新型磷脂酶A1 cDNA在酵母中的表达。
Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04317.x
Alexandre Noiriel, Pierre Benveniste, Antoni Banas, Sten Stymne, Pierrette Bouvier-Navé

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.

在寻找编码植物甾醇酰基转移酶的cdna过程中,我们从拟南芥中分离出4个全长cdna,它们编码与动物卵磷脂基本相同的蛋白质:胆固醇酰基转移酶(LCATs)。在各种酵母菌株中表达其中一种cdna, atcat3 (At3g03310),导致三酰甘油含量增加一倍。此外,对转化的野生型酵母进行全脂质分析表明,其磷脂含量低于空白质粒转化的酵母,而溶血磷脂和游离脂肪酸含量增加。当来自atlcat3转化酵母的微体与二-[1-14C]油基磷脂酰胆碱孵育时,溶血磷脂和游离脂肪酸部分都被高度标记,而与来自对照酵母的微体孵育时,这些部分的标记可以忽略不计。此外,当将转化为AtLCAT3的酵母微体与sn-1-或sn-2-[1-14C]酰基磷脂酰胆碱孵育时,游离脂肪酸和溶血磷脂酰胆碱组分之间的标记分布强烈表明AtLCAT3具有磷脂酶A1活性。通过对1-肉豆蔻酰基,2-油基磷脂酰胆碱水解的气相色谱分析证实了该磷脂酶的sn-1特异性。磷脂酰乙醇胺和磷脂酸也可被AtLCAT3水解,但效率低于磷脂酰胆碱。溶血磷脂酰胆碱是弱底物,而三棕榈酰甘油和油酸胆固醇则完全不被水解。这种新的拟蓝藻磷脂酶A1在pH 6-6.5和60-65℃时表现出最佳活性,并且似乎不受Ca2+的影响。它的序列与所有其他已知的磷脂酶无关。进一步的研究正在进行中,以阐明其生理作用。
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引用次数: 39
Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-beta gene promoter. 干扰素调节因子(IRF)-3和IRF-7在干扰素- β基因启动子激活中的转录协同作用机制。
Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04310.x
Hongmei Yang, Gang Ma, Charles H Lin, Melissa Orr, Marc G Wathelet

The interferon-beta promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-kappaB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-beta gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-beta promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-beta promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-beta gene depends on a unique promoter context and on the role played by coactivators as architectural factors.

干扰素- β启动子作为组合转录调控的模型系统已被广泛研究。在病毒感染的细胞中,转录因子ATF-2、c-Jun、干扰素调节因子(IRF)-3、IRF-7和NF-kappaB以及共激活因子p300/CBP在这一启动子和其他启动子的激活中发挥关键作用。然而,目前尚不清楚为什么AP-1、IRF和Rel蛋白的大多数其他组合不能激活干扰素- β基因。在这里,我们探讨了不同的irf如何与其他因子合作来激活转录。首先,我们发现在未分化的胚胎癌细胞中,IRF-3或IRF-7的异位表达,而不是IRF-1,足以允许干扰素- β启动子的病毒依赖性激活。此外,IRF-3和IRF-7的活性受到启动子环境的强烈影响,IRF-7优先被招募到天然干扰素- β启动子中。我们在昆虫细胞中完全重建了该启动子的激活。最大的协同作用需要IRF-3和IRF-7,但不需要IRF-1,并且强烈依赖于p300/CBP的存在,即使这些共激活因子本身仅轻微影响每个因子的活性。这些结果表明,干扰素- β基因激活的特异性取决于独特的启动子环境和共激活因子作为结构因子所起的作用。
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引用次数: 54
Ras oncogene induces beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter. Ras癌基因通过ralgef介导的信号向其管家启动子诱导β -半乳糖苷α 2,6-唾液转移酶(ST6Gal I)。
Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04284.x
Martin Dalziel, Fabio Dall'Olio, Arron Mungul, Véronique Piller, Friedrich Piller

Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection of codon 12 point mutated H-Ras increases CMP-Neu5Ac: Galbeta1,4GlcNAc alpha2,6-sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pathways (Raf, RalGEFs and PI3K) and that transcriptional control of mouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted to identify which of these parameters are involved in linking the Ras signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K-Ras or H-Ras (S12 and V12 point mutations, respectively) results in a 10-fold increase in ST6Gal I mRNA, but no alteration in the expression of related sialyltransferases. Using an inducible H-RasV12 expression system, a direct causal link between activated H-Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied by an increase of alpha2,6-sialyltransferase activity and of Neu5Acalpha2,6Gal at the cell surface. Results obtained with H-RasV12 partial loss of function mutants H-RasV12S35 (Raf signal only), H-RasV12C40 (PI3-kinase signal only) and H-RasV12G37 (RalGEFs signal only) suggest that the H-Ras induction of the mouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H-RasV12 or K-RasS12 transfection is mediated by the Siat1 housekeeping promoter P3-associated 5' untranslated exons.

已知几种致癌蛋白影响细胞糖基化。特别是,转染密码子12点突变的H-Ras增加了啮齿动物成纤维细胞中CMP-Neu5Ac: Galbeta1,4GlcNAc alpha2,6-唾液转移酶I (ST6Gal I)的活性。考虑到Ras通过至少三种次级效应通路(Raf、ralgef和PI3K)介导其作用,以及小鼠ST6Gal I的转录控制是通过选择性使用多个启动子实现的,我们试图确定这些参数中哪些参与了Ras信号与小鼠成纤维细胞中ST6Gal I基因转录的联系。人K-Ras或H-Ras(分别为S12和V12点突变)的转化导致ST6Gal I mRNA增加10倍,但相关唾液转移酶的表达没有改变。通过诱导H-RasV12表达系统,证实了H-Ras表达激活与ST6Gal I mRNA升高之间的直接因果关系。ST6Gal I转录物在Ras激活下的积累伴随着细胞表面α - 2,6-唾液基转移酶活性和neu5acα - 2,6gal活性的增加。H-RasV12部分功能缺失突变体H-RasV12S35(仅Raf信号)、H-RasV12C40(仅pi3激酶信号)和H-RasV12G37(仅RalGEFs信号)的结果表明,H-Ras诱导小鼠ST6Gal I基因(Siat1)转录主要通过RalGEFs进行。5'- cDNA末端快速扩增分析表明,H-RasV12或K-RasS12转染后ST6Gal I mRNA的增加是由Siat1管家启动子p3相关的5'未翻译外显子介导的。
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引用次数: 52
Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes. 墨西哥利什曼原虫半胱氨酸蛋白酶CPB亚型之间底物特异性的差异是由一些氨基酸变化介导的。
Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04311.x
Maria A Juliano, Darren R Brooks, Paul M Selzer, Hector L Pandolfo, Wagner A S Judice, Luiz Juliano, Morten Meldal, Sanya J Sanderson, Jeremy C Mottram, Graham H Coombs

The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.

墨西哥利什曼原虫的CPB基因编码阶段调控的组织蛋白酶l样半胱氨酸蛋白酶,该蛋白酶是重要的毒力因子,由19个基因串联而成。在本研究中,我们比较了CPB两种同工酶CPB2.8和CPB3以及后者的H84Y突变体对底物的偏好,以分析这两种同工酶之间的氨基酸差异在决定底物特异性方面所起的作用。CPB3与CPB2.8在成熟结构域只有三个残基(N60D, D61N和D64S)不同。H84Y突变模拟了另一种同工酶CPB18中存在的额外变化。利用大肠杆菌制备活性重组CPB同工酶和突变体,并利用一系列荧光肽衍生物测定S1-S3和S1'-S3'亚位点特异性,其中P3至P3'位置被天然氨基酸取代。利用肽Abz-FRAK(Dnp)-OH及其类似物观察了CPB3和H84Y的羧基二肽酶活性。CPB3、H84Y和CPB2.8对合成底物的水解动力学参数表明,在60、61、64和84氨基酸上的修饰对S3到S3'亚位的特异性有很大影响。特别值得注意的是,CPB3的水解活性在P2'位置上更倾向于Pro,这可能与L. mexicana cpb的激活机制有关。
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引用次数: 24
Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines. 不同人类线粒体dna缺失细胞系中活性氧的抗氧化防御和体内平衡。
Pub Date : 2004-09-01 DOI: 10.1111/j.1432-1033.2004.04298.x
Lodovica Vergani, Maura Floreani, Aaron Russell, Mara Ceccon, Eleonora Napoli, Anna Cabrelle, Lucia Valente, Federica Bragantini, Bertrand Leger, Federica Dabbeni-Sala

Three pairs of parental (rho+) and established mitochondrial DNA depleted (rho0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the rho0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone rho0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle rho0 cell lines but not in lung rho0 cells. GSH peroxidase activity was four times higher in all three rho0 cell lines in comparison to the parental rho+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived rho0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other rho+ and rho0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone rho0 cells originate from sources other than mitochondria.

本研究利用来自骨、肺和肌肉的三对亲本(rho+)和已建立的线粒体DNA缺失(rho0)细胞来验证核背景和缺乏有效线粒体呼吸链对细胞内活性氧(ROS)的抗氧化防御和稳态的影响。线粒体DNA缺失显著降低了所有rho0细胞系中谷胱甘肽还原酶活性和谷胱甘肽(GSH)含量,并持续改变了GSH2:氧化谷胱甘肽的比例,尽管程度不同,表明骨rho0细胞中氧化氧化还原状态最严重。骨和肌rho0细胞超氧化物歧化酶活性、基因表达和蛋白含量均下降,肺rho0细胞无明显变化。GSH过氧化物酶活性在所有3个rho0细胞系中比亲本rho+细胞系高4倍,这表明这可能是在没有功能呼吸链的情况下生存的必要适应。综上所述,这些数据表明,缺乏呼吸链会促使细胞以一种组织特异性的方式减少对抗氧化防御的需求,从而使它们面临氧化损伤的主要风险。事实上,与所有其他rho+和rho0细胞相比,骨源性rho0细胞显示出最高的稳态细胞内ROS水平(直接通过2',7'-二氯荧光素测量,或间接通过乌头酶活性测量),无论是否存在葡萄糖。线粒体和细胞质/铁调节蛋白-1乌头酸酶的分析表明,骨rho0细胞的大多数ROS来源于线粒体以外的来源。
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引用次数: 56
期刊
European journal of biochemistry
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