Proteolytic modification of tissue plasminogen activator: importance of the N-terminal part of the catalytically active B-chain for enzymatic activity.
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.
Norrman
B
B
Department of Physiological Chemistry, Umeå University, Sweden.
Ohlsson
P I
PI
Wallén
P
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D010450:Endopeptidases; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012685:Sepharose; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D012685:Sepharose; D010450:Endopeptidases; D014357:Trypsin; D010959:Tissue Plasminogen Activator; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase