2/28/2023 0 Comments Eh microsynth schematicThe minimum identity was seen in the 27 amino acid central linker region between Ca 2+ binding domains 2 and 3, which had 10 substitutions. The maximum identity was observed in the Ca 2+ binding domains with a total of 6 amino acid substitutions ( Fig. The level of similarity between the two proteins varied along the length of the sequence. Both the proteins have four typical EF-hand Ca 2+ binding domains. The amino acid sequences of the two proteins showed an identity of 78%. The conceptual translation product of EhCaBP2 was compared with EhCaBP1 and the alignment of the two sequences is shown in Fig. Of the 80 nucleotides that differed in the two genes, 28 were in the third codon position, leaving the encoded amino acids unchanged. The overall identity at the nucleotide level between EhCaBP2 and EhCaBP1 was 79% ( Fig. histolytica total cell lysate preincubated with indicated amounts of peptide(s). In competition experiments involving peptides, reactions were performed under identical conditions using E. The fluorographed gels were dried and exposed to an x-ray film. For visualization of phosphorylated products, the reaction mixture after termination was separated in a 10% SDS-PAGE. The pellet was washed with diethyl ether/ethanol (1:1) mixture and after drying the pellet, and spotting the rehydrated pellet on GF/C paper, the amount of radioactivity was determined in a scintillation counter. The reaction was terminated by adding 10% trichloroacetic acid, which precipitated total protein in the reaction mixture. The reaction was initiated by adding 100 μ m rATP (specific activity 5000 Ci/mmol, Amersham Biosciences) and allowed to proceed at 30 ☌ for 10 min. The reaction volume was adjusted to 50 μl. histolytica cell extract, protease inhibitor mixture, CaCl 2 or EGTA, and 100 pmol of peptide where indicated. The reaction mixture contained 30 m m HEPES, pH 7.5, 5 m m MgCl 2, 40 μg of histone (Type IIIS, Sigma), 30 μg of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway. Our data suggest that EhCaBP2 is a new member of a class of E. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca 2+ concentration required for their optimal functionality was also different. histolytica proteins in a Ca 2+-dependent manner. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. histolytica genome sequence data suggested that the two genes are non-allelic. Both of these genes are single copy, as revealed by Southern hybridization. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca 2+ binding domains. Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. The two isoforms are encoded by genes of the same size (402 bp). Both EhCaBPs have four canonical EF-hand Ca 2+ binding domains. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. However, the mechanistic role of Ca 2+ and calcium-binding proteins in the pathogenesis of E. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. Glycobiology and Extracellular MatricesĮntamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis.
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