alpha-chymotrypsin and tris(2-carboxyethyl)phosphine

Release of sporozoites from Eimeria oocysts/sporocysts is an essential step in the intracellular development of the parasite in its host. Little is known about this process except that elevated temperature (∼ 40 °C) plus trypsin and bile salts are required for sporozoite to escape from sporocysts. In this study, it was found that adding a reducing agent, either dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), increased the lifespan of sporozoites released from Eimeria maxima. While the addition of DTT or TCEP affected the apparent molecular weight of trypsin, it did not interfere with excystation of E. maxima, but rather had a positive effect on the number of viable sporozoites present after release. This effect was time-dependent in that the number of intact sporozoites at 15 and 30 min after excystation was similar between untreated and DTT- or TCEP-treated sporocysts. However, by 45-60 min, virtually no sporozoites were observed in excystation fluid not containing DTT or TCEP. Of interest is that this effect appeared to be Eimeria species-dependent. Eimeria acervulina and E. tenella sporozoites remained viable for at least 60 min after excystation in the absence of DTT or TCEP. The effect of DTT and TCEP on chymotrypsin was also studied with all 3 Eimeria species because there is some evidence that chymotrypsin is an effective excystation enzyme. Indeed, E. maxima sporozoites excysting from sporocysts with chymotrypsin in the presence of DTT or TCEP remained viable for at least 60 min after release, unlike excystation done in the absence of these reducing agents. Chymotrypsin was capable of excysting E. acervulina in the presence or absence of DTT or TCEP. Of interest, is that chymotrypsin was ineffective in the excystation of E. tenella. These findings suggest that trypsin and chymotrypsin have differential effects on sporozoite excystation and that reducing agents may alter sites on the enzyme that affect sporozoite viability, but not release from sporocysts.

Topics: Chymotrypsin; Dithiothreitol; Eimeria; Eimeria tenella; Oocysts; Phosphines; Reducing Agents; Sporozoites; Trypsin

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.

Topics: Amino Acid Sequence; Animals; Bombyx; Carrier Proteins; Chymotrypsin; Cyanogen Bromide; Disulfides; Indicators and Reagents; Insect Hormones; Insect Proteins; Intercellular Signaling Peptides and Proteins; Mass Spectrometry; Metalloendopeptidases; Molecular Sequence Data; Phosphines; Recombinant Proteins; Sequence Homology, Amino Acid