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1.
Regulation of alpha-ketoglutarate dehydrogenase cooperative properties in substrate binding by thiol-disulfide exchange 总被引:1,自引:0,他引:1
The influence of reducing the KGD non-cooperative form by DTT on the KG binding by the enzyme was investigated. The chemical modification of KGD by DEP has revealed that reduction of KGD cysteine residues results in the appearance of the interaction of the dimer active sites upon the enzyme-substrate complex formation. The reduction of 2 SH-groups per KGD subunit: the most reactive one and a buried one--was established to be sufficient for the appearance of KGD cooperative properties in substrate binding as well as for the change in the enzyme activity plots versus substrate concentration. It is suggested that KGD can be regulated by thiol-disulfide exchange in the cell. 相似文献
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RGD为存在于许多糖蛋白配体中的氨基酸序列,对整合素具有识别作用.此序列也发现于许多蛇毒去整合素分子中.采用基因克隆技术从大连产白眉蝮蛇的毒腺中克隆出的去整合素adinbitor是含73个氨基酸残基的去整合素,分子中含有12个半胱氨酸和RGD模体.实验证明,adinbitor作为去整合素的新成员,具有典型的抗ADP诱导的人血小板聚集作用和抗肿瘤血管新生作用.为了将adinbitor的这2种功能分开,采用PCR基因定点突变的方法,将其cDNA序列中RGD模体改变成KGD.重组adinbitor(KGD)在E.coli BL21得到表达,并通过His8226;Bind亲和层析予以纯化.实验发现,adinbitor对ADP诱导的人血小板聚集具有明显抑制作用,其IC50=85 nmol/L,明显优于adinbitor(RGD) (IC50=150 nmol/L).然而,与adinbitor(KGD)相比,adinbitor(KGD)则丧失了对血管生成的抑制作用.结果说明,adinbitor(KGD)可作为专一的抗人血小板聚集药具有潜在的开发前景. 相似文献
5.
Succinylphosphonate (SP) is a powerful inhibitor of alpha-ketoglutarate dehydrogenase (KGD). Methylation of the phosphonate reduces its inhibitory effect. The complex of KGD with SP undergoes a kinetically slow transition similar to the process observed during catalysis. alpha-Ketoglutarate binds to the enzyme-inhibitor complex, preventing its isomerisation. 相似文献
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The burst of product accumulation during the KGD reaction was investigated. It has been shown not to be the obligatory feature of catalysis, but appears when increasing the enzyme saturation by KG. Structural analogues of KG and the SH-group modification suppress the initial burst without preventing catalysis. The results obtained are in favour of the existence of the regulatory site for binding KG and its structural analogues essential for hysteretic properties of KGD. 相似文献
7.
Pathways of succinyl-Coenzyme A (succinyl-CoA) formation in various photosynthetic bacteria were investigated through several approaches, including determination of activity levels of relevant enzymes. Extracts of photosynthetically grown cells of representative Rhodospirillaceae and Chromatium vinosum showed -ketoglutarate dehydrogenase (KGD) activities sufficient to account for generation of the succinyl-CoA required for biosynthetic metabolism. Except as noted below, the observed ratios of fumarate reductase/succinate dehydrogenase activities were low, consistent with the conclusion that these organisms produce succinyl-CoA oxidatively from -ketoglutarate (KG), rather than by reductive metabolism of fumarate. On the other hand, the green bacterium Chlorobium limicola appears to produce succinyl-CoA by the reductive pathway; in this organism, KGD activity could not be detected, and a high fumarate reductase/succinate dehydrogenase ratio was observed. Results obtained with Rhodopseudomonas gelatinosa suggest that this otherwise typical member of the Rhodospirillaceae may be able to generate succinyl-CoA via both arms of the citric acid cycle, that is, oxidatively from KG, and reductively from fumarate. To further explore the several physiological roles of the conversion: KGsuccinyl-CoA in Rhodopseudomonas capsulata, a mutant (strain KGD 11) almost completely blocked in KGD activity was isolated and studied in detail. Under anaerobic photosynthetic conditions, KGD 11 grows readily on succinate as the sole carbon source; in contrast to the wild type parent, however, it cannot grow with l-glutamate as the source of carbon. The R. capsulata parental strain can grow in darkness as an aerobic heterotroph on various carbon/energy sources including pyruvate, D,L-malate, or succinate. Mutant KGD 11, however, is unable to grow aerobically on the substrates noted. These results indicate that the energy for aerobic dark growth of R. capsulata is provided by respiratory phosphorylation fueled by citric acid cycle function, and that this requires a substantial level of KGD activity. The present findings also indicate that citric acid cycle sequences in most of the Rhodospirillaceae prominently used in current research are geared to operate in the oxidative direction, as in nonphotosynthetic aerobic heterotrophs.Abbreviations CoA
coenzyme A
- FR
fumarate reductase
- KG
-ketoglutarate
- KGD
-ketoglutarate dehydrogenase
- SD
succinate dehydrogenase 相似文献
8.
