Further characterization of the major cysteine protease of Artemia franciscana cysts, and the isolation of a cDNA encoding the small subunit of the protease.

摘要

Dormant cysts (embryos) of Artemia franciscana contain large amounts of a cysteine protease which was previously believed to be cathepsin B-like, but has now been re-classified as cathepsin L-like based on the studies detailed in this thesis. The Artemia cyst cysteine protease was purified to apparent homogeneity using a protocol involving gel filtration, anion exchange, and affinity chromatography. Fast protein liquid chromatography resolved five isoforms of the protease whose subunits (28.5 kDa and 31.5 kDa) were separated using reverse phase high performance liquid chromatography. Carbohydrate moieties were found associated with both subunits of all cyst protease isoforms. Only the large subunit, however, was sensitive to endoglycosidase F, suggesting that the carbohydrate group associated with it is likely to be an N-linked high mannose, oligosaccharide, or complex biantennary carbohydrate. Assay of the enzymeu27s proteolytic activity toward synthetic peptide substrates showed that the enzyme is cathepsin L-like in this respect. A combination of enzymatic treatments and (reverse phase) high performance liquid chromatography were used to isolate a putative carboxyl-terminal peptide fragment of the small subunit of isoform #3 of the Artemia protease. The amino acid sequence information from this peptide was used to design a primer for use in polymerase chain reactions. A second primer was designed based on amino acid sequence information obtained (previously) from the amino terminus of the protein. These primers were used in a polymerase chain reaction which amplified a 435 base pair product from a bacteriophage cDNA library. (Abstract shortened by UMI.)Dept. of Biological Sciences. Paper copy at Leddy Library: Theses u26 Major Papers - Basement, West Bldg. / Call Number: Thesis1997 .A37. Source: Masters Abstracts International, Volume: 37-01, page: 0192. Adviser: Alden H. Warner. Thesis (M.Sc.)--University of Windsor (Canada), 1997.