Proteolytic modification of tissue plasminogen activator: importance of the N-terminal part of the catalytically active B-chain for enzymic activity
| Autor: | Per Ingvar Ohlsson, Bosse Norrman, Per Wallén |
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| Rok vydání: | 1988 |
| Předmět: |
Protein Conformation
Stereochemistry Molecular Sequence Data Peptide Cleavage (embryo) Biochemistry Protein structure Endopeptidases medicine Trypsin Amino Acid Sequence Peptide sequence chemistry.chemical_classification Binding Sites Activator (genetics) Sepharose Metalloendopeptidases Peptide Fragments Amino acid Kinetics chemistry Tissue Plasminogen Activator Plasminogen activator medicine.drug |
| Zdroj: | Biochemistry. 27:8325-8330 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Native one-chain tissue plasminogen activator (t-PA) was rapidly converted to the two-chain form by trypsin-Sepharose cleavage. This caused an increase in the amidolytic activity on low molecular weight peptide substrates, while plasminogen activation in the presence of fibrin markedly decreased. Cleavage sites were identified by N-terminal sequence analysis of reduced and carboxymethylated peptides. In the B-chain, the expected cleavage at Arg278-Ile279 was identified. Furthermore, a specific cleavage site was found at Arg302-Ser303, 24 amino acids from the N-terminus of the B-chain. The peptide released by this cleavage (designated B1-24) remained associated with the activator molecule by strong noncovalent interactions but could be dissociated under denaturing conditions (4 mol/L of guanidine hydrochloride), leading to a 20-fold decrease in amidolytic activity. Addition of purified B1-24 peptide to t-PA treated in this manner restored the activity in a concentration-dependent way. In contrast to trypsin, cleavage of the single-chain t-PA molecule with endoproteinase Lys-C generated a two-chain form of the activator, without simultaneous increase in the amidolytic activity. By sequence analysis, a major cleavage was identified at Lys280-Gly281, two residues into the B-chain. Together, the results presented provide additional information on the one-chain to two-chain conversion of t-PA and the role of the free N-terminus of the B-chain. |
| Databáze: | OpenAIRE |
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