Purification, characterization, and cDNA cloning of procathepsin L from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly), and its involvement in the differentiation of imaginal discs.
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.
Homma
K
K
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Kurata
S
S
Natori
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary; D004175:Diptera / Q000196:embryology / Q000201:enzymology*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D018076:DNA, Complementary; D004792:Enzyme Precursors; D012333:RNA, Messenger; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L