Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022.
BM 06.022 is a t-PA deletion variant which comprises the kringle 2 and the protease domain. Production of BM 06.022 in Escherichia coli leads to the formation of inactive inclusion bodies, which have to be refolded by an in vitro refolding process to achieve activity and proper structure of the domains. We analysed the biochemical properties of BM 06.022 to obtain some information about the structure of kringle 2 and the protease as compared with the structure of these domains in the intact t-PA molecule. The kinetic analysis of the amidolytic activity of BM 06.022 and CHO-t-PA yielded similar values for kcat (13.9 s-1 and 11.4 s-1 for the single chain forms and 33.9 s-1 and 27.1 s-1 for the two chain forms of BM 06.022 and CHO-t-PA, respectively) and for Km (2.5 mM and 2.1 mM for the single chains forms and 0.5 mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA, respectively). BM 06.022 and CHO-t-PA have the same plasminogenolytic activity in the absence of CNBr fragments of fibrinogen. However, BM 06.022 has a lower plasminogenolytic activity in the presence of CNBr fragments of fibrinogen and a lower affinity to fibrin as compared with CHO-t-PA. The affinity of BM 06.022 for fibrin is completely suppressed by 0.3 mM epsilon-aminocaproic acid, while the intact t-PA has a residual affinity of approximately 30%. The dissociation constants for the interaction with the lysine analogue epsilon-aminocaproic acid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA, respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibited by PAI-1 in a similar manner.
Kohnert
U
U
Boehringer Mannheim GmbH, Biochemical Research Center Penzberg, Germany.
Rudolph
R
R
Verheijen
J H
JH
Weening-Verhoeff
E J
EJ
Stern
A
A
Opitz
U
U
Martin
U
U
Lill
H
H
Prinz
H
H
Lechner
M
M
D000581:Amidohydrolases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005337:Fibrin / Q000494:pharmacology; D005341:Fibrinolysin / Q000378:metabolism; D002479:Inclusion Bodies; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D000581:Amidohydrolases; D008239:Lysine