Abstract
A cysteine proteinase that preferentially hydrolyzes carbobenzyloxy-Phe-Arg-MCA (MCA, methylcoumaryl-7-amide) was purified from the culture medium of NIH-Sape-4 cells. The molecular mass of this enzyme was easily changed from 50 to 35 kDa, without appreciable loss of enzyme activity. From the analysis of its cDNA, this enzyme was concluded to be a procathepsin L of Sarcophaga. Only procathepsin L was found in the medium, while the mature 35-kDa cathepsin L was found exclusively in the cells, indicating that the cells have a mechanism to secrete procathepsin L selectively. An antibody against procathepsin L was found to inhibit the differentiation of imaginal discs cultured in vitro in the presence of 20-hydroxyecdysone. In fact imaginal discs were shown to secrete procathepsin L into the culture medium. These results suggest that procathepsin L is an essential proteinase for differentiation of imaginal discs. Northern blotting analysis revealed that unfertilized eggs contain a significant amount of mRNA for the procathepsin L as a maternal mRNA.
Highlights
Procathepsin L was found in the me- is the only secretory proteinasseo far knownin Sarcophaga, we dium, while the mature 35-kDa cathepsin L was found exclusively in the cells, indicating that the cells have mechanism to secrete procathepsLinselectively.An antibody against procathepsinL was found to inhibit the differentiationof imaginal discs culturedin vitro in the ieamxaamgiinnaeld if this enzyme participates discs.When imaginal discs in the were morphogenesis cultured in the of presence of 20-hydroxyecdysone,theysecretedthesameprocathepsin L that we purified from the culture medium of NIH
Procathepsin L a n d Imaginal Disc Differentiation-We found cathepsin L into the culture medium during their differentiathat procathepsinL is essentialfor the differentiation of imagi- tion
We found by immunoblotting that the culture medium of NIH-Sape-4 cells contained only procathepsin L but thatpurified procathepsin L was converted to mature cathepsin
Summary
Denhardt's solution, 0.02% (w/v)each of Ficoll-400, bovineserum albu- purified enzyme of 50 kDa was kept at 4 "C for 24 h, it was min, and polyvinylpyrrolidone)containing 50 pg/ml denatured and sonicated salmon sperm DNA [21].After prehybridization, the filters were hybridized with 5' end-labeled probe 1 and 2 in 4 x SSC, 10 x Denhardt's solution containing 25 pg/ml denatured and sonicated salmon sperm DNA for 12 h a t 48 and 52 "C,respectively. the filters were completely convertedto a 35-kDa protein, as shown in Fig. l a , without appreciable loss of proteinase activity. MKVAVLFLCGVALAAASPSWEHFKGKYGRQWDAEEDSYRR determine the complete amino acid sequence of this 50-kDa proteinase, we isolated a cDNA clone for this enzyme. Incontrast,mature almost no differentiation ( b )or remained in the stageof early cathepsin L was detected in the imaginal discs regardless of eversion with only a small projection (e), when cultured in the their stageof differentiation (lanes 4-6) These results indicate presence of antibody against procathepsin L. Immunized or normal IgG was added at a concentration of 5 pdml, and mature 35-kDa cathepsin L was added at a concentration of 42 udml
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