Effects of tenascin-W on osteoblasts in vitro 总被引:1,自引:0,他引:1
Meloty-Kapella CV Degen M Chiquet-Ehrismann R Tucker RP 《Cell and tissue research》2008,334(3):445-455
Tenascin-W is a glycoprotein secreted into the extracellular matrix of developing bones. Here, we have examined possible roles
for tenascin-W in osteogenesis. Purified recombinant tenascin-W, like tenascin-C, increases the number of mineralized foci
in primary cultures of avian osteoblasts and increases alkaline phosphatase activity in vitro. In addition, tenascin-W in
solution promotes the migration of primary osteoblasts across fibronectin-coated filters. The sixth fibronectin type III domain
of chicken tenascin-W contains a phylogenetically conserved KGD motif that is predicted to be available to integrin binding.
To determine whether this motif is potentially functional, we have cultured osteoblasts on KGD-containing peptides and control
peptides. Osteoblasts cultured on peptides with the KGD motif acquire a multipolar phenotype with pseudopods tipped with actin-rich
ruffles, which is similar to the morphology of osteoblasts cultured on recombinant tenascin-W. Moreover, the KGD peptides,
but not the control peptides, promote proliferation in cultured osteoblasts but not alkaline phosphatase activity or migration.
Finally, explanted embryonic frontal bones are significantly thicker when cultured in the presence of tenascin-W, suggesting
that tenascin-W can accelerate the formation of new bone in a complex multicellular environment. 相似文献
9.
Koutsas C Sarigiannis Y Stavropoulos G Liakopoulou-Kyriakides M 《Protein and peptide letters》2007,14(10):1014-1020
The reaction between Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) derivatives with 3,4',5-trihydroxy-trans-stilbene (resveratrol) was investigated. Knowing that resveratrol, RGD as well as KGD analogues inhibit human platelet aggregation in vitro, it was tempting for us to examine whether their coupling products present enhanced biological activity. Here, we report on the synthesis and identification of these coupling products. The N-protected peptides were synthesized by solid phase technique, using the 2-chlorotrityl-chloride resin, by the method of carbodiimides. Coupling reactions with resveratrol took place in solution using N,N-dicyclohexylcarbodiimide (DCC) as coupling reagent and 4-dimethylaminopyridine (DMAP) as catalyst. The reaction products were purified by reversed phase HPLC and identified by ESI-MS. The mono-esterified resveratrol derivative was the main (or only) reaction product, whereas the di- and the tri-ester (to a less extent) formation was noticed in some cases. 相似文献
10.
Martelli A Salin B Dycke C Louwagie M Andrieu JP Richaud P Moulis JM 《The FEBS journal》2007,274(4):1083-1092
Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family. 相似文献
11.
The properties of alpha-ketoglutarate dehydrogenase with non-interacting active sites were investigated. The substrate and coenzyme saturation curves are found to be hyperbolic, which is consistent with the absence of cooperativity between the active sites of the enzyme. The peculiarities of KGD of this form, determining its functional properties, were revealed. Thus, 6 cysteine residues of the enzyme possess different properties in comparison with the form of the enzyme with interacting active sites. 3 Sulfhydryl groups of the "non-cooperative" enzyme form were rapidly oxidized in the process of the enzyme isolation and storage; thereafter they could not be reduced by dithiols. Three other cysteine residues are probably involved in the formation of disulfide bonds. Two of them are supposed to form intramolecular disulfide, whereas the third one is thought to be modified by some low molecular weight disulfide. The reduction of these sulfhydryl groups by dithiols is shown to be accompanied by the appearance of the kinetic cooperativity with respect to the substrate. It is suggested that the thiol/disulfide exchange in vivo can regulate a reversible conversion of the "non-cooperative" KGD form into one with interacting sites. 相似文献
12.
The contribution of ionic interactions to the stability of the collagen triple helix was studied using molecular dynamics (MD) simulations and biophysical methods. To this end, we examined the stability of a host-guest collagen model peptide, Ac-GPOGPOGPYGXOGPOGPO-NH2, substituting KGE, KGD, EGK, and DGK for the YGX sequence. All-atom, implicit solvent MD simulations show that the fraction of cross-chain ionic interactions formed is different, with the most pronounced in the KGE and KGD sequences, and the least in the DGK sequence. To test whether the fraction of cross-chain ionic interactions correlates with the stability, experimental measurements of thermostability were done using differential scanning calorimetry and circular dichroism spectroscopy. It was found that the melting temperature is very similar for KGE and KGD peptides, whereas the EGK peptide has lower thermostability and the DGK peptide is the least thermostable. A novel, to our knowledge, computational protocol termed temperature-scan MD was applied to estimate the relative stabilities of the peptides from MD simulations. We found an excellent correlation between transition temperatures obtained from temperature-scan MD and those measured experimentally. These results suggest the importance of cross-chain ionic interactions for the stability of collagen triple helix and confirm the utility of MD simulations in predicting interactions and stability in this system. 相似文献
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Neelam Keshwani Shounak Banerjee Barbara Brodsky George?I. Makhatadze 《Biophysical journal》2013,105(7):1681-1688
The contribution of ionic interactions to the stability of the collagen triple helix was studied using molecular dynamics (MD) simulations and biophysical methods. To this end, we examined the stability of a host-guest collagen model peptide, Ac-GPOGPOGPYGXOGPOGPO-NH2, substituting KGE, KGD, EGK, and DGK for the YGX sequence. All-atom, implicit solvent MD simulations show that the fraction of cross-chain ionic interactions formed is different, with the most pronounced in the KGE and KGD sequences, and the least in the DGK sequence. To test whether the fraction of cross-chain ionic interactions correlates with the stability, experimental measurements of thermostability were done using differential scanning calorimetry and circular dichroism spectroscopy. It was found that the melting temperature is very similar for KGE and KGD peptides, whereas the EGK peptide has lower thermostability and the DGK peptide is the least thermostable. A novel, to our knowledge, computational protocol termed temperature-scan MD was applied to estimate the relative stabilities of the peptides from MD simulations. We found an excellent correlation between transition temperatures obtained from temperature-scan MD and those measured experimentally. These results suggest the importance of cross-chain ionic interactions for the stability of collagen triple helix and confirm the utility of MD simulations in predicting interactions and stability in this system. 相似文献
14.
Daniel K. Shanley Patrick A. Kiely Kalyan Golla Seamus Allen Kenneth Martin Ronan T. O’Riordan Melanie Ball John D. Aplin Bernhard B. Singer Noel Caplice Niamh Moran Tom Moore 《PloS one》2013,8(2)
Pregnancy-specific glycoproteins (PSGs) are immunoglobulin superfamily members encoded by multigene families in rodents and primates. In human pregnancy, PSGs are secreted by the syncytiotrophoblast, a fetal tissue, and reach a concentration of up to 400 ug/ml in the maternal bloodstream at term. Human and mouse PSGs induce release of anti-inflammatory cytokines such as IL-10 and TGFβ1 from monocytes, macrophages, and other cell types, suggesting an immunoregulatory function. RGD tri-peptide motifs in the majority of human PSGs suggest that they may function like snake venom disintegrins, which bind integrins and inhibit interactions with ligands. We noted that human PSG1 has a KGD, rather than an RGD motif. The presence of a KGD in barbourin, a platelet integrin αIIbβ3 antagonist found in snake venom, suggested that PSG1 may be a selective αIIbβ3 ligand. Here we show that human PSG1 binds αIIbβ3 and inhibits the platelet – fibrinogen interaction. Unexpectedly, however, the KGD is not critical as multiple PSG1 domains independently bind and inhibit αIIbβ3 function. Human PSG9 and mouse Psg23 are also inhibitory suggesting conservation of this function across primate and rodent PSG families. Our results suggest that in species with haemochorial placentation, in which maternal blood is in direct contact with fetal trophoblast, the high expression level of PSGs reflects a requirement to antagonise abundant (3 mg/ml) fibrinogen in the maternal circulation, which may be necessary to prevent platelet aggregation and thrombosis in the prothrombotic maternal environment of pregnancy. 相似文献
15.
