Graduate Thesis Or Dissertation
 

Characterization of Pacific whiting proteinase P-II and partial cloning of cathepsins L and K cDNA from rainbow trout liver

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  • Proteinase P-II purified from parasitized Pacific whiting muscle was previously identified to be one form of cathepsin L. It appeared to be present in three isozymatic forms on non-denaturing PAGE gel stained for activity. Its autolytic degradation was observed on SDS-PAGE gel under its optimum condition, 55°C and pH 5.5, in the absence of substrate. Amino acid composition analysis revealed that this enzyme had a considerably greater proportion of hydrophobic amino acids than cathepsin L from other fish species, and monosaccharide analysis showed it was not glycosylated. The N-terminal amino acid sequence of the enzyme was 60-65% identical with cathepsin L from chicken and mammalian species, but only 39% identical with mammalian cathepsin B. The moderate identity of the N-terminal amino acid sequence of P-II with other cathepsin L revealed that this cysteine proteinase from Pacific whiting might be encoded by a cathepsin L-related gene. Two degenerate primers were designed to clone cathepsins cDNA from rainbow trout. The 500-bp PCR product from rainbow trout liver cDNA contained at least three different cysteine proteinase sequences, referred to as SFL2, SFL5, and SFL17. SFL5 was the partial cDNA of trout cathepsin L, which was over 80% identical with chicken cathepsin L amino acid sequence. SFL5 was labeled with Dig-11-dUTP and used to screen a trout liver cDNA library. One positive clone referred to as LC was identified and contained a 700-bp insertion overlapping with SFL5. By combining the two overlapping sequences, a 895-bp cDNA sequence was identified, which included 88% of the mature enzyme and a 307-bp 3' end untranslated part. Its deduced amino acid sequences had 83% identity, 91% similarity with chicken cathepsin L and 73% identity, 86% similarity with human cathepsin L. SFL2 might be the partial cDNA of a novel cathepsin L-related cysteine proteinase. SFL17 may be the partial cDNA of trout cathepsin K. It had 70% identity and 89% similarity with rabbit and human cathepsin K at the amino acid level.
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