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991.
The kinetics and thermodynamics of Bi(III) exchange between bismuth mononitrilotriacetate (BiL) and human serum transferrin as well as those of the interaction between bismuth-loaded transferrin and transferrin receptor 1 (TFR) were investigated at pH 7.4-8.9. Bismuth is rapidly exchanged between BiL and the C-site of human serum apotransferrin in interaction with bicarbonate to yield an intermediate complex with an effective equilibrium constant K(1) of 6 +/- 4, a direct second-order rate constant k(1) of (2.45 +/- 0.20) x 10(5) M(-1) s(-1), and a reverse second-order rate constant k(-1) of (1.5 +/- 0.5) x 10(6) M(-1) s(-1). The intermediate complex loses a single proton with a proton dissociation constant K(1a) of 2.4 +/- 1 nM to yield a first kinetic product. This product then undergoes a modification in its conformation followed by two proton losses with a first-order rate constant k(2) = 25 +/- 1.5 s(-1) to produce a second kinetic intermediate, which in turn undergoes a last modification in the conformation to yield the bismuth-saturated transferrin in its final state. This last process rate-controls Bi(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau(3)(-1) of (3 +/- 1) x 10(-2) s(-1). The mechanism of bismuth uptake differs from that of iron and probably does not involve the same transition in conformation from open to closed upon iron uptake. The interaction of bismuth-loaded transferrin with TFR occurs in a single very fast kinetic step with a dissociation constant K(d) of 4 +/- 0.4 microM, a second-order rate constant k(d) of (2.2 +/- 1.5) x 10(8) M(-1) s(-1), and a first-order rate constant k(-d) of 900 +/- 400 s(-1). This mechanism is different from that observed with the ferric holotransferrin and implies that the interaction between TFR and bismuth-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of bismuth incorporation by the transferrin receptor-mediated iron acquisition pathway is discussed. 相似文献
992.
Erbel PJ Seidel R Macintosh SE Gentile LN Amor JC Kahn RA Prestegard JH McIntosh LP Gardner KH 《Journal of biomolecular NMR》2004,29(2):199-204
We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate. 相似文献
993.
994.
Kitching R Li H Wong MJ Kanaganayakam S Kahn H Seth A 《Biochimica et biophysica acta》2003,1625(1):116-121
As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue. 相似文献
995.
The ability to rapidly and reliably develop hypotheses on the function of newly discovered protein sequences requires systematic and comprehensive analysis. Such an analysis, embodied within the DS GeneAtlas pipeline, has been used to critically evaluate the severe acute respiratory syndrome (SARS) genome with the goal of identifying new potential targets for viral therapeutic intervention. This paper discusses several new functional hypotheses on the roles played by the constituent gene products of SARS, and will serve as an example of how such assignments can be developed or extended on other systems of interest. 相似文献
996.
Valverde AM Arribas M Mur C Navarro P Pons S Cassard-Doulcier AM Kahn CR Benito M 《The Journal of biological chemistry》2003,278(12):10221-10231
To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin. 相似文献
997.
Shern JF Sharer JD Pallas DC Bartolini F Cowan NJ Reed MS Pohl J Kahn RA 《The Journal of biological chemistry》2003,278(42):40829-40836
Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis. 相似文献
998.
The platelet collagen receptor glycoprotein VI (GPVI) is structurally homologous to multisubunit immune receptors and signals through the immune receptor adaptor Fc Rgamma. Multisubunit receptors are composed of specialized subunits thought to be dedicated exclusively to ligand binding or signal transduction. However, recent studies of the intracellular region of GPVI, a ligand-binding subunit, have suggested the existence of protein-protein interactions that could regulate receptor signaling. In the present study we have investigated the signaling role of the GPVI intracellular domain by stably expressing GPVI mutants in RBL-2H3 cells, a model system that accurately reproduces the GPVI signaling events observed in platelets. Studies of mutant GPVI receptor protein-protein interaction and calcium signaling reveal the existence of discrete domains within the receptor's intracellular tail that mediate interaction with Fc Rgamma, calmodulin, and Src family tyrosine kinases. These receptor interactions are modular and mediated by non-overlapping regions of the receptor transmembrane and intracellular domains. GPVI signaling requires all three of these domains as receptor mutants able to couple to only two interacting proteins exhibited severe signaling defects despite normal surface expression. Our results demonstrate that the ligand-binding subunit of the GPVI-Fc Rgamma receptor participates directly in receptor signaling by interacting with downstream signaling molecules other than Fc Rgamma through an adaptor-like mechanism. 相似文献
999.
Phospholipid transfer protein (PLTP) activity is elevated in obese and diabetic subjects. No prospective studies have examined the effect of weight loss on PLTP activity and assessed whether the resultant changes in activity are related to changes in body weight, insulin resistance, or both. PLTP activity was measured at baseline in 46 subjects (body mass index = 19-64 kg/m2) and after diet-induced weight loss in 19 of the obese subjects. Total body fat mass (FM) by dual-energy X-ray absorptiometry, intraabdominal fat (IAF), and abdominal subcutaneous fat (SQF) by CT scan, insulin sensitivity (SI) by frequently sampled intravenous glucose tolerance test, leptin, and lipids were determined. At baseline, PLTP activity correlated with FM (r = 0.36, P = 0.02) and SQF (r = 0.31, P = 0.045), but not with IAF (r = 0.16, P = 0.32) or SI (r = 0.10, P = 0.52). With diet-induced weight loss (16 +/- 7.3 kg), PLTP activity significantly decreased 9.1% (P = 0.002). The change in PLTP activity correlated with the change in SQF (r = 0.55, P = 0.014) (33.6% decrease), but not with IAF (r = 0.09, P = 0.73) or SI (r = 0.18, P = 0.44), and was highly correlated with the change in nonesterified fatty acid (NEFA) (r = 0.71, P < 0.001). In conclusion, elevated PLTP activity in obese subjects is likely a result of increased body fat, reflected by SQF, and is influenced by NEFAs but is not directly related to insulin resistance. 相似文献
1000.
García-Sáez I Mercuri PS Papamicael C Kahn R Frère JM Galleni M Rossolini GM Dideberg O 《Journal of molecular biology》2003,325(4):651-660
The beta-lactamases are involved in bacterial resistance to penicillin and related compounds. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are thus becoming of major clinical importance. The structures of the Zn-beta-lactamase from Fluoribacter gormanii (FEZ-1) in the native and in the complex form are reported here. FEZ-1 is a monomeric enzyme, which possesses two zinc-binding sites. These structures are discussed in comparison with those of the tetrameric L1 enzyme produced by Stenotrophomonas maltophilia. From this analysis, amino acids involved in the oligomerization of L1 are clearly identified. Despite the similarity in fold, the active site of FEZ-1 was found to be significantly different. Two residues, which were previously implicated in function, are not present in L1 or in FEZ-1. The broad-spectrum substrate profile of Zn-beta-lactamases arises from the rather wide active-site cleft, where various beta-lactam compounds can be accommodated. 相似文献