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Characterization of extracellular proteinases of Tritrichomonas foetus.

The Journal of parasitology

enzymology, Amino Acid Sequence, Animals, Cathepsins, chemistry, Electrophoresis, Polyacrylamide Gel, Endopeptidases, metabolism, Gelatin, Hydrolysis, Molecular Sequence Data, Molecular Weight, Peptides, Sequence Alignment, Substrate Specificity, Tritrichomonas foetus

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      Abstract

      Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis.

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