Nykvist P Tasanen K Viitasalo T Kapyla J Jokinen J Bruckner-Tuderman L Heino J 《The Journal of biological chemistry》2001,276(42):38673-38679
Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration. 相似文献
16.
Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate. 相似文献
17.
Gorski A Dabrowska K Switala-Jeleń K Nowaczyk M Weber-Dabrowska B Boratynski J Wietrzyk J Opolski A 《Medical immunology (London, England)》2003,2(1):2
BACKGROUND: While the ability of bacteriophages to kill bacteria is well known and has been used in some centers to combat antibiotics - resistant infections, our knowledge about phage interactions with mammalian cells is very limited and phages have been believed to have no intrinsic tropism for those cells. PRESENTATION OF THE HYPOTHESIS: At least some phages (e.g., T4 coliphage) express Lys-Arg-Gly (KGD) sequence which binds beta3 integrins (primarily alphaIIbbeta3). Therefore, phages could bind beta3+ cells (platelets, monocytes, some lymphocytes and some neoplastic cells) and downregulate activities of those cells by inhibiting integrin functions. TESTING THE HYPOTHESIS: Binding of KGD+ phages to beta3 integrin+ cells may be detected using standard techniques involving phage - mediated bacterial lysis and plaque formation. Furthermore, the binding may be visualized by electron microscopy and fluorescence using labelled phages. Binding specificity can be confirmed with the aid of specific blocking peptides and monoclonal antibodies. In vivo effects of phage - cell interactions may be assessed by examining the possible biological effects of beta3 blockade (e.g., anti-metastatic activity). IMPLICATION OF THE HYPOTHESIS: If, indeed, phages can modify functions of beta3+ cells (platelets, monocytes, lymphocytes, cancer cells) they could be important biological response modifiers regulating migration and activities of those cells. Such novel understanding of their role could open novel perspectives in their potential use in treatment of cardiovascular and autoimmune disease, graft rejection and cancer. 相似文献
18.
A method was devised to search for yeast mutants impaired in peroxisome functioning, indicating cross-talk between metabolic pathways. Two mutants were isolated; they are impaired in oleate utilisation and carry mutations in the KGD1 and LIP5 genes encoding the E1 component of the mitochondrial alpha-ketoglutarate dehydrogenase complex and lipoic acid synthase, respectively. The results presented indicate that the Kgd1 and Lip5 proteins are important for the expression of genes encoding peroxisomal matrix proteins, although they are not necessary for the biogenesis of this cellular compartment. 相似文献
19.
Mj-DWD, a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus 总被引:1,自引:0,他引:1
The Mj-DWD (Marsupenaeus japonicus' double-WAP domains) gene was originally found up-regulated in virus-resistant shrimp M. japonicus by suppression subtractive hybridization (SSH). The full-length cDNA of Mj-DWD encodes a novel protein containing a KGD (Lys-Gly-Asp) motif and double WAP domains. Performed by quantitative real-time PCR, the expression level of Mj-DWD gene was consistently maintained at a high level in the newly prepared virus-resistant shrimp compared to the normal one. In addition, the Mj-DWD gene was also found to be rapidly up-regulated by WSSV infection during the early phase. Furthermore, the recombinant Mj-DWD, expressed by Pichia pastoris, showed specific protease inhibitory activity on Bacillus subtilis. These findings suggest that Mj-DWD plays an important role in the host defence system against WSSV infection in M. japonicus, possibly through its protease inhibitory activity. 相似文献
20.
An eight amino acid sequence, CAKGDWNC, from disintegrin barbourin, was introduced into an inactive human proinsulin molecule
between the B28 and A2 sites to construct a chimeric, anti-thrombosis recombinant protein. The constructed Lys-Gly-Asp (KGD)-proinsulin
gene was expressed in Escherichia coli and then purified. The KGD-proinsulin chimera protein inhibits human platelet aggregation, induced by ADP, with an IC50 value (molar concentration causing 50% inhibition of platelet aggregation) of 830 nM and demonstrates also specific affinity to glycoprotein IIb/IIIa receptor. Its insulin receptor binding activity remaines
as low as 0.04% with native insulin as a control. 相似文献