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kDa Polypeptide (kda + polypeptide)
Selected AbstractsProteolytic activation and function of the cytokine Spätzle in the innate immune response of a lepidopteran insect, Manduca sextaFEBS JOURNAL, Issue 1 2010Chunju An The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spätzle, which functions in dorsal,ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spätzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spätzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpätzle-1B that is not present in proSpätzle-1A. The proSpätzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spätzle-1, respectively. Recombinant proSpätzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpätzle-1A to release Spätzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spätzle-C108, but not proSpätzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin-6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and ,-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to ,-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spätzle-1. The resulting Spätzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection. Structured digital abstract ,,MINT-7295125: Spätzle 1A (uniprotkb:C8BMD1) and Spätzle 1A (uniprotkb:C8BMD1) bind (MI:0407) by comigration in gel electrophoresis (MI:0807) [source] Differential availability/processing of decorin precursor in arterial and venous smooth muscle cellsJOURNAL OF ANATOMY, Issue 3 2006Rafaella Franch Abstract The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of Mr 33,226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury. [source] Activity and concentration of non-proteolyzed phosphoenolpyruvate carboxykinase in the endosperm of germinating castor oil seeds: effects of anoxia on its activityPHYSIOLOGIA PLANTARUM, Issue 4 2007Mariana Martín Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) catalyses the reversible decarboxylation of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage lipids in germinating oil seeds. The enzyme is quite susceptible to limited proteolysis during extraction. Immunoblotting was used to diagnose unwanted in vitro proteolytic activity against PEPCK from germinating castor oil seeds (COS) by following the disappearance of its native 74-kDa subunit and concomitant appearance of a truncated 64-kDa polypeptide. Alkaline pH and the inclusion of thiol protease inhibitors effectively prevented COS PEPCK proteolysis during incubation of clarified COS extracts at 4°C. The carboxylating and decarboxylating activities and concentration of non-proteolyzed COS PEPCK were investigated during germination. This is the first report in which both activities catalyzed by PEPCK were measured in vitro during a whole developmental process. Carboxylating activity and the level of immunoreactive 74-kDa PEPCK polypeptides rapidly increased in parallel to maximal values by day 5 and then significantly declined over the subsequent 2 days. In contrast, decarboxylating PEPCK activity was much higher over the 7 days of growth examined. In addition, the effect on PEPCK activity while changing the endosperm from aerobic (when gluconeogenesis predominates in the tissue) to anaerobic conditions (where the tissue becomes glycolytic) was studied. While PEPCK decarboxylating activity remained almost constant, carboxylating activity declined to undetectable levels in response to anaerobiosis. These and the developmental profile results suggest that COS PEPCK may be subject to a mechanism of post-translation control that selectively inhibits the carboxylating, but not the decarboxylating activity. [source] Bifunctional indole-3-acetyl transferase catalyses synthesis and hydrolysis of indole-3-acetyl- myo -inositol in immature endosperm of Zea maysPHYSIOLOGIA PLANTARUM, Issue 2 2003Stanislaw Kowalczyk 1- O -(indole-3-acetyl)- , - d -glucose: myo -inositol indoleacetyl transferase (IA- myo -inositol synthase) is an important enzyme in IAA metabolism. This enzyme catalyses the transfer of the indole acetyl (IA) moiety from 1- O -(indole-3-acetyl)- , - d -glucose to myo -inositol to form IA- myo- inositol and glucose. IA- myo -inositol synthase was purified to an electrophoretically homogenous state from maize liquid endosperm by fractionation with ammonium sulphate, anion-exchange, adsorption on hydroxylapatite, affinity chromatography on ConA-Sepharose, preparative PAGE and isoelectric focusing. We thus obtained two enzyme preparations which differ in their Rf on 8% polyacrylamide gel. The preparation of Rf 0.36 contained a single 56.4 kDa polypeptide, whereas the preparation of Rf 0.39 consisted of two polypeptides of 56.4 and 53.5 kDa. Both purified preparations of IAInos synthase also exhibited the activity of an IAInos hydrolase, showing that the dual activity was associated with a single protein. Results of gel filtration and analytical SDS-PAGE suggest that the native enzyme exists as both a monomeric (65 kDa) and homo- or heterodimeric form (110,130 kDa). Analysis of peptide maps and amino acid sequences of two 21 amino-acid peptides showed that polypeptides of 56.4 and 53.5 kDa have the same primary structure and that the 3 kDa difference in molecular mass is probably caused by different glycosylation levels. Comparison of this partial and internal amino acid sequence with sequences of other plant acyltransferases indicated similarity to several proteins which belonged to the serine carboxypeptidase-like (SCPL) acyltransferase family. [source] Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre FractionsREPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2003E Hinsch Contents Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1,18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella. [source] Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosiscFEBS JOURNAL, Issue 16 2005Melissa C. Miles Multiple ankyrin repeat motif-containing proteins play an important role in protein,protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s). [source] | ||||||||||