Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein‐nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose‐binding protein.     

Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.     

Chromatographic approach to study the configurational stability of Ni(II) complexes of amino‐acid Schiff bases possessing stereogenic nitrogen

Herein, we disclose the design of a model Ni(II) complex of glycine Schiff base possessing single‐nitrogen stereogenic center, which was successfully used for high‐performance liquid chromatography (HPLC)‐assisted assessment of its configurational stability. The major finding is that the configurational stability of the Ni(II)‐coordinated nitrogen is profoundly dependent on the reaction conditions used, in particular the solvent, and can range from inconsequential (t_½ less than 5 min) to virtually completely stable (t_½ 90 y). The discovery reported in this study most likely to be of certain theoretical and synthetic value.     

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.     

Selective inhibition of trypsins by insect peptides: role of P6-P10 loop

PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K(i) around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K(i) 相似文献   

Enantioselective correlation between retention factor and lipophilicity index in chiral separation on cellulose and amylose tris(3,5-dimethylphenylcarbamate) CSPs in reversed mode: A case study

For a series of alkyl substituted N-arylthiazoline-2-(thi)one atropisomers 1-14, lipophilicity indexes log kw obtained by polycratic RP-HPLC were compared to ln k(+)-S and ln k(-)-R obtained on CHIRALCEL OD-R(R) (reversed mode) and CHIRALPAK AD-RH(R) (reversed mode). Linear correlations were obtained in most cases. It appears that the correlation lines for R and S enantiomers may be parallel, convergent, or divergent, accounting for the observed alpha variation in going from methyl to tert-butyl series. Some tentative hypothesis are given as future investigation routes. Copyright 2000 Wiley-Liss, Inc.