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Carboxyglutamic Acid (carboxyglutamic + acid)
Selected AbstractsNovel ,-carboxyglutamic acid-containing peptides from the venom of Conus textileFEBS JOURNAL, Issue 12 2006Eva Czerwiec The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or ,-carboxylase) and its product ,-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate ,-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1),TxVI, Gla(2),TxVI/A, Gla(2),TxVI/B and Gla(3),TxVI each have six Cys residues and belong to the O -superfamily of conotoxins. All four conopeptides contain 4- trans -hydroxyproline and the unusual amino acid 6- l -bromotryptophan. Gla(2),TxVI/A and Gla(2),TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3),TxVI were observed that differ at position +7: Gla(3),TxVI, Glu7,Gla(3),TxVI and Asp7-Gla(3),TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids ,46 to ,27) were nearly identical and highly hydrophobic. The predicted propeptide region (,20 to ,1) that contains the ,-carboxylation recognition site (,-CRS) is very similar in Gla(2),TxVI/A, Gla(2),TxVI/B and Gla(3),TxVI, but is more divergent for Gla(1),TxVI. Kinetic studies utilizing the Conus,-carboxylase and synthetic peptide substrates localized the ,-CRS of Gla(1),TxVI to the region ,14 to ,1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1),TxVI lacking a propeptide to 24 µm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2),TxVI/B reduced the Km value tenfold. [source] Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonistJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2007M. KJALKE Summary., Background:, Activation of platelets with a combination of collagen and thrombin generates a subpopulation of highly procoagulant ,coated' platelets characterized by high surface expression of fibrinogen and other procoagulant proteins. Objectives:, To analyze the interaction of recombinant factor VIIa (rFVIIa) with coated platelets. Methods and results:, rFVIIa localized to the coated platelets in flow cytometry experiments, while minimal rFVIIa was found on platelets activated with adenosine diphosphate, thrombin or via glycoprotein VI individually, and essentially no rFVIIa was found on non-stimulated platelets. Removal of the , -carboxyglutamic acid (Gla) domain of rFVIIa, and addition of EDTA, annexin V or excess prothrombin inhibited rFVIIa localization to the coated platelets, indicating that the interaction was mediated by the calcium-dependent conformation of the Gla domain and platelet exposure of negatively charged phospholipids. A reduced level of platelet fibrinogen exposure was observed at hemophilia A-like conditions in a model system of cell-based coagulation, indicating that coated platelet formation in hemophilia may be diminished. Addition of rFVIIa dose-dependently enhanced thrombin generation and partly restored platelet fibrinogen exposure. Conclusions:, The data suggest that rFVIIa localized preferentially on platelets activated with dual agonists, thereby ensuring enhanced thrombin generation localized at the site of injury where both collagen and tissue factor are exposed, the latter ensuring the formation of thrombin necessary for coated platelet formation. [source] The role of electron capture dissociation in biomolecular analysisMASS SPECTROMETRY REVIEWS, Issue 2 2005Helen J. Cooper Abstract The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant advance in the structural analysis of biomolecules. The fundamental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the fundamentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision-induced dissociation (SORI-CID), are discussed. The instrumental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, particularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfation, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligonucleotides is also discussed. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:201,222, 2005 [source] Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibrationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2002Omar Belgacem Abstract Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de- N -glycosylated form by means of MALDI mass spectrometry. The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants. Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of ±0.12 and ±0.022%, respectively. The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode. Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip® technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration. The average molecular masses of the two different forms of AT-III, namely AT-III, and AT-III,, were shown to be 57.26 and 55.04 kDa, respectively. The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135). After exhaustive de- N -glycosylation (by means of PNGase F) of the ,- and ,-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained. These values are in good agreement (,0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data. The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N - and O -glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous ,-carboxyglutamic acids). MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE. After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected. The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75. The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications. Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN. PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N -glycans. Copyright © 2002 John Wiley & Sons, Ltd. [source] | ||||||||||