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N-terminal Amino Acids (n-terminal + amino_acids)
Selected AbstractsIsocitrate dehydrogenase of Plasmodium falciparumFEBS JOURNAL, Issue 8 2003Energy metabolism or redox control? Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+ -dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 µm and 22 µm, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 µm and that of d -isocitrate was determined to be 40 µm. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites. [source] Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 geneFEBS JOURNAL, Issue 7 2001Muriel Erent The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase C,. NDP kinase C, had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase C,, based on the crystal structure of NDP kinase B, indicated that NDP kinase C, had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase C, readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo. [source] Enzymes involved in flavour formation by bacteria isolated from the smear population of surface-ripened cheeseINTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2004A G Williams Twenty-five bacterial isolates recovered from the surface population of smear-ripened cheese were assigned phenotypically as Brevibacterium spp., Corynebacterium spp. and Aureobacterium spp. using the Biolog GP2 microplate system and database. The range and activity of hydrolytic enzymes involved in the formation of cheese flavour constituents were monitored in cell-free lysates of the isolates. Esterase activity and the presence of a range of enzymes involved in amino acid release and breakdown was confirmed in all strains examined although there were pronounced interspecies and strain differences in the level of activity detected. Peptidolytic activities present in the smear bacteria included dipeptidyl peptidase and aminopeptidases that cleaved various N-terminal amino acids including proline. Subsequent breakdown of the released aromatic and branched-chain amino acids was mediated by ,-keto acid dependent aminotransferase action and several of the isolates were able to form thiols from sulphur-containing amino acid precursors. It was confirmed that the enzymic activity of the smear population could be manipulated by the use of defined starter cultures comprising selected combinations of smear isolates. The hydrolytic activities of the smear bacteria are involved in the generation of cheese flavour compounds and the enzyme profile is thus an important selection criterion for strains to be evaluated for use in defined surface smear preparations. [source] Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Sarah L. Dallas Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source] Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinaseJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 5 2004C. E. Hagemeyer Summary.,Background: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity. Objectives: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent. Methods: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPALMW), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (,15,22) in the ,-chain of human fibrin (scFv59D8). The fusion protein was purified using affinity chromatography with the ,15,22 -peptide of human fibrin. Results: Purified scFv59D8,scuPALMW migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv59D8 and one scuPALMW moiety. Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv59D8,scuPALMW is significantly faster and more potent compared with the clinically used urokinase. Conclusions: ScFv59D8,scuPALMW constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli. [source] The roles of the N-terminal portions of various tachykinins in promoting salivationORAL DISEASES, Issue 4 2001K Higa OBJECTIVES: In order to determine the active sites for salivation of various tachykinins, the regulatory roles of the N-terminal portion of various newly-synthesized tachykinins were studied after i.p. injection of rats using the submandibular glands as model organs. METHODS: N-shortened oligopeptides from kassinin, eledoisin, neurokinins A (NKA) and NKB were synthesized by the multipin peptide synthesis method. Amino acids were eliminated one by one to form octa- to undeca-peptides adjoining the inactive or less active heptapeptides and various heptapeptides, in which an amino acid in position 8 (Xaa8 ), numbering as in an undecapeptide, was replaced with Tyr, Phe, Ile or Val. RESULTS: The N-terminal amino acids in positions 1 to 4 could be activators or inhibitors, depending on whether the C-terminal heptapeptide was inactive or less active. The Xaa8 residue, in combination with amino acids in positions 5 and 6, seemed to be very important in determining the sialogogic activity of a heptapeptide. The discrimination between NKA and NKB appeared due to the N-terminal amino acid sequence in positions 1 to 4 including Phe or Ser in position 6. CONCLUSIONS: It is concluded that the N-terminal amino acids in positions 1 to 4 serve as either activators or inhibitors depending upon the sialogogic activity of the C-terminal heptapeptide, in which particular amino acids in positions 5, 6 and 8 regulate its activity. [source] Fibronectin-binding proteins secreted by Mycobacterium aviumAPMIS, Issue 9 2000HIDEKI Kitaura Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Fibronectin is an extracellular matrix protein and is a virulence factor for several extracellular pathogenic bacteria binding to mucosal surfaces. We investigated the fibronectin (FN)-binding proteins in the culture filtrate of M. avium by two-dimensional electrophoresis (2DE). Proteins in Sauton medium of M. avium after 3 weeks were separated by 2DE. The proteins were blotted onto polyvinylidene difluoride membrane and incubated with FN. FN-binding proteins were detected by Western blotting using anti-FN antibody. FN bound to five spots (33 kDa, 32 kDa, 31 kDa, 30 kDa and 25 kDa). N-terminal amino acids of these were determined. The 33 kDa spot corresponded to antigen 85 (Ag 85) C. The 32 and 31 kDa spots were either Ag 85 A or Ag 85 B. The 30 kDa spot corresponded to Ag 85 B of M. avium. The 25 kDa spot corresponded to MPA51 (M. avium MPB51). Thus, FN bound exclusively to the Ag 85 complex and MPA51. [source] Association of autoimmunity to peptidyl arginine deiminase type 4 with genotype and disease severity in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 7 2008Michelle L. Harris Objective Protein citrullination is an important posttranslational modification recognized by rheumatoid arthritis (RA),specific autoantibodies. One of the citrullinating enzymes, peptidyl arginine deiminase type 4 (PAD-4), is genetically associated with development of RA in some populations, although the mechanism(s) mediating this effect are not yet clear. There have been descriptions of anti,PAD-4 autoantibodies in different rheumatic diseases. This study was undertaken to investigate whether anti,PAD-4 antibodies are specific to RA, are associated with disease phenotype or severity, and whether PAD-4 polymorphisms influence the anti,PAD-4 autoantibody response. Methods Sera from patients with established RA, patients with other rheumatic diseases, and healthy adults were assayed for anti,PAD-4 autoantibodies by immunoprecipitation of in vitro,translated PAD-4. The epitope(s) recognized by PAD-4 autoantibodies were mapped using various PAD-4 truncations. PAD-4 genotyping was performed on RA patients with the TaqMan assay. Joint erosions were scored from hand and foot radiographs using the Sharp/van der Heijde method. Results PAD-4 autoantibodies were found in 36,42% of RA patients, and were very infrequent in controls. Recognition by anti,PAD-4 autoantibodies required the 119 N-terminal amino acids, which encompass the 3 nonsynonymous polymorphisms associated with disease susceptibility. Strikingly, the anti,PAD-4 immune response was associated with the RA susceptibility haplotype of PADI4. Anti,PAD-4 antibodies were associated with more severe joint destruction in RA. Conclusion Our findings indicate that anti,PAD-4 antibodies are specific markers of RA, independently associated with more severe disease, suggesting that an anti,PAD-4 immune response may be involved in pathways of joint damage in this disease. Polymorphisms in the PADI4 gene influence the immune response to the PAD-4 protein, potentially contributing to disease propagation. [source] | |||||||||||||