A protease-hypersensitive deletion derivative of human tissue-type plasminogen activator.
We have constructed amplified Chinese hamster ovary cell lines constitutively synthesizing human tissue-type plasminogen activator (t-PA) or a derivative in which the domains homologous to epidermal growth factor and kringle 1 have been removed [delta(G + K1)]. The properties of the secreted proteins were investigated when synthesized in the presence or absence of the serine protease inhibitor aprotinin in the medium. t-PA in the culture supernatants was either single-chain or two-chain protein. The protease activity of both forms was stimulated by fibrin. The biochemical properties of delta(G + K1) were significantly different when harvested from cells grown under different culturing conditions. Protease activity of delta(G + K1) was stimulated ten- to 20-fold by fibrin when harvested from medium with aprotinin, but was stimulated only two- to three-fold when aprotinin was absent from the serum. Characterization of the secreted proteins revealed that the heavy-chain equivalent of delta(G + K1) is degraded when serine protease inhibitor is absent in the culture medium. These results indicate that the functional and biochemical properties of restructured versions of t-PA may depend on the presence of protease(s) in the culture supernatants.
Weidle
U H
UH
Department of Genetics, Boehringer Mannheim, GmbH, Penzberg, F.R.G.
Buckel
P
P
Mattes
R
R
D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D004274:DNA, Recombinant / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D010959:Tissue Plasminogen Activator