| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Cathepsin E is a member of the aspartic proteinase superfamily. The amino-terminal structure of rat gastric cathepsin E was identified and compared with the corresponding regions of human procathepsin E and other aspartic proteinases. The alignment revealed that cathepsin E has the most extended amino-terminal structure in the superfamily, thus suggesting that the activation peptide(propeptide)of the human enzyme is 39-residues long. Analysis of oligosaccharide units suggested that rat cathepsin E possesses one N-linked carbohydrate unit, probably of the high mannose type. No evidence was obtained for the presence of 0-linked sugars in rat cathepsin E.
Macrophages labelled with[^<35> S]Met for 30 min contained mainly a single radioactive polypeptide immunoprecipitable with polyclonal antibody to rat gastric cathepsin E. The apparent molecular weight of the predominant band was 46kDa. This band was detectable even in 48hr-chased cells, but any newly formed, smaller molecule was not detected in cells chased for 1 to 48hr. When monoclonal-antibody was used as immunoprecipitant, no radioactive peptides were recovered from protein A-Cellulofine beads.
Rat gastric cathepsin E cleaved big endothelin-1, -2 and -3 to generate mature endothelins, under mildly acidic conditions. The reaction was highly for endothelin generation, as judged by amino acid analysis of HPLC separated peptides and by their cross-reactivity with monoclonal antibody to the carboxy-terminal segment of endothelin-1. It was found, however, that human, bovine or rat endothelial cells possessed no detectable amounts of cathepsin E activity, indicating that the enzyme is not responsible for the endothelin production by endothelial cells. Possible participation of cathepsin E in endothelin generation in vivo is discussed in Ref. 3.
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